Michael Schlame, M.D.

Cardiolipin Affects the Supramolecular Organization of ATP Synthase in Mitochondria
Acehan, Devrim; Malhotra, Ashim; Xu, Yang; Ren, Mindong; Stokes, David L; Schlame, Michael
2011 May 4;100(9):2184-2192, Biophysical journal
F(1)F(0) ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich in ATP synthase. Our study included a wild-type control, a cardiolipin synthase mutant with nearly complete loss of cardiolipin, and a tafazzin mutant with reduced cardiolipin levels. In the wild-type, the high-curvature edge of crista vesicles was densely populated with ATP synthase molecules that were typically organized in one or two rows of dimers. In both mutants, the density of ATP synthase was reduced at the high-curvature zone despite unchanged expression levels. Compared to the wild-type, dimer rows were less extended in the mutants and there was more scatter in the orientation of dimers. These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the crista membrane
— id: 131973, year: 2011, vol: 100, page: 2184, stat: Journal Article,

Prenatal lethality and cardiac function in a mouse model of barth syndrome
Phoon C.K.L.; Acehan D.; Schlame M.; Stokes D.L.; Edelman-Novemsky I.; Yu D.; Xu Y.; Ren M.
2011 ;24(5):B27-B28, Journal of the American Society of Echocardiography
Background: Barth syndrome (BTHS) is a rare multisystem disorder caused by mutations in tafazzin that lead to cardiolipin deficiency and mitochondrial abnormalities. Patients most commonly present with early-onset cardiomyopathy, including fetal cardiomyopathy. A newly-developed transgenic mouse induces tafazzin deficiency using a doxycycline-inducible shRNA knockdown (TAZKD). Methods: TAZKD mice and wildtype controls were fed doxycycline starting in early gestation, via the mother (gestation and pre-weanling stages) or directly. 40 MHz echocardiography (axial resolution: 40 microns) with spectral and color Doppler capabilities defined in vivo cardiac function throughout fetal, newborn, and adult ages. Functional data were correlated with cardiolipin mass spectrometry, histology, and electron microscopy. Results: Abnormal cardiolipin profiles in TAZKD mice at embryonic (E13.5) and newborn stages, confirmed high-efficiency tafazzin knockdown during development. Newborn, juvenile, and adult mice did not show an obvious cardiomyopathic phenotype through 6 months of age. However, far fewer TAZKD mice were born than the expected 50:50 Mendelian ratios (4/26 TAZKD liveborn; p<0.02). We then focused on embryonic/fetal imaging of cardiovascular function at E13.5 (N=7 wildtype, N=4 TAZKD). Notably, we found a spectrum, from entirely normal function, including systolic and diastolic function, heart rate, atrioventricular conduction and rhythm, and umbilical arterial and venous flows; to a grossly abnormal embryo predicted (then confirmed) to be TAZKD based on severe bradycardia, holodiastolic aortic flow reversal, and a systolic atrial kick that suggested elevated myocardial stiffness. Echo suggested LV noncompaction in another embryo later confirmed to be TAZKD. Histology showed qualitatively thinner TAZKD ventricular myocardium with more prominent trabeculae suggestive of LV noncompaction. Electron microscopy of TAZKD embryonic hearts, similar to echocardiography, demonstrated a spectrum from normal to severely abnormal mitochondrial structures. Notably, mitochondria from TAZKD embryonic hearts with grossly abnormal hemodynamics tended to have poorly-formed lamellar cristae and disruption of the sarcomeric organization. Conclusion: A spectrum of functional and cellular cardiomyopathic abnormalities associated with prenatal lethality is seen in this novel model of human BTHS. Experiments are ongoing to better link cellular pathophysiological processes with the whole-organ/systems hemodynamics defined by in vivo embryonic mouse echocardiography
— id: 132321, year: 2011, vol: 24, page: B27, stat: Journal Article,

Characterization of a Transgenic Short Hairpin RNA-Induced Murine Model of Tafazzin Deficiency
Soustek, Meghan S; Falk, Darin J; Mah, Cathryn S; Toth, Matthew J; Schlame, Michael; Lewin, Alfred S; Byrne, Barry J
2011 Jul;22(7):865-871, Human gene therapy
Abstract Barth's syndrome (BTHS) is an X-linked mitochondrial disease that is due to a mutation in the Tafazzin (TAZ) gene. Based on sequence homology, TAZ has been characterized as an acyltransferase involved in the metabolism of cardiolipin (CL), a unique phospholipid almost exclusively located in the mitochondrial inner membrane. Yeast, Drosophila, and zebrafish models have been invaluable in elucidating the role of TAZ in BTHS, but until recently a mammalian model to study the disease has been lacking. Based on in vitro evidence of RNA-mediated TAZ depletion, an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model has been developed (TaconicArtemis GmbH, Cologne, Germany), and herein we describe the assessment of this mouse line as a model of BTHS. Upon induction of the TAZ-specific shRNA in vivo, transgenic mouse TAZ mRNA levels were reduced by >89% in cardiac and skeletal muscle. TAZ deficiency led to the absence of tetralineoyl-CL and accumulation of monolyso-CL in cardiac muscle. Furthermore, mitochondrial morphology from cardiac and skeletal muscle was altered. Skeletal muscle mitochondria demonstrated disrupted cristae, and cardiac mitochondria were significantly enlarged and displace neighboring myofibrils. Physiological measurements demonstrated a reduction in isometric contractile strength of the soleus and a reduction in cardiac left ventricular ejection fraction of TAZKD mice compared with control animals. Therefore, the inducible TAZ-deficient model exhibits some of the molecular and clinical characteristics of BTHS patients and may ultimately help to improve our understanding of BTHS-related cardioskeletal myopathy as well as serve as an important tool in developing therapeutic strategies for BTHS
— id: 136498, year: 2011, vol: 22, page: 865, stat: Journal Article,

Genetic suppressors of Barth syndrome as potential targets for therapeutic intervention
Malhotra, A; Edelman-Novemsky, I; Xu, Y; Ma, JP; Schlame, M; Ren, MD
2010 MAR ;10(2):207-208, Mitochondrion
— id: 109829, year: 2010, vol: 10, page: 207, stat: Journal Article,

Distinct effects of tafazzin deletion in differentiated and undifferentiated mitochondria
Acehan, Devrim; Khuchua, Zaza; Houtkooper, Riekelt H; Malhotra, Ashim; Kaufman, Johanna; Vaz, Frederic M; Ren, Mindong; Rockman, Howard A; Stokes, David L; Schlame, Michael
2009 Apr;9(2):86-95, Mitochondrion
Tafazzin is a conserved mitochondrial protein that is required to maintain normal content and composition of cardiolipin. We used electron tomography to investigate the effect of tafazzin deletion on mitochondrial structure and found that cellular differentiation plays a crucial role in the manifestation of abnormalities. This conclusion was reached by comparing differentiated cardiomyocytes with embryonic stem cells from mouse and by comparing different tissues from Drosophila melanogaster. The data suggest that tafazzin deficiency affects cardiolipin in all mitochondria, but significant alterations of the ultrastructure, such as remodeling and aggregation of inner membranes, will only occur after specific differentiation
— id: 94434, year: 2009, vol: 9, page: 86, stat: Journal Article,

Role of calcium-independent phospholipase A2 in the pathogenesis of Barth syndrome
Malhotra, Ashim; Edelman-Novemsky, Irit; Xu, Yang; Plesken, Heide; Ma, Jinping; Schlame, Michael; Ren, Mindong
2009 Feb 17;106(7):2337-2341, Proceedings of the National Academy of Sciences of the United States of America
Quantitative and qualitative alterations of mitochondrial cardiolipin have been implicated in the pathogenesis of Barth syndrome, an X-linked cardioskeletal myopathy caused by a deficiency in tafazzin, an enzyme in the cardiolipin remodeling pathway. We have generated and previously reported a tafazzin-deficient Drosophila model of Barth syndrome that is characterized by low cardiolipin concentration, abnormal cardiolipin fatty acyl composition, abnormal mitochondria, and poor motor function. Here, we first show that tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility. This phenotype can be genetically suppressed by inactivation of the gene encoding a calcium-independent phospholipase A(2), iPLA2-VIA, which also prevents cardiolipin depletion/monolysocardiolipin accumulation, although in wild-type flies inactivation of the iPLA2-VIA does not affect the molecular composition of cardiolipin. Furthermore, we show that treatment of Barth syndrome patients' lymphoblasts in tissue culture with the iPLA(2) inhibitor, bromoenol lactone, partially restores their cardiolipin homeostasis. Taken together, these findings establish a causal role of cardiolipin deficiency in the pathogenesis of Barth syndrome and identify iPLA2-VIA as an important enzyme in cardiolipin deacylation, and as a potential target for therapeutic intervention
— id: 94433, year: 2009, vol: 106, page: 2337, stat: Journal Article,

Formation of molecular species of mitochondrial cardiolipin. 1. A novel transacylation mechanism to shuttle fatty acids between sn-1 and sn-2 positions of multiple phospholipid species
Malhotra, Ashim; Xu, Yang; Ren, Mindong; Schlame, Michael
2009 Apr;1791(4):314-320, Biochimica & biophysica acta
Mitochondrial cardiolipin undergoes extensive remodeling of its acyl groups to generate uniformly substituted species, such as tetralinoleoyl-cardiolipin, but the mechanism of this remodeling has not been elucidated, except for the fact that it requires tafazzin. Here we show that purified recombinant Drosophila tafazzin exchanges acyl groups between cardiolipin and phosphatidylcholine by a combination of forward and reverse transacylations. The acyl exchange is possible in the absence of phospholipase A(2) because it requires only trace amounts of lysophospholipids. We show that purified tafazzin reacts with various phospholipid classes and with various acyl groups both in sn-1 and sn-2 position. Expression studies in Sf9 insect cells suggest that the effect of tafazzin on cardiolipin species is dependent on the cellular environment and not on enzymatic substrate specificity. Our data demonstrate that tafazzin catalyzes general acyl exchange between phospholipids, which raises the question whether pattern formation in cardiolipin is the result of the equilibrium distribution of acyl groups between multiple phospholipid species
— id: 98902, year: 2009, vol: 1791, page: 314, stat: Journal Article,

Formation of molecular species of mitochondrial cardiolipin 2. A mathematical model of pattern formation by phospholipid transacylation
Schlame, Michael
2009 Apr;1791(4):321-325, Biochimica & biophysica acta
Formation of the unique molecular species of mitochondrial cardiolipin requires tafazzin, a transacylase that exchanges acyl groups between phospholipid molecular species without strict specificity for acyl groups, head groups, or carbon positions. However, it is not known whether phospholipid transacylations can cause the accumulation of specific fatty acids in cardiolipin. Here, a model is shown in linear algebra representation, in which acyl specificity emerges from the transacylation equilibrium of multiple molecular species, provided that different species have different free energies. The model defines the conditions and energy terms, under which transacylations may generate the characteristic composition of mitochondrial cardiolipin. It is concluded that acyl-specific cardiolipin patterns could arise from phospholipid transacylations in the tafazzin domain, even if tafazzin itself does not have substrate specificity
— id: 98901, year: 2009, vol: 1791, page: 321, stat: Journal Article,

The role of cardiolipin in the structural organization of mitochondrial membranes
Schlame, Michael; Ren, Mindong
2009 Oct;1788(10):2080-2083, Biochimica & biophysica acta
Considerable progress has recently been made in understanding the role of cardiolipin in mitochondria. In this brief review, we discuss new data that show how cardiolipin specifically contributes to the lateral organization of mitochondrial membranes. We argue that the function of cardiolipin has to be understood in the context of dynamic membrane assembly rather than static membrane structure, and we propose that remodeling of cardiolipin, i.e. the formation of uniformly substituted molecular species, may reduce the energy barrier of the assembly process
— id: 103149, year: 2009, vol: 1788, page: 2080, stat: Journal Article,

Characterization of tafazzin splice variants from humans and fruit flies
Xu, Yang; Zhang, Shali; Malhotra, Ashim; Edelman-Novemsky, Irit; Ma, Jinping; Kruppa, Antonina; Cernicica, Carolina; Blais, Steven; Neubert, Thomas A; Ren, Mindong; Schlame, Michael
2009 Oct 16;284(42):29230-29239, Journal of biological chemistry
The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (Delta5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Delta5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the Delta5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin
— id: 104345, year: 2009, vol: 284, page: 29230, stat: Journal Article,

