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β-Hydroxy acyl-CoA inhibition of mitochondrial ATP production
0020.7I I X:86 $3.00+ 0.00 Copyright (’ 1986 Pergamon Press Ltd
In/. J. Biochem.Vol. 18, No. 2, pp. 183-185, 1986 All rights reserved
Printed in Great Britain.
p-HYDROXY ACYL-CoA INHIBITION OF MITOCHONDRIAL ATP PRODUCTION KATHLEEN HEALY MOORE’ and FRANKLIN E. HULL’ ‘Chemistry Department, Oakland University, Rochester, MI 48063, U.S.A. [Tel. (313)370-23381 2Department of Internal Medicine, Wayne State University, School of Medicine, Detroit, MI 48201. U.S.A.
/I-HydroxypalmitoyCCoA and P-hydroxystearoyl-CoA were synthesized, purified and quantitated. 2. /I-Hydroxypalmitoyl-CoA and /I-hydroxystearoyl-CoA instantly and reversibly inhibited oxidative phosphorylation by rabbit heart mitochondria oxidizing pyruvate. 3. [8-14C]ADP uptake studies showed that the fi-hydroxy acyl-CoA species linearly inhibited the
Abstract-l.
adenine nucleotide translocase system. 4. Free fl-hydroxy fatty acids at comparable state III respiration
concentrations
INTRODUCTION
Isolated hearts oxidizing exogenous fatty acid have been shown to accumulate large pools of acyl-CoA and acylcarnitine under conditions of ischemia or hypoxia (Neely et al., 1976; Shug et al., 1975). Moore et al. (1980) have recently shown that ischemic rabbit heart accumulated significant amounts of /I -hydroxy fatty acids. These p-oxidation intermediates were found primarily as carnitine and CoA esters; particularly, the fl-hydroxy acyl-CoA content contributed significantly to the increased acyl-CoA pool (Moore et al., 1982). Several studies have shown that long-chain acyl-CoA (palmitate, stearate, or oleate) adversely affects the production of ATP by isolated mitochondria through inhibition of adenine nucleotide translocation (Harris et al., 1972; Pande et al., 1971; Shug et al., 1971 and 1975). The purpose of this study was to determine the in vitro effects of b-hydroxy acyl-CoA on isolated heart mitochondria and to compare said effects with those of nonhydroxy acyl-CoA. MATERIALS
AND METHODS
Palmitate, /I-hydroxypalmitate, and p-hydroxystearate were obtained from Applied Science, State College, Pennsylvania. PalmitovCCoA, CoASH, and other biochemicals were from Sigma Chemical Co., St Louis, Missouri. [&“‘C]ADP (sp. ac. 50 mCi/mmol) was from New England Nuclear, Boston, Massachusetts.
(0.005 mM) did not affect ADP uptake
or
titated by the hydroxamate ester test (Rapport and Alonzo. 1955) and by thiol group assay subsequent to hydrolysis (Grunert and Phillips, 1951). Commercial palmitoyl-CoA and CoASH were the respective standards. In addition, the fi-hydroxy acyl-CoA products were further quantitated by gas chromatography-mass spectrometry analysis of the /3-hydroxy fatty acyl chains as developed by Moore Ed al. (1980). The CoA ester concentrations obtained by all methods correlated within a range of 2%.
