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Acylation of monolysocardiolipin in rat heart

1999; Elsevier BV; Volume: 40; Issue: 10 Linguagem: Inglês

10.1016/s0022-2275(20)34900-2

1539-7262

J. Brian, William A. Taylor, Vernon W. Dolinsky, Grant M. Hatch,

Endoplasmic Reticulum Stress and Disease

Cardiolipin is a major mitochondrial membrane glycerophospholipid in the mammalian heart. In this study, the ability of the isolated intact rat heart to remodel cardiolipin and the mitochondrial enzyme activities that reacylate monolysocardiolipin to cardiolipin in vitro were characterized. Adult rat heart cardiolipin was found to contain primarily linoleic and oleic acids. Perfusion of the isolated intact rat heart in the Langendorff mode with various radioactive fatty acids, followed by analysis of radioactivity incorporated into cardiolipin and its immediate precursor phosphatidylglycerol, indicated that unsaturated fatty acids entered into cardiolipin mainly by deacylation followed by reacylation. The in vitro mitochondrial acylation of monolysocardiolipin to cardiolipin was coenzyme A-dependent with a pH optimum in the alkaline range. Significant activity was also present at physiological pH. With oleoyl-coenzyme A as substrate, the apparent Km for oleoyl-coenzyme A and monolysocardiolipin were 12.5 μm and 138.9 μm, respectively. With linoleoyl-coenzyme A as substrate, the apparent Km for linoleoyl-coenzyme A and monolysocardiolipin were 6.7 μm and 59.9 μm, respectively. Pre-incubation at 50°C resulted in different profiles of enzyme inactivation for the two activities. Both activities were affected similarly by phospholipids, triacsin C, and various lipid binding proteins but were affected differently by various detergents and myristoyl-coenzyme A. [3H]cardiolipin was not formed from monolyso[3H]cardiolipin in the absence of acyl-coenzyme A. Monolysocardiolipin acyltransferase activities were observed in mitochondria prepared from various other rat tissues. In summary, the data suggest that the isolated intact rat heart has the ability to rapidly remodel cardiolipin and that rat heart mitochondria contain coenzyme A-dependent acyl-transferase(s) for the acylation of monolysocardiolipin to cardiolipin. A simple and reproducible in vitro assay for the determination of acyl-coenzyme A-dependent monolysocardiolipin acyltransferase activity in mammalian tissues with exogenous monolysocardiolipin substrate is also presented.—Ma, B. J., W. A. Taylor, V. W. Dolinsky, and G. M. Hatch. Acylation of monolysocardiolipin in rat heart. J. Lipid Res. 1999. 40: 1837–1845. Cardiolipin is a major mitochondrial membrane glycerophospholipid in the mammalian heart. In this study, the ability of the isolated intact rat heart to remodel cardiolipin and the mitochondrial enzyme activities that reacylate monolysocardiolipin to cardiolipin in vitro were characterized. Adult rat heart cardiolipin was found to contain primarily linoleic and oleic acids. Perfusion of the isolated intact rat heart in the Langendorff mode with various radioactive fatty acids, followed by analysis of radioactivity incorporated into cardiolipin and its immediate precursor phosphatidylglycerol, indicated that unsaturated fatty acids entered into cardiolipin mainly by deacylation followed by reacylation. The in vitro mitochondrial acylation of monolysocardiolipin to cardiolipin was coenzyme A-dependent with a pH optimum in the alkaline range. Significant activity was also present at physiological pH. With oleoyl-coenzyme A as substrate, the apparent Km for oleoyl-coenzyme A and monolysocardiolipin were 12.5 μm and 138.9 μm, respectively. With linoleoyl-coenzyme A as substrate, the apparent Km for linoleoyl-coenzyme