A linear algebra model of the tafazzin reaction
Schlame, M
2008 AUG ;154(6):S28-S29, Chemistry & physics of lipids
— id: 86827, year: 2008, vol: 154, page: S28, stat: Journal Article,

Mechanism and significance of acyl remodeling of mitochondrial cardiolipin
Schlame, M; Malhotra, A; Acehan, D; Ren, MD; Xu, Y
2008 AUG ;154(6):S28-S28, Chemistry & physics of lipids
— id: 86826, year: 2008, vol: 154, page: S28, stat: Journal Article,

Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes
Schlame, Michael
2008 Aug;49(8):1607-1620, Journal of lipid research
In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease
— id: 83099, year: 2008, vol: 49, page: 1607, stat: Journal Article,

Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography
Acehan, Devrim; Xu, Yang; Stokes, David L; Schlame, Michael
2007 Jan;87(1):40-48, Laboratory investigation
Barth syndrome (BTHS) is a mitochondrial disorder that is caused by mutations in the tafazzin gene, which affects phospholipid composition. To determine whether this defect leads to alterations in the internal three-dimensional organization of mitochondrial membranes, we applied electron microscopic tomography to lymphoblast mitochondria from BTHS patients and controls. Tomograms were formed from 50 and 150 nm sections of chemically fixed lymphoblasts and the data were used to manually segment volumes of relevant structural details. Normal lymphoblast mitochondria contained well-aligned, lamellar cristae with slot-like junctions to the inner boundary membrane. In BTHS, mitochondrial size was more variable and the total mitochondrial volume per cell increased mainly due to clusters of fragmented mitochondria inside nuclear invaginations. However, mitochondria showed reduced cristae density, less cristae alignment, and inhomogeneous cristae distribution. Three-dimensional reconstruction of BTHS mitochondria revealed zones of adhesion of the opposing inner membranes, causing obliteration of the intracrista space. We found small isolated patches of adhesion as well as extended adhesion zones, resulting in sheets of collapsed cristae packaged in multiple concentric layers. We also found large tubular structures (diameter 30-150 nm) that appeared to be derivatives of the adhesion zones. The data suggest that mitochondrial abnormalities of BTHS involve adhesions of inner mitochondrial membranes with subsequent collapse of the intracristae space
— id: 71408, year: 2007, vol: 87, page: 40, stat: Journal Article,

De novo biosynthesis of the late endosome lipid, bis(monoacylglycero)phosphate
Hullin-Matsuda, Francoise; Kawasaki, Kiyoshi; Delton-Vandenbroucke, Isabelle; Xu, Yang; Nishijima, Masahiro; Lagarde, Michel; Schlame, Michael; Kobayashi, Toshihide
2007 Sep;48(9):1997-2008, Journal of lipid research
Bis(monoacylglycero)phosphate (BMP) is a unique lipid enriched in the late endosomes participating in the trafficking of lipids and proteins through this organelle. The de novo biosynthesis of BMP has not been clearly demonstrated. We investigated whether phosphatidylglycerol (PG) and cardiolipin (CL) could serve as precursors of de novo BMP synthesis using two different cellular models: CHO cells deficient in phosphatidylglycerophosphate (PGP) synthase, the enzyme responsible for the first step of PG synthesis; and human lymphoblasts from patients with Barth syndrome (BTHS), characterized by mutations in tafazzin, an enzyme implicated in the deacylation-reacylation cycle of CL. The biosynthesis of both PG and BMP was reduced significantly in the PGP synthase-deficient CHO mutants. Furthermore, overexpression of PGP synthase in the deficient mutants induced an increase of BMP biosynthesis. In contrast to CHO mutants, BMP biosynthesis and its fatty acid composition were not altered in BTHS lymphoblasts. Our results thus suggest that in mammalian cells, PG, but not CL, is a precursor of the de novo biosynthesis of BMP. Despite the decrease of de novo synthesis, the cellular content of BMP remained unchanged in CHO mutants, suggesting that other pathway(s) than de novo biosynthesis are also used for BMP synthesis
— id: 94642, year: 2007, vol: 48, page: 1997, stat: Journal Article,

Assays of cardiolipin levels
Schlame, Michael
2007 ;80:223-240, Methods in cell biology
— id: 72030, year: 2007, vol: 80, page: 223, stat: Journal Article,

Evaluation of aromatherapy in treating postoperative pain: pilot study
Kim, Jung T; Wajda, Michael; Cuff, Germaine; Serota, David; Schlame, Michael; Axelrod, Deborah M; Guth, Amber A; Bekker, Alex Y
2006 Dec;6(4):273-277, Pain practice
This study compared the analgesic efficacy of postoperative lavender oil aromatherapy in 50 patients undergoing breast biopsy surgery. Twenty-five patients received supplemental oxygen through a face mask with two drops of 2% lavender oil postoperatively. The remainder of the patients received supplemental oxygen through a face mask with no lavender oil. Outcome variables included pain scores (a numeric rating scale from 0 to 10) at 5, 30, and 60 minutes postoperatively, narcotic requirements in the postanesthesia care unit (PACU), patient satisfaction with pain control, as well as time to discharge from the PACU. There were no significant differences in narcotic requirements and recovery room discharge times between the two groups. Postoperative lavender oil aromatherapy did not significantly affect pain scores. However, patients in the lavender group reported a higher satisfaction rate with pain control than patients in the control group (P = 0.0001)
— id: 69409, year: 2006, vol: 6, page: 273, stat: Journal Article,

Barth syndrome, a human disorder of cardiolipin metabolism
Schlame, Michael; Ren, Mindong
2006 Oct 9;580(23):5450-5455, FEBS letters
Barth syndrome is an X-linked recessive disease caused by mutations in the tafazzin gene. Patients have reduced concentration and altered composition of cardiolipin, the specific mitochondrial phospholipid, and they have variable clinical findings, often including heart failure, myopathy, neutropenia, and growth retardation. This article provides an overview of the molecular basis of Barth syndrome. It is argued that tafazzin, a phospholipid acyltransferase, is involved in acyl-specific remodeling of cardiolipin, which promotes structural uniformity and molecular symmetry among the cardiolipin molecular species. Inhibition of this pathway leads to changes in mitochondrial architecture and function
— id: 69247, year: 2006, vol: 580, page: 5450, stat: Journal Article,

A Drosophila model of Barth syndrome
Xu, Yang; Condell, Morgan; Plesken, Heide; Edelman-Novemsky, Irit; Ma, Jinping; Ren, Mindong; Schlame, Michael
2006 Aug 1;103(31):11584-11588, Proceedings of the National Academy of Sciences of the United States of America
Barth syndrome is an X-linked disease presenting with cardiomyopathy and skeletal muscle weakness. It is caused by mutations in tafazzin, a putative acyl transferase that has been associated with altered metabolism of the mitochondrial phospholipid cardiolipin. To investigate the molecular basis of Barth syndrome, we created Drosophila melanogaster mutants, resulting from imprecise excision of a P element inserted upstream of the coding region of the tafazzin gene. Homozygous flies for that mutation were unable to express the full-length isoform of tafazzin, as documented by RNA and Western blot analysis, but two shorter tafazzin transcripts were still present, although the expression levels of their encoded proteins were too low to be detectable by Western blotting. The tafazzin mutation caused an 80% reduction of cardiolipin and a diversification of its molecular composition, similar to the changes seen in Barth patients. Other phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were not affected. Flies with the tafazzin mutation showed a reduced locomotor activity, measured in flying and climbing assays, and their indirect flight muscles displayed frequent mitochondrial abnormalities, mostly in the cristae membranes. Thus, tafazzin mutations in Drosophila generated a Barth-related phenotype, with the triad of abnormal cardiolipin, pathologic mitochondria, and motor weakness, suggesting causal links between these findings. We conclude that a lack of full-length tafazzin is responsible for the cardiolipin deficiency, which is integral to the disease mechanism, leading to mitochondrial myopathy
— id: 68981, year: 2006, vol: 103, page: 11584, stat: Journal Article,

The enzymatic function of tafazzin
Xu, Yang; Malhotra, Ashim; Ren, Mindong; Schlame, Michael
2006 Dec 22;281(51):39217-39224, Journal of biological chemistry
Tafazzin is a putative enzyme that is involved in cardiolipin metabolism, it may carry mutations responsible for Barth syndrome. To identify the biochemical reaction catalyzed by tafazzin, we expressed the full-length isoform of Drosophila melanogaster tafazzin in a baculovirus-Sf9 insect cell system. Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria, namely 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine:monolysocardiolipin linoleoyltransferase. We also found evidence for the reverse reaction, because tafazzin expression caused transfer of acyl groups from phospholipids to 1-[14C]palmitoyl-2-lyso-phosphatidylcholine. An affinity-purified tafazzin construct, tagged with the maltose-binding protein, catalyzed both forward and reverse transacylations between cardiolipin and phosphatidylcholine, but was unable to utilize CoA or acyl-CoA as substrates. Whereas tafazzin supported transacylations between various phospholipid-lysophospholipid pairs, it showed the highest rate for the phosphatidylcholine-cardiolipin transacylation. Transacylation activities were about 10-fold higher for linoleoyl groups than for oleoyl groups, and they were negligible for arachidonoyl groups. The data show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid transacylase with substrate preference for cardiolipin and phosphatidylcholine
— id: 70309, year: 2006, vol: 281, page: 39217, stat: Journal Article,

Binding of 10-N-nonyl acridine orange to cardiolipin-deficient yeast cells: implications for assay of cardiolipin
Gohil, Vishal M; Gvozdenovic-Jeremic, Jelena; Schlame, Michael; Greenberg, Miriam L
2005 Aug 15;343(2):350-352, Analytical biochemistry
— id: 72031, year: 2005, vol: 343, page: 350, stat: Journal Article,

Molecular symmetry in mitochondrial cardiolipins
Schlame, Michael; Ren, Mindong; Xu, Yang; Greenberg, Miriam L; Haller, Ivan
2005 Dec;138(1-2):38-49, Chemistry & physics of lipids
Cardiolipin is a unique mitochondrial phospholipid with an atypical fatty acid profile, but the significance of its acyl specificity has not been understood. We explored the enormous combinatorial diversity among cardiolipin species, which results from the presence of four fatty acids in each molecule, by integrated use of high-performance liquid chromatography, mass spectrometry, diacylglycerol species analysis, fatty acid analysis, and selective cleavage of fatty acids by phospholipase A2. The most abundant cardiolipin species from various organisms and tissues (human heart, human lymphoblasts, rat liver, Drosophila, sea urchin sperm, yeast, mung bean hypocotyls) contained only one or two types of fatty acids, which generated a high degree of structural uniformity and molecular symmetry. However, an exception was found in patients with Barth syndrome, in whom an acyltransferase deficiency led to loss of acyl selectivity and formation of multiple molecular species. These results suggest that restriction of the number of fatty acid species, rather than the selection of a particular kind of fatty acid, is the common theme of eukaryotic cardiolipins. This limits the structural diversity of the cardiolipin species and creates molecular symmetry with implications for the stereochemistry of cardiolipin
— id: 62637, year: 2005, vol: 138, page: 38, stat: Journal Article,

Vitamin E differentially regulates the expression of peroxiredoxin-1 and -6 in alveolar type II cells
Tolle, Angelika; Schlame, Michael; Charlier, Nico; Guthmann, Florian; Rustow, Bernd
2005 May 15;38(10):1401-1408, Free radical biology & medicine
Vitamin E is the primary lipophilic antioxidant in mammals. Lack of vitamin E may lead to an increase of cytotoxic phospholipid-peroxidation products (PL-Ox). However, we could previously show that alimentary vitamin E-depletion in rats did not change the concentrations of dienes, hydroperoxides, and platelet-activating factor-related oxidation products in alveolar type II cells (TII cells). We hypothesized that vitamin E deficiency increases the activity of enzymes involved in the degradation of PL-Ox. Degradation of PL-Ox may be catalyzed by phospholipase A2, PAF-acetylhydrolase, or peroxiredoxins (Prx's). Alimentary vitamin E deficiency in rats increased the expression of Prx-1 at the mRNA and protein levels and the formation of Prx-SO3, but it did not change the expression of Prx-6 or the activity of phospholipase A2 and PAF-acetylhydrolase in TII cells. H2O2-induced oxidative stress in isolated TII cells activated protein kinase Calpha (PKCalpha) and increased the expression of Prx-1 and Prx-6. Inhibition of PKCalpha in isolated TII cells by long-time incubation with PMA inhibited PKCalpha and Prx-1 but not Prx-6. We concluded that the expression of Prx-1 and -6 is selectively regulated in TII cells; PKCalpha regulates the expression of Prx-1 but not Prx-6. Prx-6 expression may be closely linked to lipid peroxidation
— id: 72032, year: 2005, vol: 38, page: 1401, stat: Journal Article,