Mitochondrial isolation and incubation Male New Zealand White rabbits (Oryctolagus cuniculus) were sacrificed; their hearts were rapidly removed and flushed with ice-cold saline. Mitochondria were released by Polytron disruption of a suspension of finely minced heart in ice-cold isolation medium (0.18 M KCl, 0.1 mM EGTA, pH 7.2) according to the method of Sordahl and Schwartz (1967). Mitochondrial protein content was determined by the biuret procedure (Gornall et al., 1949) after solubilization with deoxycholate. Mitochondrial incubations were carried out at 30°C in a medium of 0.23 M mannitol. 0.07 M sucrose, 0.02 M Tris-HCI, 0.005 M K,HPO,. and 0.02 mM EDTA at pH 7.4. State IV (Chance) respiration was initiated by addition of 5 mM pyruvate and I mM r.-malate; state III (Chance) respiration was initiated by addition of 0.29 mM ADP. Respiration was monitored with a recording oxygen polarograph. RESULTS
Inhibition
of state III respiration
Addition of palmitoyl-, fi-hydroxypalmitoyl-, /Ihydroxystearoyl-CoA to incubating mitochondria instantly inhibited the rate of state III respiration as compared to the rate in the absence of acyl-CoA (Fig. 1). Extrapolation of the inhibition curves indicated that, under the conditions used, 50% inhibition of respiration was obtained with l-3 micromolar concentrations of CoA esters. The inhibitions were reversible and were attenuated by t_-carnitine (2 mM), albumin (O.S%, w/v), or increased amounts of mitochondria and/or ADP. Inhibition was also relieved by uncoupling with 2,4-dinitrophenol (0.029 mM). In contrast to the CoA esters. unesterified palmitate,
Synthesis of fi-hydroxy acyI-CoA Long-chain /?-hydroxy acyl-CoA esters are not commercially available; therefore, it was necessary to synthesize and quantitate the appropriate esters in this laboratory. CoA esters of palmitate, /3-hydroxypalmitate, and p-hydroxystearate were synthesized by the mixed anhydride procedure of Goldman and Vagelos (1961). The reaction mixture was ether-extracted to remove free fatty acids and was purified by thin-layer chromatography (Pullman, 1973). Assay of the product for free thiol groups was negative (Grunert and Phillips, 1951). The purified acyl-CoA products were quan183
KATHLtt.\
H~AI_Y M(x)K~ and
FRANKLIN
E.
HULL
loor __.
Acyl - CoA (mlcromolor
)
InhibitIon of State III respiration by addition of palmltoyl-CoA (--) /~-hydroxypalmttoyCCoA ( ~~ ). and fi-hydroxystearoyl-CoA (--). Oxygraph studies were carried out at 30 C in buffer (0.23 M mannitol. 0.07 M sucrose. 0.02 M Tris--HCl. 5 mM K?HPO,. and 0.02 mM EDTA) at pH 7.4. State IV (Chance) respiration was initiated by addition of 5 mM pyruvate and I mM L-malate: state 111 (Chance) respiration was initiated by addition of 0.29 mM ADP. Volume of sample was 3.5 ml; each sample contained 4mg of mltochondrial protein For each run. state III rates were determined in the absence and presence of the respective acyl-CoA and compared. Shown are the means of two or more experiments.
P-hydroxypalmitate, and /I-hydroxystearate did not inhibit state III respiration at comparable concentrations (0.005 mM); however, at tenfold higher concentrations the free acids caused irreversible inhibition (i.e. not reversed by uncoupling) presumably by disruption of mitochondrial membrane structure. Inhibition
of‘ ADP uptake
Specific binding of the CoA esters to the adenine nucleotide translocase system was assayed by the [14C]ADP binding technique of Wojtczak and Zaluska (I 967). The assays were performed in triplicate at 0°C. The twice-washed mitochondrial pellet was dissolved in scintillation cocktail containing Triton X-100 (25% v/v); aliquots of combined supernatants were counted to assure complete recovery. All three CoA esters inhibited [‘4C]ADP binding to the adenine nucleotide translocase system (Fig. 2). Under the conditions used, 50% inhibition of ADP uptake was effected by S-IO micromolar acyl-CoA. The free acids exhibited no effect on [14C]ADP uptake at concentrations of to 0.025 mM; higher concentrations gave erratic results. DISCCSSION
From the above results it is clear that b-hydroxypalmitoyl-CoA and /j-hydroxystearoyl-CoA are similar to palmitoyl-CoA in terms of their effects on mitochondrial ATP production. The slight differences in inhibitory effects observed with P-hydroxypalmitoyl-CoA and p-hydroxystearoyl-CoA may be a consequence of chain length (Shrago et al., 1974). Lack of inhibition by free acids at corresponding concentrations supports earlier findings that the CoA portion of the ester is essential to inhibition of the adenine nucleotide translocase (Pande and Blanchaer, 1971). Free CoASH has also been shown to be incapable of the type of inhibition observed here
Acyl - CoA
( mlcromolorl
Fig. 2. Inhibition of mitochondrial [“CIADP binding b! palmitoyl-CoA (--). p-hydroxypalmitoyl-CoA (~). and /I-hydroxystearoyl-CoA (- --). Triplicate I-ml samples containing buffer (100 mM KCI. I mM MgClz, 40 mM Tris HCI. pH 7.4). CoA ester. and mitochondria (2 mg) were preequilibrated at 0 C for 4 min. Binding reaction was initiated by addition of 0. IO mM [R-“C]ADP (20 nCi): after 2 mm incubation. reaction was terminated by addition of atractyloside (0.003 mM). Shown are the means of two or more experiments.