A and monolysocardiolipin were 6.7 μm and 59.9 μm, respectively. Pre-incubation at 50°C resulted in different profiles of enzyme inactivation for the two activities. Both activities were affected similarly by phospholipids, triacsin C, and various lipid binding proteins but were affected differently by various detergents and myristoyl-coenzyme A. [3H]cardiolipin was not formed from monolyso[3H]cardiolipin in the absence of acyl-coenzyme A. Monolysocardiolipin acyltransferase activities were observed in mitochondria prepared from various other rat tissues. In summary, the data suggest that the isolated intact rat heart has the ability to rapidly remodel cardiolipin and that rat heart mitochondria contain coenzyme A-dependent acyl-transferase(s) for the acylation of monolysocardiolipin to cardiolipin. A simple and reproducible in vitro assay for the determination of acyl-coenzyme A-dependent monolysocardiolipin acyltransferase activity in mammalian tissues with exogenous monolysocardiolipin substrate is also presented.—Ma, B. J., W. A. Taylor, V. W. Dolinsky, and G. M. Hatch. Acylation of monolysocardiolipin in rat heart. J. Lipid Res. 1999. 40: 1837–1845. Cardiolipin (CL), the first polyglycerophospholipid ever discovered, was initially isolated from beef heart by Mary Pangborn in 1942 (1Pangborn M. Isolation and purification of a serologically active phospholipid from beef heart.J. Biol. Chem. 1942; 143: 247-256Google Scholar). CL is a major membrane glycerophospholipid of mammalian mitochondria and, in the rat heart, CL comprises approximately 15% of the entire cardiac glycerophospholipid mass (for reviews see 2–5). CL is characteristically associated with the inner mitochondrial membrane where it may constitute as much as 21% of the total membrane glycerophospholipid mass of that organelle (6Stoffel W. Schiefer H-G. Biosynthesis and composition of phosphatides in outer and inner mitochondrial membranes.Hoppe-Seyler's Z. Physiol. Chem. 1968; 349: 1017-1026Google Scholar). In addition to its inner membrane localization some CL has been identified in the mitochondrial outer membrane (7Hovius R. Lambrechts H. Nicolay K. de Kruijff B. Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane.Biochim. Biophys. Acta. 1990; 1021: 217-226Google Scholar). CL was shown to be required for the reconstituted activity of a number of key mammalian mitochondrial enzymes involved in cellular energy metabolism including cytochrome c oxidase (8Vik S.B. Georgevich G. Capaldi R.A. Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase.Proc. Natl. Acad. Sci. USA. 1981; 78: 1456-1460Google Scholar), carnitine palmitoyltransferase (9Fiol C.J. Bieber L.L. Sigmoidal kinetics of purified beef heart mitochondrial carnitine palmitoyltransferase.J. Biol. Chem. 1984; 259: 13084-13088Google Scholar), creatine phosphokinase (10Muller M. Moser R. Cheneval D. Carafoli E. Cardiolipin is the membrane receptor for creatine phosphokinase.J. Biol. Chem. 1985; 260: 3839-3843Google Scholar), pyruvate translocator (11Hutson S.M. Roten S. Kaplan R.S. Solubilization and reconstitution of the branched-chain-alpha-keto acid transporter from rat heart mitochondria.Proc. Natl. Acad. Sci. USA. 1990; 87: 1028-1031Google Scholar), tricarboxylate carrier (12Kaplan R.S. Mayor J.A. Johnston N. Oliveira D.L. Purification and characterization of the reconstitutively active tricarboxylate transporter from rat liver mitochondria.J. Biol. Chem. 1990; 265: 13379-13385Google Scholar), mitochondrial glycerol-3-phosphate dehydrogenase (13Belezani Z.S. Janesik V. Role of cardiolipin in the functioning of the L-glycerol-3-phosphate dehydrogenase.Biochem. Biophys. Res. Commun. 