Characterization of lymphoblast mitochondria from patients with Barth syndrome
Xu, Yang; Sutachan, John J; Plesken, Heide; Kelley, Richard I; Schlame, Michael
2005 Jun;85(6):823-830, Laboratory investigation
Barth syndrome (BTHS) is a multisystem disorder of individuals who carry mutations in tafazzin, a putative phospholipid acyltransferase. We investigated the hypothesis that BTHS is caused by specific impairment of the mitochondrial lipid metabolism. The fatty acid composition of all major mitochondrial phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL), changed in lymphoblasts from BTHS patients. These changes were most extensive in CL and least extensive in PE. The complementary nature of the fatty acid alterations in CL and PC suggested that fatty acid transfer between these two lipids was inhibited in BTHS. Fluorescence staining and electron microscopy showed abnormal proliferation of mitochondria in BTHS lymphoblasts. The mitochondrial membrane potential, monitored with the fluorescence probe JC-1, was reduced in BTHS lymphoblasts. However, mitochondrial ATP formation of permeabilized lymphoblasts remained unaffected in BTHS. The data suggest that phospholipid abnormalities of BTHS mitochondria led to partial uncoupling of oxidative phosphorylation and that lymphoblasts compensated for this deficiency by expanding the mitochondrial compartment.Laboratory Investigation (2005) 85, 823-830, advance online publication, 4 April 2005; doi:10.1038/labinvest.3700274
— id: 56153, year: 2005, vol: 85, page: 823, stat: Journal Article,

Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent
Gohil, Vishal M; Hayes, Paulette; Matsuyama, Shigemi; Schagger, Hermann; Schlame, Michael; Greenberg, Miriam L
2004 Oct 8;279(41):42612-42618, Journal of biological chemistry
Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes. We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants. In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis. Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants. CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively. The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression. These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the DeltapH component of the proton-motive force generated by the functional electron transport chain. This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH
— id: 49192, year: 2004, vol: 279, page: 42612, stat: Journal Article,

Vitamin E deficiency reduces surfactant lipid biosynthesis in alveolar type II cells
Guthmann, Florian; Kolleck, Ingrid; Schachtrup, Christian; Schlame, Michael; Spener, Friedrich; Rustow, Bernd
2003 Mar 15;34(6):663-673, Free radical biology & medicine
Reactive oxygen species play an important role in development of lung injury. Neonates exhibit a high risk of developing acute and/or chronic lung disorder, often associated with surfactant deficiency, and in parallel they show low vitamin E concentration. We investigated whether the vitamin E status of adult rats affects the content of phospholipids (PL) in bronchoalveolar lavage and alveolar type II cells. Phosphatidylcholine (PtdCho) is the dominant and functional most important PL in lung surfactant. Therefore, we determined its formation via de novo synthesis and reacylation of lyso-PtdCho in type II cells. Vitamin E depletion caused a decrease of PL content in bronchoalveolar lavage and type II cells and decreased glycerol-3-phosphate O-acyltransferase (G3P-AT) activity, de novo synthesis of PtdCho, and reacylation of lyso-PtdCho in type II cells. Preincubation of type II cell homogenates with dithiothreitol restored the activity of G3P-AT and de novo synthesis but inhibited reacylation. Reacylation was strongly reduced by chelerythrine-mediated inhibition of protein kinase C. We conclude that antioxidant and PKC-modulating properties of vitamin E regulate de novo synthesis of PtdCho and reacylation of lyso-PtdCho in alveolar type II cells. Vitamin E depletion reduced the two pathways of PL synthesis and caused a decrease of PL content in alveolar surfactant of rats
— id: 49193, year: 2003, vol: 34, page: 663, stat: Journal Article,

Phospholipid abnormalities in children with Barth syndrome
Schlame, Michael; Kelley, Richard I; Feigenbaum, Annette; Towbin, Jeffrey A; Heerdt, Paul M; Schieble, Thomas; Wanders, Ronald J A; DiMauro, Salvatore; Blanck, Thomas J J
2003 Dec 3;42(11):1994-1999, Journal of the American College of Cardiology
OBJECTIVES: We sought to identify characteristic lipid abnormalities in patients with Barth syndrome (BTHS) and to correlate the lipid profile to phenotype and genotype. BACKGROUND: Barth syndrome typically includes cardiomyopathy, skeletal myopathy, neutropenia, growth retardation, and 3-methylglutaconic aciduria, and it is commonly associated with mutations in the tafazzin (TAZ) gene, whose products are homologous to phospholipid acyltransferases. However, clinical features of BTHS have also been found in patients with normal TAZ gene. METHODS: We analyzed molecular species of phospholipids in left and right ventricle, skeletal muscle, platelets, lymphoblasts, and fibroblasts from 19 children with BTHS (positive TAZ mutation), 6 children with BTHS-like syndromes (wild-type TAZ), 4 children with isolated cardiomyopathy (wild-type TAZ), and various controls. RESULTS: Cardiolipin, the specific lipid found only in mitochondria, was decreased in all tissues from BTHS patients, whereas concentrations of other phospholipids were normal. The molecular composition of cardiolipin was altered in all tissues from BTHS patients. The molecular compositions of phosphatidylcholine and phosphatidylethanolamine were altered in the heart. Cardiolipin abnormalities were only found in children with true BTHS, not in children with BTHS-like disease or with isolated cardiomyopathy. The degree of cardiolipin deficiency was tissue-specific but did not correlate with severity or specific phenotypic expression of BTHS. CONCLUSIONS: Abnormal cardiolipin is a specific diagnostic marker of cardiomyopathies caused by TAZ mutations. These mutations lead to alterations in the fatty acid composition of several phospholipids, supporting the idea that TAZ encodes a human acyltransferase
— id: 45505, year: 2003, vol: 42, page: 1994, stat: Journal Article,

Remodeling of cardiolipin by phospholipid transacylation
Xu, Yang; Kelley, Richard I; Blanck, Thomas J J; Schlame, Michael
2003 Dec 19;278(51):51380-51385, Journal of biological chemistry
Mitochondrial cardiolipin (CL) contains unique fatty acid patterns, but it is not known how the characteristic molecular species of CL are formed. We found a novel reaction that transfers acyl groups from phosphatidylcholine or phosphatidylethanolamine to CL in mitochondria of rat liver and human lymphoblasts. Acyl transfer was stimulated by ADP, ATP, and ATP gamma S, but not by other nucleotides. Coenzyme A stimulated the reaction only in the absence of adenine nucleotides. Free fatty acids were not incorporated into CL under the same incubation condition. The transacylation required addition of exogenous CL or monolyso-CL, whereas dilyso-CL was not a substrate. Transacylase activity was decreased in lymphoblasts from patients with Barth syndrome (tafazzin deletion), and this was accompanied by drastic changes in the molecular composition of CL. In rat liver, where linoleic acid was the most abundant residue of CL, only linoleoyl groups were transferred into CL, but not oleoyl or arachidonoyl groups. We demonstrated complete remodeling of tetraoleoyl-CL to tetralinoleoyl-CL in rat liver mitochondria and identified the intermediates linoleoyl-trioleoyl-CL, dilinoleoyl-dioleoyl-CL, and trilinoleoyl-oleoyl-CL by high-performance liquid chromatography. The data suggest that CL is remodeled by acyl specific phospholipid transacylation and that tafazzin is an acyltransferase involved in this mechanism
— id: 45506, year: 2003, vol: 278, page: 51380, stat: Journal Article,

Bioactive oxidized lipids in the plasma of cardiac surgical intensive care patients
Frey, Bettina; Johnen, Wolfram; Haupt, Renate; Kern, Hartmut; Rustow, Bernd; Kox, Wolfgang J; Schlame, Michael
2002 Jul;18(1):14-17, Shock
Critical illness is associated with increased oxidative stress that may give rise to the formation of lipid hydroperoxides (LOOH) and various secondary degradation products such as fragmented phosphatidylcholine (FPC) and lipids related to the platelet-activating factor (PAF). Because some oxidized phospholipids are potent proinflammatory agents, we measured the concentration of LOOH, FPC, and PAF-like activity in blood plasma of 36 patients who had undergone cardiac surgery and developed postoperative complications associated with systemic inflammatory response syndrome (SIRS) or multiple organ failure (MOF). These patients were compared to two control groups, namely preoperative patients scheduled for cardiac surgery (n = 13), and postoperative patients without complications (n = 19). Postoperative patents had higher concentrations of LOOH and lower concentrations of FPC than preoperative patients (P < 0.01). However, SIRS and MOF had no significant effect on the concentration of oxidatively modified lipids. This is despite the fact that MOF patients showed evidence of increased lipid peroxidation (7-fold higher ratio of alpha-tocoquinone/alpha-tocopherol compared to control). LOOH correlated positively with the white blood cell count. Postoperative patients had 4-fold higher plasma activities of phospholipase A2 and this activity was further increased in patients with SIRS (P < 0.04). Phospholipase A2 activity correlated negatively with the concentration of FPC. The data suggest that oxidatively modified lipids do not accumulate in patients with SIRS and MOF, perhaps because enhanced peroxidation of lipids is offset by enhanced lipolytic activity
— id: 49194, year: 2002, vol: 18, page: 14, stat: Journal Article,

Disease-specific remodeling of cardiac mitochondria after a left ventricular assist device
Heerdt, Paul M; Schlame, Michael; Jehle, Roswitha; Barbone, Alessandro; Burkhoff, Daniel; Blanck, Thomas J J
2002 Apr;73(4):1216-1221, Annals of thoracic surgery
BACKGROUND: Failing hearts can exhibit elements of structural and molecular 'reverse remodeling' after support with a left ventricular assist device (LVAD). The present study examined LVAD-induced remodeling of cardiac mitochondria. METHODS: Left ventricular tissue from 20 failing and 21 LVAD-supported hearts, catagorized as ischemic (ICM) or dilated (DCM) cardiomyopathy and four nonfailing hearts were studied. Myocyte mitochondrial ultrastructure was assessed by high-performance liquid chromatography determination of cardiolipin, a specific lipid component of the inner membrane, and its three major molecular species: L4, L3O, and L2O2. RESULTS: Both failing and LVAD-supported hearts exhibited a reduction in cardiolipin content that was independent of the type of cardiomyopathy. However, in failing/ICM hearts, there was a 25% increase in the L4/L3O ratio and a 70% increase in the L4/L2O2 ratio, indicating a change in cardiolipin composition. These alterations were normalized by LVAD support. In sharp contrast, molecular species ratios in DCM hearts were the same as those in nonfailing hearts regardless of whether LVAD support had been used or not. CONCLUSIONS: These data demonstrate LVAD-induced reverse remodeling of myocyte cardiolipin composition in ICM but not DCM hearts
— id: 45508, year: 2002, vol: 73, page: 1216, stat: Journal Article,

Cellular cholesterol stimulates acute uptake of palmitate by redistribution of fatty acid translocase in type II pneumocytes
Kolleck, Ingrid; Guthmann, Florian; Ladhoff, Axel-M; Tandon, Narendra N; Schlame, Michael; Rustow, Bernd
2002 May 21;41(20):6369-6375, Biochemistry
Cholesterol is an abundant lipid of lung surfactant, where its concentration changes relative to phospholipids in response to certain physiological conditions. We investigated the effect of the cellular cholesterol content on uptake and esterification of palmitic acid, and on cellular distribution of fatty acid translocase (FAT/CD36) in alveolar type II cells. Incubation of type II cells with methyl-beta-cyclodextrin-cholesterol complexes increased the cholesterol content of lamellar bodies. The palmitate uptake of type II cells increased in parallel with the cellular cholesterol content. The content of FAT/CD36 increased in membranes and decreased in cytosol in type II cells. The detergent-insoluble fraction (DIGs), isolated from type II cells, was enriched in FAT/CD36 and caveolin-1 after increasing the cellular cholesterol. The total incorporation of labeled palmitic acid into glycerolipids and cholesterol ester (CE) increased by a factor of about 10 when the amount of unbound (14)C-palmitic acid added to type II cells was increased by a factor of about 1000. Under these conditions, a small but significant increase of the palmitate incorporation into PL occurred. Independent from the amount of added palmitate, palmitate incorporation into triacylglycerol decreased and palmitate incorporation into cholesterol ester increased about 40-65-fold. The beta-oxidation of palmitate significantly decreased. We conclude that alveolar type II cells respond to an increase of the cholesterol level with (i) cellular redistribution of FAT/CD36 into DIGs causing enhanced palmitate uptake and increased cholesterol ester-formation, (ii) storage of cholesterol in lamellar bodies, and (iii) induction of the formation of caveolae-like microdomains in the surface membrane, a structure possibly involved in a lamellar body-independent efflux of free cholesterol via the high-density lipoprotein-specific pathway
— id: 49197, year: 2002, vol: 41, page: 6369, stat: Journal Article,