(Pande and Blanchaer. I97 1: Christiansen and Davis. 1978). Long-chain acyl-CoA molecules may be important physiological effecters. In addition to the inhibition of adenine nucleotide translocase (Pande and Blanchaer, 1971; Shug et al., 197l), in citro inhibition or perturbation of a variety of processes such as energy-linked Ca’+-mediated mitochondrial transport (Beatrice and Pfeiffer, 1981) and sarcolemmal Na+,K+-ATPase (Owens et al., 1982) have been reported. Attempts to extrapolate these in vitro observations to the in uiuo situation have resulted in diverse predictions. Idell-Wenger et al. (1978) showed that the mitochondrial and cytosolic concentrations of long-chain acyl-CoA in ischemic rat heart were great enough to allow for in vice inhibition of adenine nucleotide translocation based on K, values determined with isolated mitochondria. Recently LaNoue et al., (1981) have demonstrated that the apparent K, for acyl-CoA under in viva conditions is dramatically increased due to the high concentration of mitochondrial protein in the cell; therefore, long-chain acyl-CoA levels in the ischemic heart may not be sufficient to inhibit oxidative phosphorylation. In either case, this report has shown that the long-chain /?-hydroxyl acyl-CoA species which accumulate during ischemia (Moore et al., 1982) must also be considered as potential inhibitors of mitochondrial energy production. REFERENCES Beatrice M. C. and Pfeiffer D. R. (1981) The mechanism of palmitoyl-CoA inhibition of Cal’ uptake in liver and heart mitochondria. Biochem. J. 194, 71 77. Christiansen E. N. and Davis E. J. (1978) The effect of coenzyme A and carnitine on steady state ATP,‘ADP ratios and the rate of long chain free fatty acid oxidation in liver mitochondria. Biochim. biophys. Acta 502, 17-28. Goldman P. and Vagelos P. R. (1961) The specificity of triglyceride synthesis from diglycerides in chicken adipose tissue. J. bid. Chem. 236, 262&2623. Gornall A. G.. Bardawill C. S. and David M. M. (1949)
fi-Hydroxy
acyl-CoA
Determination of serum proteins by means of the biuret reaction, J. hiol. C/tern. 177, 751-766. Grunert R. R. and Phillips P. H. (1951) A modification of the nitroprusside method of analysis for glutathione. Arch. Biochem. 30, 217-225. Harris R. A., Farmer B. and Ozawa T. (1972) Inhibition of the mitochondrial adenine nucleotide transport system by olevl CoA. Archs Biochem. Bioahrs. 150, 199.-209. Idell-Wenger J. A.. Grotyohann’ L: W. and Neely J. R. (1978) Coenzyme A and carnitine distribution in normal and ischemic hearts. J. hi@/. Cfienr. 253, 43104318. LaNoue K. F., Watts J. A. and Koch C. D. (1981) Adenine nucleotide transport during cardiac ischemia. Am. J. Ph~siol. 241, H663-H671. Moore K. H., Radloff J. F., Hull F. E. and Sweeley C. C. (1980) Incomplete fatty acid oxidation by ischemic heart: /I-hydroxy fatty acid production. Am. J. Physiol. 239, H257--H265. Moore K. H., Koen A. E. and Hull F. E. (1982) a-hydroxy fatty acid production by ischemic rabbit heart: distribution and chemical states. 1. c/in. Inwsr. 69, 377-383. Neely J. R., Rovetto M. J. and Whitmer J. T. (1976) Rate-limiting steps of carbohydrate and fatty acid metabolism in ischemic’heart. Acta Med. .ycand. S.&l. 587,9-l 5. Owens K.. Kennett F. F. and Wealicki W. B. (1982) Effects of fatty acid intermediates on Ni ’ -K +-ATPase activity in cardiac sarcolemma. Am. J. Physiol. 242, H456H461.