1989; 159: 132-139Google Scholar), phosphate transporter (14Kadenbach B. Mende P. Kolbe H.V. Stipani I. Palmieri F. The mitochondrial phosphate carrier has an essential requirement for cardiolipin.FEBS Lett. 1982; 139: 109-112Google Scholar), ADP/ATP carrier (15Hoffman B. Stockl A. Schlame M. Beyer K. Klingenberg M. The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants.J. Biol. Chem. 1994; 269: 1940-1944Google Scholar), and the ATP synthase (16Eble K.S. Coleman W.B. Hantgan R.R. Cunningham C.C. Tightly associated cardiolipin in bovine heart mitochondrial ATP synthase as analyzed by 31P nuclear magnetic resonance spectroscopy.J. Biol. Chem. 1990; 265: 19434-19440Google Scholar). CL interaction with the above proteins appeared to be specific as substitution with other mitochondrial phospholipids (for example, phosphatidylcholine and phosphatidylethanolamine) did not fully reconstitute activity (for review see 5). Under experimental conditions in which CL was removed, denaturation and complete loss in activity of many of these proteins was observed. Thus, the appropriate content of CL may be an important requirement for activation of enzymes involved in mitochondrial respiration. CL was shown to be synthesized via the cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol (CDP-DG) pathway in the isolated rat heart (17Hatch G.M. Cardiolipin biosynthesis in the isolated heart.Biochem. J. 1994; 297: 201-208Google Scholar). In this pathway, phosphatidic acid (PA) was converted to CDP-DG. CDP-DG was then condensed with sn-glycerol-3-phosphate to form phosphatidylglycerol (PG) phosphate which was rapidly converted to PG. Finally, PG condensed with another CDP-DG molecule to form CL. CL contains four fatty acid side chains and the molecular composition of CL appeared to be in part dependent upon the type of fatty acids provided in the diet (2Hostetler K.Y. Polyglycerophospholipids: phosphatidylglycerol, diphosphatidylglycerol and bis(monoacylglycerol)phosphate.in: Hawthorne J.N. Ansell G.B. Phospholipids. Elsevier Press, Amsterdam1982: 215-261Google Scholar, 3Daum G. Lipids of mitochondria.Biochim. Biophys. Acta. 1985; 822: 1-42Google Scholar, 4Hatch G.M. Cardiolipin: biosynthesis, remodeling and trafficking in the heart and mammalian tissues.Int. J. Mol. Med. 1998; 1: 33-41Google Scholar, 5Hoch F.L. Cardiolipins and biomembrane function.Biochim. Biophys. Acta. 1992; 1113: 71-133Google Scholar, 18Berger A. German J.B. Phospholipid fatty acid composition of various mouse tissues after feeding α-linoleate or eicosatrienoate.Lipids. 1990; 25: 473-480Google Scholar). The proportion of CL symmetrical molecular species was determined to be 50–65% and the four acyl positions were shown to be occupied by monounsaturated and diunsaturated chains of 16–18 carbons in length (19Schlame M. Brody S. Hostetler K.Y. Mitochondrial cardiolipin in diverse eukaryotes: comparison of biosynthetic reactions and molecular acyl species.Eur. J. Biochem. 1993; 212: 727-735Google Scholar). The hydrophobic double unsaturated linoleic diacylglycerol species appeared to be an important structural requirement for the high protein binding affinity of CL (20Schlame M. Hovath L. Vigh L. Relationship between lipid saturation and lipid-protein interaction in liver mitochondria modified by catalytic hydrogenation with reference to cardiolipin molecular species.Biochem. J. 1990; 265: 79-85Google Scholar). Dietary modification of the molecular species composition of CL was shown to alter the oxygen consumption in cardiac mitochondria (21Yamaoka S. Urade R. Kido M. Cardiolipin molecular species in rat heart mitochondria are sensitive to essential fatty acid-deficient dietary lipids.J. Nutr. 1990; 120: 415-421Google Scholar, 22Yamaoka-Koseki S. Urade R. Kito