HDL-holoparticle uptake by alveolar type II cells: effect of vitamin E status
Kolleck, Ingrid; Wissel, Heide; Guthmann, Florian; Schlame, Michael; Sinha, Pranav; Rustow, Bernd
2002 Jul;27(1):57-63, American journal of respiratory cell & molecular biology
Alveolar type II cells accumulate vitamin E preferentially from high-density lipoproteins (HDL) and express at least three receptors that are specific for HDL. The expression of these receptors increases in response to vitamin E deficiency. Beside receptors for specific lipid transfer from HDL, cubilin and megalin, several other receptors that mediate HDL-particle uptake were found in the lung. We hypothesize that alveolar type II cells also exhibit the HDL-particle uptake and that this process can be regulated by the vitamin E status. By confocal laser microscopy and flow cytometry we showed that type II cells accumulate protein-labeled HDL-particle. Vitamin E depletion in rats increased HDL-particle uptake in alveolar type II cells and the expression of megalin. The expression of cubilin did not change. Refeeding with vitamin E reversed HDL-particle uptake and megalin expression. Long-time incubation of type II cells with phorbol myristyl acetate (PMA) reduced HDL-holoparticle uptake and megalin expression. We assume that alveolar type II cells exhibit HDL-holoparticle uptake mediated by megalin and cubilin. Megalin represents the regulated element of the megalin/cubilin receptor-cooperation and can be modulated by protein kinase C
— id: 49195, year: 2002, vol: 27, page: 57, stat: Journal Article,

Deficiency of tetralinoleoyl-cardiolipin in Barth syndrome
Schlame, Michael; Towbin, Jeffrey A; Heerdt, Paul M; Jehle, Roswitha; DiMauro, Salvatore; Blanck, Thomas J J
2002 May;51(5):634-637, Annals of neurology
Barth syndrome is an X-linked cardiac and skeletal mitochondrial myopathy. Barth syndrome may be due to lipid alterations because the product of the mutated gene is homologous to phospholipid acyltransferases. Here we document that a single mitochondrial phospholipid species, tetralinoleoyl-cardiolipin, was lacking in the skeletal muscle (n = 2), right ventricle (n = 2), left ventricle (n = 2), and platelets (n = 6) of 8 children with Barth syndrome. Tetralinoleoyl-cardiolipin is specifically enriched in normal skeletal muscle and the normal heart. These findings support the notion that Barth syndrome is caused by alterations of mitochondrial lipids
— id: 45507, year: 2002, vol: 51, page: 634, stat: Journal Article,

Vitamin E deficiency sensitizes alveolar type II cells for apoptosis
Sinha, Pranav; Kolleck, Ingrid; Volk, Hans Dieter; Schlame, Michael; Rustow, Bernd
2002 Jun 13;1583(1):91-98, Biochimica & biophysica acta
Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells
— id: 49196, year: 2002, vol: 1583, page: 91, stat: Journal Article,

Platelet-activating factor (PAF)-acetylhydrolase and PAF-like compounds in the lung: effects of hyperoxia
Jehle R; Schlame M; Buttner C; Frey B; Sinha P; Rustow B
2001 May 31;1532(1-2):60-66, Biochimica & biophysica acta
Platelet-activating factor (PAF)-acetylhydrolase is the enzyme modulating in tissues and biological fluids the concentration of the proinflammatory factors PAF and PAF-like oxidation products of phospholipids (PAF-like compounds). We investigated whether there is a relation between PAF-acetylhydrolase activity and the concentration of PAF-like compounds in bronchoalveolar lavage (BAL). We found that alveolar type II cells are an additional source of PAF-acetylhydrolase in BAL beside macrophages. Secretion of PAF-acetylhydrolase was stimulated by phorbol ester in alveolar type II cells but not in macrophages. Studies in BAL suggested that secreted PAF-acetylhydrolase was bound to alveolar surfactant. Exposure of rats to high oxygen concentration reduced the activity of PAF-acetylhydrolase in BAL and macrophages, but not in plasma or alveolar type II cells. In contrast, hyperoxia increased the concentration of PAF-like-compounds, lipid hydroperoxides and malonedialdehyde in plasma but not in BAL. Therefore, we conclude that neither the oxidant-induced decrease of the PAF-acetylhydrolase activity nor the direct peroxidation of surfactant lipids in the alveoli provide a likely mechanism for hyperoxia-induced lung injury. Instead, lung injury is apparently caused by lipid peroxidation in plasma rather than by high oxygen pressure in the alveoli
— id: 49198, year: 2001, vol: 1532, page: 60, stat: Journal Article,

Effect of cardiolipin oxidation on solid-phase immunoassay for antiphospholipid antibodies
Schlame M; Haller I; Sammaritano LR; Blanck TJ
2001 Dec;86(6):1475-1482, Thrombosis & haemostasis
Diagnostic assays for antiphospholipid antibodies are routinely performed on microtitre plates coated with cardiolipin. Here we show that contact between cardiolipin and NUNC-Immuno plates leads to extensive oxidation, generating a series of peroxy-cardiolipins which were identified by electrospray ionization mass spectrometry. To investigate the impact of oxidation on the antibody assay. cardiolipin was resolved into 12 molecular species, including oxidized species and non-oxidized species with different degrees of unsaturation. All 12 species reacted under anaerobic conditions with serum from patients with primary antiphospholipid syndrome. Immune reactivity was similar for tetralinoleoyl-cardiolipin, trilinoleoyl-oleoyl-cardiolipin, and peroxycardiolipins, but somewhat lower for tristearoyl-oleoyl-cardiolipin. Oxidative treatment of cardiolipin with air, cytochrome c, or Cu2+/tert-butylhydroperoxide, either before or during the assay, did not enhance immune reactivity. Similar results were obtained with a monoclonal IgM from lupus-prone mice, that binds cardiolipin in the absence of protein cofactors. We conclude that the solid-phase assay for antiphospholipid antibodies can be supported by various oxidized and non-oxididized molecular species of cardiolipin
— id: 32233, year: 2001, vol: 86, page: 1475, stat: Journal Article,

Increase in fragmented phosphatidylcholine in blood plasma by oxidative stress
Frey B; Haupt R; Alms S; Holzmann G; Konig T; Kern H; Kox W; Rustow B; Schlame M
2000 Jul;41(7):1145-1153, Journal of lipid research
Oxidatively modified phospholipids with fragmented acyl chains have attracted much interest because of their proinflammatory activity and their potential involvement in atherosclerosis. They can be formed in vitro by free radical treatment of unsaturated phospholipids but it is not known under which conditions they accumulate in vivo. We assayed one species of fragmented phosphatidylcholine (PC) in human blood plasma by high performance liquid chromatography after precolumn derivatization with chloromethylanthracene. Structural analysis suggested that fragmented PC was a diacyl species with a palmitoyl group and a short oxidized residue, which most likely had four carbons. The concentration of fragmented PC was higher in elderly individuals with coronary heart disease than in young healthy controls. Smoking one cigarette acutely increased the concentration of fragmented PC in healthy adults. Fragmented PC also increased in the reperfusion period after treatment with cardiopulmonary bypass. The increase coincided with a surge of circulating neutrophils. In rats, the plasma concentration of fragmented PC was elevated by vitamin E deficiency and exposure to high oxygen.The data demonstrate that fragmented PC increases in blood plasma in response to various forms of oxidative stress
— id: 49200, year: 2000, vol: 41, page: 1145, stat: Journal Article,

Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function
Jiang F; Ryan MT; Schlame M; Zhao M; Gu Z; Klingenberg M; Pfanner N; Greenberg ML
2000 Jul 21;275(29):22387-22394, Journal of biological chemistry
Cardiolipin (CL) is a unique phospholipid which is present throughout the eukaryotic kingdom and is localized in mitochondrial membranes. Saccharomyces cerevisiae cells containing a disruption of CRD1, the structural gene encoding CL synthase, have no CL in mitochondrial membranes. To elucidate the physiological role of CL, we compared mitochondrial functions in the crd1Delta mutant and isogenic wild type. The crd1Delta mutant loses viability at elevated temperature, and prolonged culture at 37 degrees C leads to loss of the mitochondrial genome. Mutant membranes have increased phosphatidylglycerol (PG) when grown in a nonfermentable carbon source but have almost no detectable PG in medium containing glucose. In glucose-grown cells, maximum respiratory rate, ATPase and cytochrome oxidase activities, and protein import are deficient in the mutant. The ADP/ATP carrier is defective even during growth in a nonfermentable carbon source. The mitochondrial membrane potential is decreased in mutant cells. The decrease is more pronounced in glucose-grown cells, which lack PG, but is also apparent in membranes containing PG (i.e. in nonfermentable carbon sources). We propose that CL is required for maintaining the mitochondrial membrane potential and that reduced membrane potential in the absence of CL leads to defects in protein import and other mitochondrial functions
— id: 49202, year: 2000, vol: 275, page: 22387, stat: Journal Article,

Heparin induces release of phospholipase A(2) into the splanchnic circulation
Kern H; Johnen W; Braun J; Frey B; Rustow B; Kox WJ; Schlame M
2000 Sep;91(3):528-532, Anesthesia & analgesia
Cardiopulmonary bypass results in increased plasma activity of phospholipase A(2) (PLA(2)) that appears to be caused by the administration of heparin. High PLA(2) activity may be responsible for increased production of eicosanoids and, thus, may be implicated in various pathophysiologic events associated with cardiac surgery. To investigate the site of PLA(2) secretion, blood samples were simultaneously collected from the radial artery, the pulmonary artery, and the hepatic vein at 2, 4, 6, and 20 min after systemic heparinization (350 U/kg). Within 2 min of the heparin injection, plasma activity of PLA(2) increased 4- to 9-fold and remained so for at least 20 min. Two minutes after the heparin injection, PLA(2) was significantly higher in the hepatic vein than in the radial artery (P: < 0.01). No such difference was detected between pulmonary and radial arteries. When heparin was added to blood samples in vitro (5-100 U/mL), plasma activity of PLA(2) did not increase, which suggests that the enzyme was not secreted by blood cells. IMPLICATIONS: Heparin, given in the dosage required for cardiopulmonary bypass, caused release of phospholipase A(2) into the splanchnic circulation
— id: 49199, year: 2000, vol: 91, page: 528, stat: Journal Article,

The biosynthesis and functional role of cardiolipin
Schlame M; Rua D; Greenberg ML
2000 May;39(3):257-288, Progress in lipid research
— id: 49201, year: 2000, vol: 39, page: 257, stat: Journal Article,

HDL is the major source of vitamin E for type II pneumocytes
Kolleck I; Schlame M; Fechner H; Looman AC; Wissel H; Rustow B
1999 Oct;27(7-8):882-890, Free radical biology & medicine
The alveolar surfactant is a prime target of reactive oxygen species present in air. Alveolar surfactant is supplemented with vitamin E during its assembly in type II pneumocytes. However, it is unknown which of the lipoproteins supply type II pneumocytes with vitamin E. The measurement of the uptake kinetics indicates that HDL might be the primary source of the vitamin E uptake by type II pneumocytes. Vitamin E depletion of rats caused an increase of vitamin E uptake by isolated type II pneumocytes from HDL but not from LDL or VLDL. We demonstrated that type II pneumocytes express the scavenger receptor class B type 1 (SR-B1), an HDL-specific receptor. Vitamin E depletion caused an increased expression of SR-B1 by a post-transcriptional mechanism. The increased vitamin E uptake from HDL and the increased expression of the SR-B1 were reversed by refeeding the vitamin. We propose that HDL is the primary source of vitamin E for type II pneumocytes. The rate of uptake of vitamin E by this cell type might be regulated by the expression of SR-B1
— id: 49203, year: 1999, vol: 27, page: 882, stat: Journal Article,