inhibition
185
Pande S. V. and Blanchaer M. C. (1971) J. hi&. Chem. 246, 402m~4I I. Pullman M. E. (I 973) A convenient and versatile method for purification of CoA thiol esters. Anal??. Biochem. 54, 188~198. Rapport M. M. and Alonzo N. (1955) Photometric determination of fatty acid ester groups in phospholipids. J. biol. Chem. 217, 193.-197. Shrago E., Shug A., Elson C, Spennetta T. and Crosby C. (1974) Regulation of metabolite transport in rat and guinea pig liver mitochondria by long chain fatty acyI coenzyme A esters. Biol. Chem. 249, 5269-5214. Shug A., Lerner E., Elson C. and Shrago E. (1971) The inhibition of adenine nucleotide translocase activity by oleyl CoA and it’s reversal in rat liver mitochondria. Biochem. hiophys. Res. Commun. 43, 557-563. Shug A. L., Shrago E., Bittar N., Felts J. and Koke J. R. (1975) Acyl-CoA inhibition of adenine nucleotide translocation in ischemic myocardium Am. J. Phy~M. 228, 689-692. Sordahl D. A. and Schwartz A. (1967) Effects of dipyridmole on heart muscle mitochondria. M&c. fharmaco(. 3, 509-5 15. Wojtczak L. and Zaluska H. (1967) The inhibition of translocation of adenine nucleotides through mitochondrial membranes by oleate. Bioc~re~?z.hiophys. Res. C~)rn~~~~z. 28, 768 I
In/. J. Biochem.Vol. 18, No. 2, pp. 183-185, 1986 All rights reserved
Printed in Great Britain.
p-HYDROXY ACYL-CoA INHIBITION OF MITOCHONDRIAL ATP PRODUCTION KATHLEEN HEALY MOORE’ and FRANKLIN E. HULL’ ‘Chemistry Department, Oakland University, Rochester, MI 48063, U.S.A. [Tel. (313)370-23381 2Department of Internal Medicine, Wayne State University, School of Medicine, Detroit, MI 48201. U.S.A.
/I-HydroxypalmitoyCCoA and P-hydroxystearoyl-CoA were synthesized, purified and quantitated. 2. /I-Hydroxypalmitoyl-CoA and /I-hydroxystearoyl-CoA instantly and reversibly inhibited oxidative phosphorylation by rabbit heart mitochondria oxidizing pyruvate. 3. [8-14C]ADP uptake studies showed that the fi-hydroxy acyl-CoA species linearly inhibited the
Abstract-l.
adenine nucleotide translocase system. 4. Free fl-hydroxy fatty acids at comparable state III respiration
concentrations
INTRODUCTION
Isolated hearts oxidizing exogenous fatty acid have been shown to accumulate large pools of acyl-CoA and acylcarnitine under conditions of ischemia or hypoxia (Neely et al., 1976; Shug et al., 1975). Moore et al. (1980) have recently shown that ischemic rabbit heart accumulated significant amounts of /I -hydroxy fatty acids. These p-oxidation intermediates were found primarily as carnitine and CoA esters; particularly, the fl-hydroxy acyl-CoA content contributed significantly to the increased acyl-CoA pool (Moore et al., 1982). Several studies have shown that long-chain acyl-CoA (palmitate, stearate, or oleate) adversely affects the production of ATP by isolated mitochondria through inhibition of adenine nucleotide translocation (Harris et al., 1972; Pande et al., 1971; Shug et al., 1971 and 1975). The purpose of this study was to determine the in vitro effects of b-hydroxy acyl-CoA on isolated heart mitochondria and to compare said effects with those of nonhydroxy acyl-CoA. MATERIALS
AND METHODS
Palmitate, /I-hydroxypalmitate, and p-hydroxystearate were obtained from Applied Science, State College, Pennsylvania. PalmitovCCoA, CoASH, and other biochemicals were from Sigma Chemical Co., St Louis, Missouri. [&“‘C]ADP (sp. ac. 50 mCi/mmol) was from New England Nuclear, Boston, Massachusetts.