M. Cardiolipins from rats fed different diets affect bovine heart cytochrome c oxidase activity.J. Nutr. 1991; 121: 956-958Google Scholar). In addition, the activity of delipidated rat liver cytochrome c oxidase was reconstituted by the addition of CL. The specific activity of the reconstituted cytochrome c oxidase varied markedly and significantly with different molecular species of CL. Thus, in addition to CL content, the appropriate molecular composition of CL may be critical for optimum mitochondrial respiratory performance. The deacylation–reacylation cycle for the molecular remodeling of glycerophospholipids was first described by Lands (23Lands W.E.M. Metabolism of glycerides II. The acylation of lysolecithin.J. Biol. Chem. 1960; 253: 2233-2237Google Scholar, 24Choy P.C. Skrzypczak M. Lee D. Jay F.T. Acyl-GPC and alkenyl/alkyl-GPC:acyl-CoA acyltransferases.Biochim. Biophys. Acta. 1997; 1348: 124-133Google Scholar). The acyltransferase (AT) activities for mammalian phosphatidylcholine remodeling have been extensively investigated (for review see 24). However, limited information was available on the ATs that are involved in the molecular remodeling of CL. An acyl-coenzyme A-dependent reacylation of dilysocardiolipin in rat liver microsomes was reported (25Eichberg J. The reacylation of deacylated derivatives of diphosphatidylglycerol by microsomes and mitochondria from rat liver.J. Biol. Chem. 1974; 249: 3423-3429Google Scholar). This process was not specific for linoleoyl-coenzyme A and was inactive in rat liver mitochondria. More recently, a deacylation–reacylation cycle for the molecular remodeling of endogenous CL in rat liver mitochondria was proposed (26Schlame M. Rustow B. Lysocardiolipin formation and regulation in isolated rat liver mitochondria.Biochem. J. 1990; 272: 589-595Google Scholar). Endogenous CL was deacylated to monolysocardiolipin (MLCL) and then reacylated with linoleoyl-coenzyme A, derived from phosphatidylcholine, to form CL. Such a deacylation followed by reacylation scheme for CL seems logical as mitochondrial phospholipase A2 was shown to readily hydrolyze endogenous and exogenous CL (27De Winter J.M. Lenting H.B.M. Neys F.W. Van Den Bosch H. Hydrolysis of membrane-associated phosphoglycerides by mitochondrial phospholipase A2.Biochim. Biophys. Acta. 1987; 917: 169-177Google Scholar, 28Buckland A.G. Kinkaid A.R. Wilton D.C. Cardiolipin hydrolysis by human phospholipases A2: The multiple enzymatic activities of human cytosolic phospholipase A2.Biochim. Biophys. Acta. 1998; 1390: 65-72Google Scholar, 29Waite M. Sisson P. Partial purification and characterization of the phospholipase A2 from rat liver mitochondria.Biochemistry. 1971; 10: 2377-2383Google Scholar, 30Hostetler K.Y. Zenner B.D. Morris H.P. Altered subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase in the Morris 7777 hepatoma.J. Lipid Res. 1978; 19: 553-560Google Scholar). However, AT(s) activities involved in the reacylation of MLCL to CL had never been identified or characterized in any mammalian tissue. In this study, we show that the isolated intact rat heart has the ability to rapidly and readily remodel CL with unsaturated fatty acids. In addition, we have characterized the enzyme activities that acylate MLCL to CL in rat heart mitochondrial fractions. To our knowledge this study represents the first detailed characterization of mitochondrial acyl-coenzyme A-dependent MLCL AT activity assayed with exogenous MLCL. We present a simple and reproducible in vitro assay for the determination of this activity. A preliminary report of this work was published in abstract form (31Taylor W.A. Dolinsky V.W. Hatch G.M. Physiological regulation of cardiolipin remodeling in