Microanalysis of cardiolipin in small biopsies including skeletal muscle from patients with mitochondrial disease
Schlame M; Shanske S; Doty S; Konig T; Sculco T; DiMauro S; Blanck TJ
1999 Sep;40(9):1585-1592, Journal of lipid research
Cardiolipin is a specific mitochondrial phospholipid that is present in mammalian tissues in low concentration. To measure cardiolipin in small biopsies from patients with mitochondrial disease, we developed a new technique that can detect subnanomolar levels of well-resolved molecular species, the most abundant of which are tetralinoleoyl-cardiolipin (L(4)) and trilinoleoyl-oleoyl-cardiolipin (L(3)O). To this end, a fluorescence-labeled derivative of cardiolipin (2-[naphthyl-1'-acetyl]-cardiolipin dimethyl ester) was formed and analyzed by high performance liquid chromatography. Cardiolipin was measured in skeletal muscle biopsies from 8 patients with mitochondrial disease and in 17 control subjects. In 5 patients with mitochondrial disease, cardiolipin content was higher than normal (2. 4;-7.0 vs. 0.4;-2.2 nmol/mg protein). In 3 patients with mitochondrial disease, the L(4)/L(3)O ratio was lower than normal (2;-4 vs. 4;-6). Cardiolipin was also measured in various rat and dog muscle tissues. The L(4)/L(3)O ratio was higher in condensed 'muscle' type mitochondria (heart ventricle, skeletal muscle, ratios 4;-7) than in orthodox 'liver' type mitochondria (liver, smooth muscle, heart auricular appendage, H9c2 myoblasts, ratios 0.4;-3), suggesting that the L(4)/L(3)O proportion is important for cristae membrane structure. We concluded that the L(4)/L(3)O ratio is a tissue-specific variable that may change in the presence of mitochondrial disease. The new method is suitable to measure cardiolipin in muscle biopsies in order to estimate concentration of mitochondria
— id: 45511, year: 1999, vol: 40, page: 1585, stat: Journal Article,

alpha- and delta-tocopherol induce expression of hepatic alpha-tocopherol-transfer-protein mRNA
Fechner H; Schlame M; Guthmann F; Stevens PA; Rustow B
1998 Apr 15;331 ( Pt 2):577-581, Biochemical journal
alpha-Tocopherol transfer protein (alpha-TTP) supplements nascent very-low-density lipoprotein (VLDL) preferentially with alpha-tocopherol by selecting the alpha-isomers against other stereoisomers of tocopherol. It is exclusively expressed in liver. We investigated whether the expression of the hepatic alpha-TTP can be induced by dietary tocopherols. Vitamin E-depleted rats were fed with a diet containing alpha- and delta-tocopherol (ratio 1:3). The expression of alpha-TTP mRNA was measured in liver tissue. The ratio of tocopherol stereoisomers was determined in plasma, plasma lipoproteins and tissues to measure the metabolic action of alpha-TTP. Refeeding a diet containing either alpha- or delta-tocopherol, or both, caused a steady increase of the expression of alpha-TTP mRNA. In parallel the alpha/delta-tocopherol ratio increased in plasma, VLDL, high-density lipoprotein and low-density lipoprotein as well as in liver tissue, when the diet was fed containing both isomers. The alpha-tocopherol/delta-tocopherol ratio of heart, kidney, lung, lamellar bodies of lung and in lung lavage showed no or a comparatively low increase. The data show that both tocopherol isomers were able to induce alpha-TTP mRNA in rat liver and, thus, the ability of liver to select for the alpha-isomer. On the other hand, tocopherol depletion did not change the expression of hepatic alpha-TTP mRNA in the rat
— id: 49205, year: 1998, vol: 331 ( Pt 2), page: 577, stat: Journal Article,

Stimulation of monocytes and platelets by short-chain phosphatidylcholines with and without terminal carboxyl group
Kern H; Volk T; Knauer-Schiefer S; Mieth T; Rustow B; Kox WJ; Schlame M
1998 Oct 2;1394(1):33-42, Biochimica & biophysica acta
Oxidation of unsaturated phosphatidylcholine (PC) produces fragmented phospholipids which have similar bioactivities as the platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-PC). Since a large number of molecular species are produced upon PC oxidation, the active ingredients have not been identified. We synthesized several short-chain PCs which are known to be characteristic PC oxidation products to test their PAF-like activity. The synthetic PCs contained palmitoyl or hexadecyl residues (both C16) in sn-1 position, and propionyl (C3), valeroyl (C5), succinyl (C4 with omega-carboxyl), glutaroyl (C5 with omega-carboxyl), or suberoyl (C8 with omega-carboxyl) residues in sn-2 position. Biological activity was measured by: (1) increase of intracellular calcium in human monocytes; (2) [3H]serotonin release from rabbit platelets; and (3) aggregation of human platelets. Specificity of the cellular response was tested by inhibition with the PAF-receptor antagonists BN 52021 and WEB 2086. Synthetic PC oxidation products activated both monocytes and platelets in a PAF-specific manner. The effective concentration varied with respect to assay system and chemical structure. In general, 1-hexadecyl-PCs were more effective than 1-palmitoyl-PCs, while increasing chain length in sn-2 position lowered biological activity. However, several 1-palmitoyl-PCs activated monocytes in concentrations between 10-8 and 10-6 M. In contrast, platelets were less susceptible to 1-palmitoyl-PCs. No significant difference was found between 2-valeroyl-PC (C5 with omega-methyl) and 2-glutaroyl-PC (C5 with omega-carboxyl). The data suggest that typical products of PC oxidation, containing propionyl, succinyl, or glutaroyl residues in sn-2 position, display PAF-like activity at micromolar concentrations
— id: 49204, year: 1998, vol: 1394, page: 33, stat: Journal Article,

Study of platelet-activating factor acetylhydrolase in the perioperative period of patients undergoing cardiac surgery
Schlame M; Schmid AB; Haupt R; Rustow B; Kox WJ
1998 May;9(5):313-319, Shock
After operations with cardiopulmonary bypass, patients often show early symptoms of the systemic inflammatory response syndrome (SIRS). Potential mediators of SIRS include the platelet-activating factor (PAF), which has been linked to septic shock and multiple organ dysfunction. We studied the effect of cardiac surgery on PAF acetylhydrolase, the PAF-degrading plasma enzyme, as well as the relationship between the enzyme and the postoperative state of the patients. PAF acetylhydrolase activity decreased by 38+/-8% after instituting cardiopulmonary bypass because of plasma dilution and returned to near-preoperative levels within 6 h postsurgery. After that, enzyme levels decreased again, resulting in a 24+/-12% reduction until at least 3 days postsurgery. Patients in poor postoperative condition (Acute Physiology Score >9) had a lower preoperative PAF acetylhydrolase activity than did normal patients (12+/-4 vs. 17+/-4 nmol min(-1) mL(-1); p < .05). Likewise, patients who developed postoperative SIRS had a lower preoperative PAF acetylhydrolase activity than did patients without SIRS (12+/-3 vs. 17+/-4 nmol min(-1) mL(-1); p < .05). The data suggest that PAF acetylhydrolase deficiency is among the factors associated with postoperative distress after cardiac surgery
— id: 49206, year: 1998, vol: 9, page: 313, stat: Journal Article,

Cardiolipin synthase is associated with a large complex in yeast mitochondria
Zhao M; Schlame M; Rua D; Greenberg ML
1998 Jan 23;273(4):2402-2408, Journal of biological chemistry
The phospholipid cardiolipin (CL) is ubiquitous in eucaryotes and is unique in structure, subcellular localization, and potential function. Previous studies have shown that CL is associated with major respiratory complexes in the mitochondrial membrane. To determine whether CL biosynthesis requires the presence of intact respiratory complexes, we measured activity of CL synthase, which catalyzes the synthesis of CL from cytidine diphosphate diacylglycerol and phosphatidylglycerol, in Saccharomyces cerevisiae strains with genetic defects in the oxidative phosphorylation system. Assembly mutants of cytochrome oxidase had significantly reduced CL synthase activity, while assembly mutants of respiratory complex III and the F0F1-ATPase were less inhibited. To obtain further information on the activity of CL synthase, we purified the enzyme and compared the size of the catalytic protein with the functional molecular mass. The enzyme was solubilized by Triton X-100 from KSCN-extracted mitochondrial membranes of S. cerevisiae. The functional molecular mass of Triton-solubilized CL synthase, determined by radiation inactivation, was 150-240 kDa, indicating that the functional enzyme was a large complex. After partial purification, the enzyme eluted from a Superose 12 gel filtration column with an apparent molecular mass of 70 kDa. CL synthase was further purified by hydroxylapatite and cytidine diphosphate diacylglycerol affinity chromatographies, Mono Q anion exchange FPLC, and preparative gel electrophoresis. These steps led to identification of a 28-kDa protein, which had catalytic activity when eluted from an SDS-polyacrylamide gel. This 28-kDa protein also reacted with an antiserum that inactivated the enzyme. We conclude that yeast CL synthase is a 28-kDa protein, which forms an oligomeric complex whose biogenesis and/or activity is influenced by the assembly of cyto-chrome oxidase
— id: 49207, year: 1998, vol: 273, page: 2402, stat: Journal Article,

Cardiolipin synthase from yeast
Schlame M; Greenberg ML
1997 Sep 4;1348(1-2):201-206, Biochimica & biophysica acta
Cardiolipin synthase catalyzes the synthesis of the mitochondrial phospholipid cardiolipin. Cardiolipin synthase is a unique membrane-bound enzyme in that it utilizes two phospholipids, both insoluble in water, as substrates. Kinetic analysis suggests that the enzyme forms a ternary complex with the two lipid substrates, and that a divalent metal ion directly associates with cardiolipin synthase to form the active enzyme. While little is known about the regulation of cardiolipin synthase in yeast, activity is reduced in mutants in which the mitochondrial genome is deleted, and in mutants with defective respiratory complexes. In p0 mutants, which contain no mitochondrial DNA and are defective in the assembly of many mitochondrial membrane protein complexes, cardiolipin synthase activity is reduced by 50%. Mutants defective in respiratory complexes, particularly those incapable of cytochrome oxidase assembly, also have reduced cardiolipin synthase activity. Thus it is likely that respiration and cardiolipin formation are interdependent. The enzyme was recently purified from the budding yeast Saccharomyces cerevisiae. Enzyme activity was associated with a 25-30-kDa protein. The amino acid sequence of this protein, combined with the availability of the complete yeast genome sequence, will hopefully lead to the identification of the structural gene for this enzyme in the near future
— id: 49209, year: 1997, vol: 1348, page: 201, stat: Journal Article,

Cardiolipin synthase from mammalian mitochondria
Schlame M; Hostetler KY
1997 Sep 4;1348(1-2):207-213, Biochimica & biophysica acta
Cardiolipin was first isolated from beef heart and was shown to contain an unusually high content of linoleic acid ester residues. Cardiolipin is found throughout the eukaryotes including animals, plants and fungi. In mammalian tissue and in yeast, cardiolipin is found exclusively in mitochondria. Mitochondrial synthesis of cardiolipin utilizes phosphatidylglycerol and CDP-diacylglycerol as substrates in a reaction which requires a divalent cation (Mg2+, Mn2+ or Co2+). Cardiolipin synthase has been purified to near-homogeneity from rat liver by solubilization with Zwittergent 3-14 followed by FPLC anion exchange, gel permeation and chromatofocusing steps. Cardiolipin synthase has a molecular mass of 50 kDa, a pH optimum of 8.0, and requires added phospholipids (phosphatidylethanolamine and cardiolipin) and 4 mM Co2+ for optimal activity. Except for the effects of divalent cations and the requirement for phospholipids, little is known about the regulation of cardiolipin synthase. Cardiolipin deficiency in aging mitochondria has been linked to decreased metabolite transport across the inner membrane. Both cardiolipin levels and cardiolipin synthase activity are increased in hyperthyroidism and decreased in hypothyroidism suggesting regulation by thyroid hormone. Mammalian cardiolipin synthase has not been sequenced or cloned and its biological role in mitochondria is not yet fully understood
— id: 49208, year: 1997, vol: 1348, page: 207, stat: Journal Article,