(0.005 mM) did not affect ADP uptake
or
titated by the hydroxamate ester test (Rapport and Alonzo. 1955) and by thiol group assay subsequent to hydrolysis (Grunert and Phillips, 1951). Commercial palmitoyl-CoA and CoASH were the respective standards. In addition, the fi-hydroxy acyl-CoA products were further quantitated by gas chromatography-mass spectrometry analysis of the /3-hydroxy fatty acyl chains as developed by Moore Ed al. (1980). The CoA ester concentrations obtained by all methods correlated within a range of 2%.
Mitochondrial isolation and incubation Male New Zealand White rabbits (Oryctolagus cuniculus) were sacrificed; their hearts were rapidly removed and flushed with ice-cold saline. Mitochondria were released by Polytron disruption of a suspension of finely minced heart in ice-cold isolation medium (0.18 M KCl, 0.1 mM EGTA, pH 7.2) according to the method of Sordahl and Schwartz (1967). Mitochondrial protein content was determined by the biuret procedure (Gornall et al., 1949) after solubilization with deoxycholate. Mitochondrial incubations were carried out at 30°C in a medium of 0.23 M mannitol. 0.07 M sucrose, 0.02 M Tris-HCI, 0.005 M K,HPO,. and 0.02 mM EDTA at pH 7.4. State IV (Chance) respiration was initiated by addition of 5 mM pyruvate and I mM r.-malate; state III (Chance) respiration was initiated by addition of 0.29 mM ADP. Respiration was monitored with a recording oxygen polarograph. RESULTS
Inhibition
of state III respiration
Addition of palmitoyl-, fi-hydroxypalmitoyl-, /Ihydroxystearoyl-CoA to incubating mitochondria instantly inhibited the rate of state III respiration as compared to the rate in the absence of acyl-CoA (Fig. 1). Extrapolation of the inhibition curves indicated that, under the conditions used, 50% inhibition of respiration was obtained with l-3 micromolar concentrations of CoA esters. The inhibitions were reversible and were attenuated by t_-carnitine (2 mM), albumin (O.S%, w/v), or increased amounts of mitochondria and/or ADP. Inhibition was also relieved by uncoupling with 2,4-dinitrophenol (0.029 mM). In contrast to the CoA esters. unesterified palmitate,
Synthesis of fi-hydroxy acyI-CoA Long-chain /?-hydroxy acyl-CoA esters are not commercially available; therefore, it was necessary to synthesize and quantitate the appropriate esters in this laboratory. CoA esters of palmitate, /3-hydroxypalmitate, and p-hydroxystearate were synthesized by the mixed anhydride procedure of Goldman and Vagelos (1961). The reaction mixture was ether-extracted to remove free fatty acids and was purified by thin-layer chromatography (Pullman, 1973). Assay of the product for free thiol groups was negative (Grunert and Phillips, 1951). The purified acyl-CoA products were quan183
KATHLtt.\
H~AI_Y M(x)K~ and
FRANKLIN
E.
HULL
loor __.
Acyl - CoA (mlcromolor
)
InhibitIon of State III respiration by addition of palmltoyl-CoA (--) /~-hydroxypalmttoyCCoA ( ~~ ). and fi-hydroxystearoyl-CoA (--). Oxygraph studies were carried out at 30 C in buffer (0.23 M mannitol. 0.07 M sucrose. 0.02 M Tris--HCl. 5 mM K?HPO,. and 0.02 mM EDTA) at pH 7.4. State IV (Chance) respiration was initiated by addition of 5 mM pyruvate and I mM L-malate: state 111 (Chance) respiration was initiated by addition of 0.29 mM ADP. Volume of sample was 3.5 ml; each sample contained 4mg of mltochondrial protein For each run. state III rates were determined in the absence and presence of the respective acyl-CoA and compared. Shown are the means of two or more experiments.