rat heart.Can. Fed. Biol. Soc. 1998; 41: P003Google Scholar). Adult male Sprague-Dawley rats (150–300 g body weight) were maintained on Purina rat chow and tap water ad libitum, in a light- and temperature-controlled room. Treatment of animals conformed to the Guidelines of the Canadian Council on Animal Care. Rat heart fatty acid binding protein (FABP) and mouse adipocyte FABP were obtained from Dr. Judith Storch (Rutgers University). Adipocyte and keratinocyte lipid binding protein (LBP) were obtained from Dr. David Bernlohr (University of Minnesota). Rat liver acyl CoA binding protein (ACBP) was obtained from Dr. Jens Knudsen (Odense University, Denmark). H9c2 cells were obtained from the American Type Culture Collection. [1-14C]oleic acid, [1-14C]linoleic acid, [1-14C]palmitic acid, [1-14C]oleoyl-coenzyme A, and [1,3-3H]glycerol were obtained from Mandel Scientific (Missassauga, Ontario, Canada). [1-14C] linoleoyl-coenzyme A and [1-14C]palmitoyl-coenzyme A were obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO). Oleic acid, linoleic acid, palmitic acid and acyl-coenzyme As were obtained from Serdary Research Laboratories (Englewood Cliffs, NJ). MLCL (a mixture of 1′(1-acyl-sn-glycerol-3-phosphoryl)-3′(1′′,2′′:-diacyl-sn-glycerol-3-phosphoryl)glycerol and 1′ (1,2-diacyl-sn-glycerol-3-phosphoryl)-3′(1′′-acyl-sn-glycerol-3-phosphoryl)glycerol, produced by phospholipase A2 hydrolysis of bovine heart CL, was obtained from Avanti Polar Lipids (Alabaster, AL). Purity of the MLCL substrate was checked by two-dimensional thin-layer chromatography as described (17Hatch G.M. Cardiolipin biosynthesis in the isolated heart.Biochem. J. 1994; 297: 201-208Google Scholar). The fatty acyl molecular species composition of the MLCL substrate was examined as described (32Tardi P.G. Mukherjee J.J. Choy P.C. The quantitation of long chain acyl-CoA in mammalian tissues.Lipids. 1992; 27: 65-67Google Scholar) and was comprised mainly of linoleic (90.3%) and oleic (8.6%) acids. Dilysocardiolipin was synthesized from CL as described (28Buckland A.G. Kinkaid A.R. Wilton D.C. Cardiolipin hydrolysis by human phospholipases A2: The multiple enzymatic activities of human cytosolic phospholipase A2.Biochim. Biophys. Acta. 1998; 1390: 65-72Google Scholar). Ecolite scintillation cocktail was obtained from ICN Biochemicals (Costa Mesa, CA) and thin-layer plates (silica gel 60, 0.25 mm thickness) were obtained from Fisher Scientific (Winnipeg, Manitoba, Canada). N. mocambique mocambique phospholipase A2 and all other biochemicals were of analytical grade and obtained from either Fisher Scientific (Edmonton, Alberta, Canada) or Sigma Chemical Company (St. Louis, MO). For fatty acid perfusion experiments, the animal was killed by decapitation and the heart was quickly removed and cannulated via the aorta using a modified syringe needle (18 gauge). The remaining blood in the coronary circulation was removed by injecting the heart with freshly prepared Krebs-Henseleit buffer (33Krebs H.A. Henseleit K. Untersuchungen uber die Harstoffibildung in Tierkorper.Hoppe Seyler's Z. Physiol. Chem. 1932; 210: 33-36Google Scholar) using a 10 cm3 syringe. The heart was placed on a perfusion apparatus and perfused for 5 min or until electrical stabilization was achieved. The viability of the heart throughout the perfusion experiment was monitored via electrocardiac analysis. Subsequent to stabilization, the heart was perfused for 30 min in the Langendorff mode (34Langendorff O. Untersuchungen am uberlebenden saugetierberzen.Pfluegers Arch. 1896; 61: 291-332Google Scholar) with 12.5 ml of Krebs-Henseleit buffer containing 0.1 mm [1-14C]oleic acid (0.4 μCi/ml) bound to bovine serum albumin in a 1:1 molar ratio (35Cao S.G. Hatch G.M. Oleate stimulation of incorporation of exogenous glycerol into cardiolipin does not involve direct activation of the CDP-DG pathway.Biochem. Cell Biol. 1995; 73: 299-305Google Scholar, 36Arvidson G.A.E. Structural and metabolic heterogeneity of rat liver glycerophospholipids.Eur. J. Biochem. 