Effect of hyperoxia on the composition of the alveolar surfactant and the turnover of surfactant phospholipids, cholesterol, plasmalogens and vitamin E
Tolle A; Kolleck I; Schlame M; Wauer R; Stevens PA; Rustow B
1997 Jun 2;1346(2):198-204, Biochimica & biophysica acta
Experimental and clinical studies have provided evidence for the involvement of oxygen free radicals in development of acute and chronic lung diseases. Hyperoxia is very often an indispensable therapeutic intervention which seems to impose oxidative stress on lung tissue. We measured the effect of hyperoxia (80% O2 for 20 h) (1) on the lipid composition of pulmonary surfactant treated in vitro, (2) on surfactant lipid synthesis and secretion of type II pneumocytes in primary culture, (3) on the lipid composition and on the SP-A content of rat lung lavages and (4) on the turnover of phospholipids, cholesterol, plasmalogens and vitamin E in type II pneumocytes, lamellar bodies and lavages of adult rat lungs. (1) Hyperoxia of lung lavages in vitro reduces the vitamin E content significantly but does not change the relative proportion of PUFA or the content of plasmalogens. (2) Hyperoxia does not affect the biosynthesis or secretion of surfactant lipids and plasmalogens by type pneumocytes in primary culture. (3) Hyperoxic treatment of rats increases the SP-A content and reduces the vitamin E content significantly but does not change the concentration of other lipid components of lung lavage. (4) The vitamin E turnover, measured in type II pneumocytes, lamellar bodies and lung lavages, is increased 2-fold in these fractions. In contrast, the turnover of surfactant cholesterol and surfactant lipids does not change. (5) Hyperoxia caused an increase of the vitamin E uptake by type II pneumocytes resulting in a vitamin E enrichment of lamellar bodies. From these results we conclude that type II pneumocytes are able to regulate the turnover of lipophilic constituents of the alveolar surfactant independently of each other. Hyperoxia caused type II pneumocytes to increase the vitamin E content of lamellar bodies. The lipid and SP-A content of alveolar fluid can be regulated independently each other
— id: 49210, year: 1997, vol: 1346, page: 198, stat: Journal Article,

Identification of short-chain oxidized phosphatidylcholine in human plasma
Schlame M; Haupt R; Wiswedel I; Kox WJ; Rustow B
1996 Dec;37(12):2608-2615, Journal of lipid research
Oxidized phospholipids have been recognized as potentially important compounds that carry biological activities similar to the platelet-activating factor, but their presence in biological tissue has not been firmly established. We developed a novel technique for the quantitative analysis of phospholipids with oxidized acyl chains. The method involves 1) lipid extraction, 2) chromatographic enrichment of phospholipids with short acyl chains, 3) derivatization with 9-(chloromethyl)anthracene, 4) solid-phase extraction of the derivatives, and 5) reversed-phase HPLC with fluorescence detection. The technique was capable of measuring dicarboxylate-containing phosphatidylcholines (PCs) at the picomole level. The method was suited to monitor the generation of oxidized phospholipids from 1-palmitoyl-2-arachidonoyl-PC in the presence of Fe21/ascorbate. The new procedure was used to isolate lipids from human plasma that were identified as anthracene derivatives of short-chain oxidized PC on the basis of chromatographic enzymatic, and spectroscopic evidence. The plasma concentration, determined with an internal standard (1-palmitoyl-2-suberoyl-PC), was 0.6 +/- 0.2 microM (n = 11). The analytical method did not produce oxidation antifacts in significant amount. We concluded that human blood contains oxidatively fragmented PC in submicromolar concentration
— id: 49211, year: 1996, vol: 37, page: 2608, stat: Journal Article,

CHEB, a convulsant barbiturate, evokes calcium-dependent spontaneous glutamate release from rat cerebrocortical synaptosomes
Wei L; Schlame M; Downes H; Hemmings HC
1996 Jun;35(6):695-701, Neuropharmacology
CHEB [5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid] is a potent convulsant barbiturate that causes direct neuronal excitation by an unknown mechanism. We have analyzed the effects of CHEB on the release of endogenous glutamate from rat cerebrocortical synaptosomes using an on-line enzyme-coupled fluorimetric assay. CHEB evoked spontaneous Ca(2+)-dependent glutamate release with an EC50 = 14.2 microM and an Emax = 3.2 mumol/min/mg. The non-convulsant barbiturates pentobarbital and phenobarbital evoked significantly less glutamate release at high concentrations. CHEB (30 microM) increased intrasynaptosomal [Ca2+] by 58 +/- 4 nM (p < 0.01; n = 4) above baseline compared to an increase of 5 +/- 4 nM (NS; n = 4) produced by pentobarbital (30 microM). CHEB-evoked glutamate release was inhibited by pentobarbital, phenobarbital, EGTA, CoCl2/CdCl2 and flunarizine, but not by local anesthetics, tetrodotoxin, nitrendipine or omega-conotoxin GVIA. These results demonstrate that CHEB acts as a potent and effective secretogogue for glutamate by a pre-synaptic mechanism that does not require activation of Na+ channels or of L-type or N-type Ca2+ channels. Stimulation of spontaneous glutamate release may contribute to the convulsant properties of CHEB
— id: 49212, year: 1996, vol: 35, page: 695, stat: Journal Article,

Alveolar surfactant subfractions differ in their lipid composition
Guthmann F; Haupt R; Schlame M; Stevens PA; Rustow B
1995 Oct;27(10):1021-1026, International journal of biochemistry & cell biology
Alveolar surfactant consists of subfractions which are generated during normal lung function. Although subfractions obtained by differential centrifugation of lung lavage differ in structure, function and protein content, the phospholipid-pattern shows only minor differences. To correlate possible differences in composition between subfractions to their functional properties we did a more detailed analysis of lipid pattern. Subfractions of lung lavages from Wistar rats were obtained by differential centrifugation, lipid classes were separated by thin layer chromatography (TLC). Fatty acids and plasmalogens were determined as methylester and dimethylacetals by gas chromatography, respectively. Cholesterol and vitamin E were determined enzymatically and by HPLC, respectively. The patterns of fatty acids of total lipids and of the molecular species of phosphatidylcholine and phosphatidylethanolamine were very similar among the subfractions. The distribution of individual lavage lipids varied considerably. Three types of subfractions can be distinguished: The two dense subfractions (1000 g and 60,000 g) contain 70-88% of total phospholipids, dipalmitoylphosphatidylcholine, polyunsaturated phospholipids and polyunsaturated fatty acids present in lung lavage. The less dense subfraction (100,000 g) contains 44-60% of total cholesterol, choline plasmalogen, ethanolamine plasmalogen and vitamin E. The 100,000 g supernatant contains 40-50% of total tri-, diacylglycerols and free fatty acids. Our results support the concept that the 1000 g subfraction contains freshly secreted surfactant. The 60,000 g subfraction likely contains the monolayer and freshly secreted surfactant. The 100,000 g pellet probably contains material 'squeezed out' from the monolayer at expiration. Most likely, the supernatant contains material destined for removal from the airspace
— id: 49213, year: 1995, vol: 27, page: 1021, stat: Journal Article,

Inhibition by volatile anesthetics of endogenous glutamate release from synaptosomes by a presynaptic mechanism
Schlame M; Hemmings HC Jr
1995 Jun;82(6):1406-1416, Anesthesiology
BACKGROUND: Synaptic transmission is more sensitive than axonal conduction to the effects of general anesthetics. Previous studies of the synaptic effects of general anesthetics have focused on postsynaptic sites of action. We now provide direct biochemical evidence for a presynaptic effect of volatile anesthetics on neurotransmitter release. METHODS: Rat cerebrocortical synaptosomes (isolated presynaptic nerve terminals) were used to determine the effects of general anesthetics on the release of endogenous L-glutamate, the major fast excitatory neurotransmitter. Basal and evoked (by 4-aminopyridine, veratridine, increased KCl, or ionomycin) glutamate release were measured by continuous enzyme-coupled fluorometry. RESULTS: Clinical concentrations of volatile halogenated anesthetics, but not of pentobarbital, inhibited 4-aminopyridine-evoked Ca(2+)-dependent glutamate release. Halothane also inhibited veratridine-evoked glutamate release but not basal, KCl-evoked, or ionomycin-evoked glutamate release. Halothane inhibited both the 4-aminopyridine-evoked and the KCl-evoked increase in free intrasynaptosomal [Ca2+]. CONCLUSIONS: Inhibition of glutamate release from presynaptic nerve terminals is a potential mechanism of volatile anesthetic action. Comparison of the sensitivity of glutamate release evoked by secretogogues that act at various steps in the neurotransmitter release process suggests that halothane does not affect Ca(2+)-secretion coupling or vesicle exocytosis but inhibits glutamate release at a step proximal to Ca2+ influx, perhaps by blocking presynaptic Na+ channels. Synaptosomal glutamate release evoked by 4-aminopyridine should provide a useful system for further characterization of the presynaptic effects of anesthetics
— id: 49215, year: 1995, vol: 82, page: 1406, stat: Journal Article,

Kinetic analysis of cardiolipin synthase: a membrane enzyme with two glycerophospholipid substrates
Schlame M; Zhao M; Rua D; Haldar D; Greenberg ML
1995 Jul;30(7):633-640, Lipids
Mitochondrial cardiolipin synthase catalyzes the transfer of a phosphatidyl moiety from phosphatidyl-CMP (PtdCMP) to phosphatidylglycerol (PtdGro) in the presence of specific divalent cations. The synthase was solubilized from Saccharomyces cerevisiae mitochondria and purified about 300-fold. The partially enzyme was part of a medium-size, mixed micelle which had to bind to a foreign substrate/detergent micelle before catalysis could occur. The kinetics of cardiolipin synthase were studied by changing the molar fraction of substrate in the micelles. The enzyme obeyed Michaelis-Menten kinetics in relation to PtdCMP with a Km of 0.03 mol%. PtdGro caused sigmoidal kinetics with a low apparent affinity. It is speculated that it was involved in docking the enzyme to the substrate/detergent micelle. Cardiolipin synthase did not catalyze isotope exchange between [14C]CMP and PtdCMP, virtually excluding a ping-pong catalytic mechanism. Mg2+ stimulated the activity by increasing the turnover number rather than the substrate affinity, a mechanism which was also found for the Co(2+)-activation of rat liver cardiolipin synthase. It is concluded that a direct association of the metal ion and the enzyme forms the active cardiolipin synthase which has a very high affinity for PtdCMP and a lower affinity for PtdGro
— id: 49214, year: 1995, vol: 30, page: 633, stat: Journal Article,

The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants
Hoffmann B; Stockl A; Schlame M; Beyer K; Klingenberg M
1994 Jan 21;269(3):1940-1944, Journal of biological chemistry
Although the site-directed C73S mutation in the ADP/ATP carrier (AAC) AAC2 gene from Saccharomyces cerevisiae produced a glycerol-positive strain, indicating that the mutant AAC is active, on isolation and reconstitution in egg yolk phosphatidylcholine, the C73S AAC had no transport activity, whereas the wild-type AAC was fully active. Only on addition of cardiolipin was an exchange activity with the C73S AAC obtained. The AACs isolated from the other cysteine mutants did not (C244S) or only marginally (C271S) require cardiolipin for transport on reconstitution. [3H]Carboxyatractylate binding as a measure of incorporated AAC molecules was unchanged on addition of cardiolipin in all mutants, indicating that cardiolipin does not increase the incorporation of the AAC. It also shows that cardiolipin is required only for translocation and not for binding. The activity of the C73S mutant AAC shows half-saturation with cardiolipin at 2% by weight or at 1.15 mol % in the phosphatidylcholine vesicles. Other acidic phospholipids tested such as phosphatidylserine and phosphatidic acid did not activate. Among various cardiolipin derivatives, the selectivity for cardiolipin is high. Only monolysocardiolipin still retains 12% activity. After removal of the bulk of phospholipid, the content of bound phospholipids was assayed by 31P NMR. By unmasking with SDS, in the wild-type AAC and in the C73S AAC, 6.4 mol and only 1.3 and 2.9 mol of bound cardiolipin/mol of AAC dimer are found, respectively. Presumably, on isolation, cardiolipin is lost from the more labile C73S mutant AAC. Although the absolute requirement for cardiolipin is unique for the C73S AAC, it is concluded that in this mutant, the unmasking of the cardiolipin requirement demonstrates a general cardiolipin requirement of the wild-type AAC and of AACs from other sources
— id: 49216, year: 1994, vol: 269, page: 1940, stat: Journal Article,