1968; 4: 478-486Google Scholar, 37Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding.Anal. Biochem. 1976; 72: 248-254Google Scholar). In some experiments, the heart was perfused with 0.1 mm [1-14C]linoleic acid (0.4 μCi/ml) or 0.1 mm [1-14C]palmitic acid (0.4 μCi/ml) bound 1:1 to bovine serum albumin as above. The lipids were extracted and radioactivity incorporated into PG and CL was determined exactly as described [17]. For assay of MLCL AT activities, a 10% (w/v) rat heart mitochondrial fraction (or mitochondria from other rat tissues) was prepared by homogenizing the heart (Polytron 20 sec burst) in 0.25 m sucrose, 10 mm Tris-HCL, pH 7.4, 2 mm EDTA followed by differential centrifugation. The homogenate was centrifuged at 1,000 g for 5 min and the resulting supernatant was centrifuged at 12,000 g for 10 min. The pellet obtained from this centrifugation was washed once in homogenizing buffer and then resuspended in 1–2 ml of homogenizing buffer using a tight-fitting Dounce A homogenizer and designated the mitochondrial fraction. Rat heart mitochondrial fractions (50 μg protein) were incubated for 30 min at 25°C in 50 mm Tris-HCl, pH 9.0, 93 μm [1-14C]oleoyl-coenzyme A (6,700 dpm/nmol), or 33 μm [1-14C]linoleoyl-coenzyme A (68,700 dpm/nmol) at pH 8.0, or 33 μm [1-14C]palmitoyl-coenzyme A (80,200 dpm/nmol) at pH 8.0, 300 μm MLCL in a final volume of 0.35 ml. Under these optimum assay conditions the reactions were linear up to 150 μg protein and at least 40 min. The MLCL substrate in chloroform was dried under nitrogen and resuspended in double distilled water via sonication in a bath sonicator for 45 min prior to addition to the assay mixture. The temperature of the bath sonicator was maintained at 4°C by ice. The reaction was initiated by the addition of the radioactive acyl-coenzyme A substrate and terminated by the addition of 3 ml of chloroform–methanol 2:1 (v/v). To facilitate phase separation, 0.8 ml of 0.9% KCl was added. The aqueous phase was removed and the organic phase was washed with 2 ml of chloroform–methanol–0.9% NaCl 3:48:47 (v/v/v). The resulting organic fraction was dried under nitrogen and resuspended in 25 μl of chloroform–methanol 2:1 (v/v). A 20-μl aliquot of the resuspended organic phase was placed on a thin-layer plate and CL was separated from other phospholipids in a solvent system containing chloroform–hexane–methanol–acetic acid 50:30:10:5 (v/v/v/v). [3H]CL was prepared as described below and after addition of 0.1 μm [3H]CL (1,000 dpm/nmol) to the incubation mixture the recovery of labeled CL was 93% (average of two determinations) indicating little loss of the final product during the incubation. In some experiments, separation of CL from other lipids was confirmed using a two-dimensional thin-layer chromatography system described previously [17]. The silica gel corresponding to CL was removed and placed in a plastic scintillation vial and 5 ml of scintillant added. Radioactivity incorporated into CL was examined approximately 24 h later using a liquid scintillation counter. MLCL AT activity was taken as radioactivity incorporated into CL in the presence of the MLCL substrate minus radioactivity incorporated into CL in the absence of the MLCL substrate. In some experiments, 0.5 mm phospholipid, or 0.05% detergent was included in the assay mixture. In other experiments, mitochondrial fractions were preincubated for 5 min with 14 μm triacsin C prior to assay. In other experiments, 16.5 or 33 μm rat heart FABP, mouse adipocyte FABP, keratinocyte LBP, adipocyte LBP, rat liver ACBP, or albumin were included in the incubation mixture. In other experiments, 10 mm ATP, 1.0 mm coenzyme A, and 33 μm [1-14C]oleic acid (11,944 dpm/nmol) were added in place of [1-14C]oleoyl-coenzyme A (or 33 μm [1-14C]linoleic acid (13,907 dpm/nmol) added in place of [1-14C]linoleoyl-coenzyme A). In acyl-coenzyme A competition experiments, 33 μm of the competing acyl-coenzyme A was added at a 1:1 molar ratio with the appropriate radioactive acyl-coenzyme A substrate. H9c2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated newborn calf serum, 100 U/ml penicillin G, 10 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. Cell cultures were maintained at 37°C saturated with humidified air/5% carbon dioxide. H9c2 cells were incubated for 24 h with [1,3-3H]glycerol and the radioactive CL was isolated by thin-layer chromatography as described [17]. The radioactive CL was removed from the silica gel [36] and treated with 0.5 units of phospholipase A2 for 30 min in 0.5 ml buffer containing 50 mm Tris-HCl, pH 8.0, 3 mm CaCl2 at 37°C. The [3H]MLCL formed was isolated by thin-layer chromatography as described [17] and removed from the silica gel as above. The [3H]MLCL preparation was added to assay incubation mixtures (20,000 dpm/tube) and the formation of [3H]CL was monitored in the standard assay system described above minus acyl-coenzyme A. For fatty acid analysis, PG and CL were extracted from the silica gel as described [36] and fatty acid methyl esters were prepared and analyzed as described [32]. Protein was determined as described [37]. To examine whether the isolated intact rat heart could reacylate MLCL to CL, we initially examined the molecular composition of CL and its immediate precursor PG in rat heart. As seen in Table 1, CL was comprised of mainly linoleate (18:2) and oleate (18:1) molecular species. In contrast, PG was comprised of mainly palmitic and oleic acids with significant amounts of stearic and linoleic acids. Thus, the molecular composition of rat heart CL differed markedly from its immediate phospholipid precursor PG. We then perfused isolated rat hearts in the Langendorff mode for 30 min with oleic, linoleic, and palmitic acids (0.1 mm) bound to albumin in a 1:1 molar ratio. As seen in Table 2 perfusion of the isolated rat heart with labeled fatty acids resulted in considerable incorporation of these fatty acids into CL and PG. The incorporation of radioactive unsaturated fatty acids (oleate, 18:1; linoleate, 18:2) into CL was approximately 3- to 4-times higher than the saturated fatty acid (palmitate, 16:0). In addition, incorporation of radioactive unsaturated fatty acids into CL was higher than PG. In contrast, incorporation of radioactive palmitic acid into PG was much greater than into CL. Because saturated fatty acids such as palmitic acid enter into glycerophospholipids mainly by glycerophospholipid de novo biosynthetic pathways (38Akesson B. Initial esterification and conversion of intraportally injected [1-14C]linoleic acid in rat liver.Biochim. Biophys. Acta. 1970; 218: 57-70Google Scholar), the data suggest that cardiac CL is remodeled to obtain its appropriate molecular composition in vivo.TABLE 1.Molecular composition of rat heart cardiolipin and phosphatidylglycerolRelative PercentageFatty AcidCardiolipinPhosphatidylglycerolPalmitic acid (16:0)1.136.8Stearic acid (18:0)<1.015.5Oleic acid (18:1)7.735.9Linoleic acid (18:2)88.210.1All others combined2.02.7CL and PG were isolated from rat heart and the fatty acid composition was determined as described in Materials and Methods. Values