Mitochondrial cardiolipin in diverse eukaryotes. Comparison of biosynthetic reactions and molecular acyl species
Schlame M; Brody S; Hostetler KY
1993 Mar 15;212(3):727-735, European journal of biochemistry
Cardiolipin, a unique dimeric phospholipid of bacteria and mitochondria, can be synthesized by two alternative pathways discovered in rat and Escherichia coli, respectively. In mitochondrial preparations from fungi (Saccharomyces cerevisiae, Neurospora crassa), higher plants (Phaseolus aureus), molluscs (Mytilus edulis) and mammals (rat liver, bovine adrenal gland), cardiolipin was synthesized from CDP-diacylglycerol and phosphatidylglycerol, suggesting a common eukaryotic mechanism of cardiolipin formation which is in contrast to the prokaryotic biosynthesis from two molecules of phosphatidylglycerol. All mitochondrial cardiolipin synthases were inhibited by lysophosphatidylglycerol, were insensitive to N-ethylmaleimide and required divalent cations, although they had different cation specificities. The molecular species of cardiolipin from rat liver, bovine heart, S. cerevisiae and N. crassa were analysed by high-performance liquid chromatography of the derivative 1,3-bis[3'-sn-phosphatidyl]-2-benzoyl-sn-glycerol dimethyl ester. Cardiolipins from these organisms contained mainly monounsaturated or diunsaturated chains with 16 or 18 carbon atoms, resulting in a relatively homogeneous distribution of double bonds and carbon numbers among the four acyl positions. About half of the molecular species were symmetrical, i.e. they combined two identical diacylglycerol moieties. In N. crassa, the same species pattern was found at growth temperatures of 25 degrees C and 37 degrees C. Tentative molecular models were created for the most abundant molecular species and subjected to energy minimization. Geometric data, derived from these models, suggested similarities in the gross structure of the major cardiolipin species from different sources
— id: 49217, year: 1993, vol: 212, page: 727, stat: Journal Article,

Cardiolipin is synthesized on the matrix side of the inner membrane in rat liver mitochondria
Schlame M; Haldar D
1993 Jan 5;268(1):74-79, Journal of biological chemistry
In the mitochondrial inner membrane, cardiolipin is a specific lipid component associated with various protein complexes. The assembly of such complexes has been studied, and it seems that most protein subunits enter the inner membrane from the matrix side, but nothing is known about the path of cardiolipin. In this paper, the topography of cardiolipin biosynthesis is investigated. Cardiolipin synthase, a membrane-bound protein, could not be released by sonication or 1 M KCl. In sucrose density gradient subfractionation, cardiolipin synthase co-migrated with the inner membrane marker cytochrome oxidase. no indication was obtained for a preferential localization of this enzyme at contact sites between the outer and inner membranes. Protease digestion experiments showed that cardiolipin synthase exposed protease-susceptible domains mainly to the matrix side of the inner membrane. In intact mitochondria, the Mn(2+)-dependent stimulation of cardiolipin synthesis was abolished when the Mn2+ influx into the matrix was blocked by ruthenium red. 1-Decanoyl-sn-glycero-3-phosphorylcholine, a water-soluble inhibitor of cardiolipin synthase, was only effective after disintegration of mitochondria. The metabolic precursor of cardiolipin, CDP-diacylglycerol, was synthesized by an inner membrane enzyme whose protease-susceptible domains were mainly exposed to the matrix side. It is concluded that cardiolipin is synthesized in the inner leaflet of the mitochondrial inner membrane
— id: 49218, year: 1993, vol: 268, page: 74, stat: Journal Article,

Studies on the formation of dipalmitoyl species of phosphatidylcholine and phosphatidylethanolamine in pulmonary type II cells
Rustow B; Schlame M; Haupt R; Wilhelm D; Kunze D
1992 Mar 1;282 ( Pt 2):453-458, Biochemical journal
Endogenous content of and incorporation of labelled glycerol into alkenylacyl-, alkylacyl- and diacyl-glycerol, -glycerol-3-phosphocholine and -glycero-3-phosphoethanolamine of pulmonary type II cells were measured. On prolonged incubation of type II cells with labelled glycerol, the proportion of label incorporated into the diacyl subclass of these glycerolipids increased and the proportion of label incorporated into the ether lipids declined. Endogenous phosphatidylcholine (PtdCho) of type II cells contained 38.4% of the dipalmitoyl species, but endogenous phosphatidylethanolamine (PtdEtn) only 2.5%. In contrast, similar proportions of labelled glycerol were incorporated into dipalmitoyl-PtdCho and -PtdEtn after short-time incubation but, with prolonged incubation time the proportion of labelled dipalmitoyl-PtdCho increased from 11.3 to 18.8%, whereas that of dipalmitoyl-PtdEtn did not change significantly. Type II cell membranes were found to exhibit cofactor-independent and CoA-mediated transacylations of [1-14C]palmitoyl-lyso-PtdCho and -lyso-PtdEtn. The distribution of label among the palmitic acid-containing species of PtdCho and PtdEtn formed by both transacylation activities was determined. Cofactor-independent and CoA-mediated transacylation showed a strong selectivity for palmitate and arachidonate and a strong discrimination against oleate. The amount (nmol) of dipalmitoyl-PtdEtn formed by both transacylation activities after short-time incubation (2 min) decreased with prolonged incubation time (60 min). In contrast, the nmol of dipalmitoyl-PtdCho formed by cofactor-independent transacylation remains nearly the same after short-time and longer incubation. The nmol of dipalmitoyl-PtdCho formed by CoA-mediated transacylation increased strongly in the same time interval. Beside synthesis de novo via the CDP-choline pathway and reacylation of lyso-PtdCho with palmitoyl-CoA, the CoA-mediated transacylation of lyso-PtdCho may be an effective pathway for the formation of dipalmitoyl-PtdCho in pulmonary type II cells
— id: 49219, year: 1992, vol: 282 ( Pt 2), page: 453, stat: Journal Article,

Mammalian cardiolipin biosynthesis
Schlame M; Hostetler KY
1992 ;209:330-337, Methods in enzymology
— id: 49220, year: 1992, vol: 209, page: 330, stat: Journal Article,

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?
Schlame M; Beyer K; Hayer-Hartl M; Klingenberg M
1991 Jul 15;199(2):459-466, European journal of biochemistry
Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment
— id: 49223, year: 1991, vol: 199, page: 459, stat: Journal Article,

Solubilization, purification, and characterization of cardiolipin synthase from rat liver mitochondria. Demonstration of its phospholipid requirement
Schlame M; Hostetler KY
1991 Nov 25;266(33):22398-22403, Journal of biological chemistry
Cardiolipin is a specific and functionally important phospholipid of mitochondria, and its biosynthesis is considered to be crucial for the assembly of this organelle. However, little information is available about the enzyme cardiolipin synthase, largely because it has not yet been isolated. We solubilized cardiolipin synthase from rat liver mitochondrial membranes with Zwittergent 3-14 and purified it by Mono Q anion exchange chromatography, Superose 12 gel filtration, and Mono P chromatofocusing. Cardiolipin synthase is one of the most acidic mitochondrial proteins (isoelectric point, pH 4-5) and appears as a 50-kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme requires CO2+ for activity, has an alkaline pH optimum (pH 8-9), and exhibits Km values of 45 and 1.6 microM for phosphatidylglycerol and CDP-diacylglycerol, respectively. Cardiolipin synthase loses activity during purification, and the activity can be partially reconstituted by the addition of phospholipids. The most effective phospholipid is phosphatidylethanolamine which reactivates in a cooperative manner. Cardiolipin reactivates hyperbolically at low concentrations but inhibits the enzyme at higher concentrations. In addition, cardiolipin shifts the sigmoidal reactivation curve of phosphatidylethanolamine toward lower concentrations. It is suggested that cardiolipin synthase requires interaction with several molecules of phosphatidylethanolamine and at least one molecule of cardiolipin for full enzymatic activity
— id: 49221, year: 1991, vol: 266, page: 22398, stat: Journal Article,

Analysis of cardiolipin molecular species by high-performance liquid chromatography of its derivative 1,3-bisphosphatidyl-2-benzoyl-sn-glycerol dimethyl ester
Schlame M; Otten D
1991 Jun;195(2):290-295, Analytical biochemistry
Cardiolipin (CL, 1,3-bisphosphatidyl-sn-glycerol) is a four-acyl-chain phospholipid whose molecular species composition cannot be analyzed by standard procedures. Here we report a method to resolve the molecular species of CL by high-performance liquid chromatography of its derivative 1,3-bisphosphatidyl-2-benzoyl-sn-glycerol dimethyl ester. The CL derivative was characterized by 1H nuclear magnetic resonance spectroscopy, ultraviolet (uv) spectroscopy, thin-layer chromatography, and fatty acid analysis. The derivatization procedure did not change the fatty acid profile and provided a virtually complete conversion to the highly apolar, uv-visible product. In HPLC separations, recorded by 228 nm absorbance, a linear correlation was found between the area of individual peaks and their amount of lipid phosphorus. Bovine heart CL was resolved into 11 molecular species of which 6 (together accounting for 97 mol%) could be identified. The molecular species of bovine heart CL feature a linear relationship between their logarithmic retention time and their double bond number
— id: 49224, year: 1991, vol: 195, page: 290, stat: Journal Article,

Cytidine diphosphate diglyceride analogs of antiretroviral dideoxynucleosides: evidence for release of dideoxynucleoside-monophosphates by phospholipid biosynthetic enzymes in rat liver subcellular fractions
van Wijk GM; Hostetler KY; Schlame M; van den Bosch H
1991 Oct 15;1086(1):99-105, Biochimica & biophysica acta
We recently synthesized phospholipid analogs with antiviral nucleosides in the polar headgroup and demonstrated their antiretroviral activity in vitro in human immunodeficiency virus-infected cells (Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.B.M., van den Bosch, H. and Richman, D.D. (1990) J. Biol. Chem. 265, 6112-6117). Dideoxynucleoside analogs of cytidine diphosphate diglyceride (CDP-DG) represent one class of such phospholipid prodrugs from which the antiviral active principle may be released through established pathways of cellular phospholipid metabolism. We now demonstrate that the liponucleotides of dideoxycytidine, 3'-deoxythymidine and 3'-azido-3'-deoxythymidine (AZT, Zidovudine) can substitute to varying extents for CDP-DG in the biosynthesis of phosphatidylinositol, phosphatidylglycerol or diphosphatidylglycerol by rat liver subcellular fractions. In all three biosynthetic pathways dideoxycytidine diphosphate diglyceride was the most active donor of the phosphatidyl unit. The nearly stoichiometric formation of dideoxycytidine-5'-monophosphate during phosphatidylinositol biosynthesis supports the rationale that the antiretroviral liponucleotides may provide cells with a depot form from which the antiviral drug can be released in 5'-monophosphorylated form, thus bypassing the initial phosphorylation of free dideoxynucleosides
— id: 49222, year: 1991, vol: 1086, page: 99, stat: Journal Article,

Intramembraneous hydrogenation of mitochondrial lipids reduces the substrate availability, but not the enzyme activity of endogenous phospholipase A. The role of polyunsaturated phospholipid species
Schlame M; Horvath I; Torok Z; Horvath LI; Vigh L
1990 Jun 28;1045(1):1-8, Biochimica & biophysica acta
(1) Isolated rat liver mitochondria were subjected to catalytic hydrogenation using a water-soluble Pd complex and molecular H2. This treatment resulted in a reduction of double bonds on phospholipid acyl chains as judged by gas chromatography of fatty acid methyl esters and HPLC of dinitrobenzoyldiacylglycerols. (2) After hydrogenation, mitochondria lost their ability to hydrolyze endogenous phospholipids in alkaline, Ca2+ containing medium, while phospholipase A2 retained full activity against exogenous substrates, regardless of whether those substrates were hydrogenated or not. (3) Inhibition by hydrogenation of endogenous phospholipid hydrolysis correlated with the loss of polyunsaturated fatty acyls, rather than with changes of the bulk membrane fluidity as measured by ESR and fluorescence studies. (4) These data suggest that the unsaturation of mitochondrial membrane lipids might be important for regulation of phospholipid breakdown by endogenous phospholipases. In particular, polyunsaturated molecular species seem to be involved in making phospholipids accessible to phospholipase A-mediated hydrolysis
— id: 49226, year: 1990, vol: 1045, page: 1, stat: Journal Article,