are means of four hearts and are expressed as a relative percentage of total fatty acid. Open table in a new tab TABLE 2.Incorporation of [1-14C]palmitate, [1-14C]oleate, and [1-14C]linoleate into rat heart cardiolipin and phosphatidylglycerolRadioactivity incorporated into Cardiolipin and PhosphatidylglycerolFatty acidCardiolipinPhosphatidylglyceroldpm × 104/g freeze-dried heartPalmitic acid (16:0)0.4 ± 0.11.5 ± 0.2Oleic acid (18:1)1.4 ± 0.41.0 ± 0.3Linoleic acid (18:2)1.7 ± 0.30.7 ± 0.2Isolated hearts were perfused for 30 min in the Langendorff mode with Krebs-Henseleit buffer containing [1-14C]palmitic or [1-14C]oleic or [1-14C]linoleic acids and the radioactivity incorporated into CL and PG was determined as described in Materials and Methods. Values are means ± standard deviation of three hearts. Open table in a new tab CL and PG were isolated from rat heart and the fatty acid composition was determined as described in Materials and Methods. Values are means of four hearts and are expressed as a relative percentage of total fatty acid. Isolated hearts were perfused for 30 min in the Langendorff mode with Krebs-Henseleit buffer containing [1-14C]palmitic or [1-14C]oleic or [1-14C]linoleic acids and the radioactivity incorporated into CL and PG was determined as described in Materials and Methods. Values are means ± standard deviation of three hearts. MLCL AT activity had not been demonstrated or characterized in the heart. We thus examined the ability of rat heart mitochondria to reacylate MLCL to CL in vitro. MLCL AT activities were determined in freshly prepared isolated rat heart mitochondrial fractions. Oleoyl-coenzyme A and linoleoyl-coenzyme A were used as the acyl-coenzyme A substrates in this study as these were the major molecular species found in rat heart CL. The acylation of MLCL to CL was found to have a pH optimum in the alkaline range using oleoyl-coenzyme A and linoleoyl-coenzyme A as substrates (Fig. 1). When oleoyl-coenzyme A was used as substrate, the pH optimum range for the reaction was 8.0–9.0. With linoleoyl-coenzyme A as substrate, the pH optimum range for the reaction was 7.5–8.0. A gradual loss of activity with both substrates was observed when the pH was decreased (data not shown). However, with both substrates significant MLCL AT activity was observed at physiological pH. When dilysocardiolipin was added to these incubations, in place of MLCL, [14C]CL formation was not observed. In the presence of [1-14C]oleic acid, ATP and coenzyme A, MLCL and mitochondrial protein, some formation of [14C]CL was observed (9 pmol/min·mg protein). In addition, in the presence of [1-14C]linoleic acid, ATP and coenzyme A, MLCL and mitochondrial protein, some formation of [14C]CL was observed (4 pmol/min· mg protein). However, [14C]CL was not formed when [1-14C]oleic acid or [1-14C]linoleic acid was added alone, indicating the reaction was coenzyme A-dependent. When [1-14C]palmitoyl-coenzyme A was used as substrate, the formation of [14C]CL was 13 ± pmol/min·mg protein (average of three determinations) and was approximately 5% of the activity observed with [1-14C]oleoyl-coenzyme A as substrate. We examined whether MLCL could be converted to CL by transacylase activity. [3H]MLCL was synthesized by phospholipase A2 hydrolysis of [3H]CL isolated from H9c2 cells and added directly to assay incubation mixtures in the absence of acyl-coenzyme A. No significant formation of [3H]CL was observed, suggesting that transacylase activity does not contribute significantly to the acylation of MLCL in the heart. A previous study had shown that fatty acid binding proteins increased the incorporation of oleoyl-coenzyme A into phosphatidic acid (39Hubbell T. Behnke W.D.