Relationship between lipid saturation and lipid-protein interaction in liver mitochondria modified by catalytic hydrogenation with reference to cardiolipin molecular species
Schlame M; Horvath L; Vigh L
1990 Jan 1;265(1):79-85, Biochemical journal
Lipid acyl double bonds in isolated mitochondrial membranes were gradually reduced by palladium-complex-catalysed hydrogenation, and the resulting saturation was monitored by fatty acid analysis of phosphatidylcholine, phosphatidylethanolamine and cardiolipin. The courses of hydrogenation of these phospholipids suggested that cardiolipin is in a membrane compartment which is less accessible to the applied catalyst. Native cardiolipin and its hydrogenation products were further characterized by analysis of their molecular diacylglycerol species. A decrease in the double bond content was accompanied by an increased amount of motionally restricted lipids at the hydrophobic interface of proteins as measured by two different spin-labelled lipids (C-14 positional isomers of spin-labelled stearic acid and phosphatidylcholine analogues). The protein-immobilized fraction of spin-labelled stearic acid increased in parallel with the hydrogenation of cardiolipin rather than of phosphatidylcholine or phosphatidylethanolamine. These data are interpreted in terms of a tight association of cardiolipin with membrane proteins, which becomes looser upon double bond reduction leading to the replacement of cardiolipin by spin-labelled stearic acid in the solvation shell. Thus the hydrophobic moiety of cardiolipin, characterized by double-unsaturated C18-C18 diacylglycerol species, seems to be an important structural requirement for the high protein affinity of this compound
— id: 49227, year: 1990, vol: 265, page: 79, stat: Journal Article,

Lysocardiolipin formation and reacylation in isolated rat liver mitochondria
Schlame M; Rustow B
1990 Dec 15;272(3):589-595, Biochemical journal
Liver mitochondrial cardiolipin (CL) is distinguished from other phospholipids by the presence of linoleoyl in almost all molecular species, and the biosynthesis of these species is not yet understood. The present study was carried out in order to test the hypothesis that the linoleoyl proportion of CL may be specifically enriched by a deacylation-reacylation cycle. Incorporation of [14C]glycerol 3-phosphate into the metabolites of the CL pathway was accompanied by formation of 14C-labelled monolyso- and dilyso-CL. Labelling of dilyso-CL was increased or decreased by stimulation or inhibition respectively of mitochondrial phospholipase A2. These data suggest a rapid deacylation of newly formed [14C]CL by phospholipase A2, whereas endogenous mitochondrial CL was very resistant to hydrolytic degradation. Unlike dilyso-CL, monolyso-CL could be reacylated by [14C]linoleoyl residues. [14C]Linoleoyl incorporation into CL was also observed when exogenous CL was added instead of monolyso-CL, thus indicating the concerted action of de- and re-acylation. Although 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine was a suitable acyl donor under experimental conditions, the reaction was not a transacylation but required splitting of [14C]linoleic acid from phosphatidylcholine and formation of [14C]linoleoyl-CoA as an intermediate. The [14C]linoleoyl was mainly bound to the sn-2(2') position of CL, and a small portion (about 20%) to the sn-1(1') position. It is concluded that a cycle, comprising CL deacylation and monolyso-CL reacylation by linoleoyl-CoA, provides a potential mechanism for the remodelling of molecular species of newly formed CL
— id: 49225, year: 1990, vol: 272, page: 589, stat: Journal Article,

Species pattern of phosphatidic acid, diacylglycerol, CDP-diacylglycerol and phosphatidylglycerol synthesized de novo in rat liver mitochondria
Rustow B; Schlame M; Rabe H; Reichmann G; Kunze D
1989 Apr 3;1002(2):261-263, Biochimica & biophysica acta
Rat liver mitochondria were incubated with [3H]glycerol 3-phosphate, ATP, CTP and coenzyme A allowing acylatin of glycerophosphate with endogenous fatty acids and the further conversion of labelled phosphatidic acid (PA) to diacylglycerol (DG), CDP-diacylglycerol (CDP-DG) and phosphatidylglycerol (PG). In these glycerolipids, the distribution of label among the individual molecular species was found to be similar, with 16:0-18:1, 16:0-18:2 and 18:0-18:2/16:0-16:0 being the main species. It was concluded that mitochondrial enzymes involved in the de novo synthesis of these glycerolipids exhibited no acyl selectivity for their substrates. The pattern of molecular species of mitochondrial PA, DG and CDP-DG closely approached that of the same glycerolipids synthesized de novo in isolated rat liver microsomes
— id: 49228, year: 1989, vol: 1002, page: 261, stat: Journal Article,

Synthesis of phosphatidylcholine and phosphatidylglycerol in rat lung mitochondria
Schlame M; Rustow B; Kunze D
1989 Feb 21;85(2):115-122, Molecular & cellular biochemistry
The mitochondrial fraction of adult rat lung contains choline phosphotransferase (EC 2.7.8.2) activity which can not be explained by microsomal contamination estimated on the basis of marker enzyme distribution. Mitochondrial (14C)glycerol-3-phosphate incorporation into PC (phosphatidylcholine) can be distinguished from the microsomal incorporation by different sensitivity to N-ethylmaleimide inhibition. The data indicate that rat lung mitochondria have the intrinsic capability to synthesize PC. Both synthesis of PC and PG (phosphatidylglycerol) are susceptible to isotonic tryptic attack against the cytoplasmic face of isolated rat lung mitochondria, suggesting the outer membrane location of crucial activities involved in the formation of these phospholipids. Rat liver mitochondria are different from rat lung mitochondria with respect to their capability to synthesize PC, their rate of (14C)glycerol-3-phosphate incorporation into PG as well as the submitochondrial site of PG formation
— id: 49229, year: 1989, vol: 85, page: 115, stat: Journal Article,

Molecular species of phosphatidylcholine and phosphatidylglycerol in rat lung surfactant and different pools of pneumocytes type II
Schlame M; Casals C; Rustow B; Rabe H; Kunze D
1988 Jul 1;253(1):209-215, Biochemical journal
It is not yet completely understood how a cell is able to export specific phospholipids, like dipalmitoylphosphatidylcholine (dipalmitoyl-PC), which is secreted by pneumocytes type II, into pulmonary surfactant. The acyl species composition of [3H]PC which was synthesized in type II cells in the presence of [2-3H]glycerol resembled the species composition of PC localized in intracellular pneumocyte membranes. This species pattern was different from the pattern of PC of lamellar bodies, i.e., intracellularly stored surfactant, by a higher proportion of dipalmitoyl-PC mainly at expense of 1-palmitoyl-2-oleoyl-PC. Lamellar body PC in turn showed the same species distribution as surfactant PC. The data suggest that subcellular compartmentation and/or intracellular transfer of PC destined to storage in lamellar bodies, but not secretion of lamellar bodies, involves an enrichment of dipalmitoyl-PC and a depletion of 1-palmitoyl-2-oleoyl-PC. In contrast, the acyl species pattern of phosphatidylglycerol does not seem to undergo gross changes on the path from synthesis to secretion
— id: 49230, year: 1988, vol: 253, page: 209, stat: Journal Article,

Molecular species of mitochondrial phosphatidylcholine in rat liver and lung
Schlame M; Rabe H; Rustow B; Kunze D
1988 Feb 19;958(3):493-496, Biochimica & biophysica acta
The acyl species of mitochondrial phosphatidylcholine from rat liver and lung were analysed by HPLC separation of the 1,2-diacyl-3-naphthylurethane derivatives. Comparison of phosphatidylcholine species patterns in microsomal, mitochondrial and submitochondrial fractions revealed only minor differences, whereas mitochondria from liver and lung differed markedly in the molecular composition of their respective phosphatidylcholine species
— id: 49231, year: 1988, vol: 958, page: 493, stat: Journal Article,

Dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space of rat-heart mitochondria
Gellerich FN; Schlame M; Bohnensack R; Kunz W
1987 Feb 11;890(2):117-126, Biochimica & biophysica acta
To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space
— id: 49232, year: 1987, vol: 890, page: 117, stat: Journal Article,

Phosphatidylglycerol of rat lung. Intracellular sites of formation de novo and acyl species pattern in mitochondria, microsomes and surfactant
Schlame M; Rustow B; Kunze D; Rabe H; Reichmann G
1986 Nov 15;240(1):247-252, Biochemical journal
The subcellular site of phosphatidylglycerol (PG) formation for lung surfactant has not been convincingly clarified. To approach this problem we analysed the acyl species pattern of lung PG in mitochondria, microsomes and surfactant by h.p.l.c. separation of its 1,2-diacyl-3-naphthylurethane derivatives. Both mitochondrial and microsomal PG proved identical with surfactant PG, containing the major species 1-palmitoyl-2-oleoyl-PG and 1,2-dipalmitoyl-PG. The fatty acid composition of mitochondrial PG differs markedly from that of diphosphatidylglycerol. This may be taken as an indication that mitochondrial PG is synthesized on purpose to form surfactant, rather than being only the precursor of diphosphatidylglycerol. In vitro, sn-[U-14C]glycerol 3-phosphate incorporation into PG of mitochondria or microsomes occurs in the presence of CTP, ATP and CoA but independently of the supply of exogenous lipoidic precursors. Although the rate in vitro of autonomous PG synthesis, and the endogenous PG content, are higher in mitochondria than in microsomes, it is assumed that both subcellular fractions are involved in PG formation for surfactant
— id: 49233, year: 1986, vol: 240, page: 247, stat: Journal Article,

Association of creatine kinase with rat heart mitochondria: high and low affinity binding sites and the involvement of phospholipids
Schlame M; Augustin W
1985 ;44(7-8):1083-1088, Biomedica biochimica acta
Cleavage of mitochondrial phosphatidylethanolamine (PE), phosphatidylcholine (PC) and cardiolipin (CL) by phospholipase A2 but not selective degradation of PE and PC by phospholipase C dissociates creatine kinase from rat heart mitochondria. Creatine kinase exhibits a high resistance against Triton X-100 solubilization up to concentrations of 0.05-0.1%. Scatchard plot of rebinding experiments using mitoplasts revealed the presence of both low and high affinity binding sites; the latter may account for the originally bound creatine kinase activity. It is suggested that creatine kinase is specifically bound to a CL containing domain of the inner mitochondrial membrane
— id: 49234, year: 1985, vol: 44, page: 1083, stat: Journal Article,

Creatine kinase of heart mitochondria: no changes in its kinetic properties after inhibition of the adenine nucleotide translocator
Gellerich FN; Schlame M; Saks VA
1983 ;42(10):1335-1337, Biomedica biochimica acta
The kinetic properties of heart mitochondrial creatine kinase were measured with and without inhibitors of the adenine nucleotide translocator. No significant differences were observed suggesting that mitochondrial creatine kinase is not acting together with the adenine nucleotide translocator as a functional multienzyme complex. Adenine nucleotides from the bulk phase are able to enter the active center of creatine kinase without the necessity of transport via the adenine nucleotide translocator
— id: 49235, year: 1983, vol: 42, page: 1335, stat: Journal Article,

Localization of hexokinase in mitochondria from rabbit reticulocytes and its relation to mitochondrial ATP-formation studied by measurement of 32P-fluxes
Schlame M; Gellerich FN; Augustin W
1981 ;40(4-5):617-623, Acta biologica & medica Germanica
The submitochondrial distribution of hexokinase was studied by repeated specific solubilizations and by tryptic digestion of isolated rabbit reticulocyte mitochondria. Whereas most of the enzyme is dissociably bound to the outer side of outer mitochondrial membrane, a small tightly bound portion is localized more internally. Electrophoretic separations did not reveal a specific isoenzyme pattern of the internal mitochondrial enzyme. Relationships between mitochondrial hexokinases and the intramitochondrial ATP pool, generated by oxidative phosphorylation, were studied by measuring 32P-fluxes following gamma-32P-ATP pulses on phosphorylating and non-phosphorylating mitochondria. Under both conditions, the specific activities in deoxyglucose-6-phosphate correspond closely to that of total gamma-ATP, thus not supporting a preferential use of intramitochondrial generated ATP by part of the mitochondrial hexokinases
— id: 49236, year: 1981, vol: 40, page: 617, stat: Journal Article,