P-hydroxypalmitate, and /I-hydroxystearate did not inhibit state III respiration at comparable concentrations (0.005 mM); however, at tenfold higher concentrations the free acids caused irreversible inhibition (i.e. not reversed by uncoupling) presumably by disruption of mitochondrial membrane structure. Inhibition
of‘ ADP uptake
Specific binding of the CoA esters to the adenine nucleotide translocase system was assayed by the [14C]ADP binding technique of Wojtczak and Zaluska (I 967). The assays were performed in triplicate at 0°C. The twice-washed mitochondrial pellet was dissolved in scintillation cocktail containing Triton X-100 (25% v/v); aliquots of combined supernatants were counted to assure complete recovery. All three CoA esters inhibited [‘4C]ADP binding to the adenine nucleotide translocase system (Fig. 2). Under the conditions used, 50% inhibition of ADP uptake was effected by S-IO micromolar acyl-CoA. The free acids exhibited no effect on [14C]ADP uptake at concentrations of to 0.025 mM; higher concentrations gave erratic results. DISCCSSION
From the above results it is clear that b-hydroxypalmitoyl-CoA and /j-hydroxystearoyl-CoA are similar to palmitoyl-CoA in terms of their effects on mitochondrial ATP production. The slight differences in inhibitory effects observed with P-hydroxypalmitoyl-CoA and p-hydroxystearoyl-CoA may be a consequence of chain length (Shrago et al., 1974). Lack of inhibition by free acids at corresponding concentrations supports earlier findings that the CoA portion of the ester is essential to inhibition of the adenine nucleotide translocase (Pande and Blanchaer, 1971). Free CoASH has also been shown to be incapable of the type of inhibition observed here
Acyl - CoA
( mlcromolorl
Fig. 2. Inhibition of mitochondrial [“CIADP binding b! palmitoyl-CoA (--). p-hydroxypalmitoyl-CoA (~). and /I-hydroxystearoyl-CoA (- --). Triplicate I-ml samples containing buffer (100 mM KCI. I mM MgClz, 40 mM Tris HCI. pH 7.4). CoA ester. and mitochondria (2 mg) were preequilibrated at 0 C for 4 min. Binding reaction was initiated by addition of 0. IO mM [R-“C]ADP (20 nCi): after 2 mm incubation. reaction was terminated by addition of atractyloside (0.003 mM). Shown are the means of two or more experiments.
(Pande and Blanchaer. I97 1: Christiansen and Davis. 1978). Long-chain acyl-CoA molecules may be important physiological effecters. In addition to the inhibition of adenine nucleotide translocase (Pande and Blanchaer, 1971; Shug et al., 197l), in citro inhibition or perturbation of a variety of processes such as energy-linked Ca’+-mediated mitochondrial transport (Beatrice and Pfeiffer, 1981) and sarcolemmal Na+,K+-ATPase (Owens et al., 1982) have been reported. Attempts to extrapolate these in vitro observations to the in uiuo situation have resulted in diverse predictions. Idell-Wenger et al. (1978) showed that the mitochondrial and cytosolic concentrations of long-chain acyl-CoA in ischemic rat heart were great enough to allow for in vice inhibition of adenine nucleotide translocation based on K, values determined with isolated mitochondria. Recently LaNoue et al., (1981) have demonstrated that the apparent K, for acyl-CoA under in viva conditions is dramatically increased due to the high concentration of mitochondrial protein in the cell; therefore, long-chain acyl-CoA levels in the ischemic heart may not be sufficient to inhibit oxidative phosphorylation. In either case, this report has shown that the long-chain /?-hydroxyl acyl-CoA species which accumulate during ischemia (Moore et al., 1982) must also be considered as potential inhibitors of mitochondrial energy production. REFERENCES Beatrice M. C. and Pfeiffer D. R. (1981) The mechanism of palmitoyl-CoA inhibition of Cal’ uptake in liver and heart mitochondria. Biochem. J. 194, 71 77. Christiansen E. N. and Davis E. J. (1978) The effect of coenzyme A and carnitine on steady state ATP,‘ADP ratios and the rate of long chain free fatty acid oxidation in liver mitochondria. Biochim. biophys. Acta 502, 17-28. Goldman P. and Vagelos P. R. (1961) The specificity of triglyceride synthesis from diglycerides in chicken adipose tissue. J. bid. Chem. 236, 262&2623. Gornall A. G.. Bardawill C. S. and David M. M. (1949)
fi-Hydroxy
acyl-CoA
Determination of serum proteins by means of the biuret reaction, J. hiol. C/tern. 177, 751-766. Grunert R. R. and Phillips P. H. (1951) A modification of the nitroprusside method of analysis for glutathione. Arch. Biochem. 30, 217-225. Harris R. A., Farmer B. and Ozawa T. (1972) Inhibition of the mitochondrial adenine nucleotide transport system by olevl CoA. Archs Biochem. Bioahrs. 150, 199.-209. Idell-Wenger J. A.. Grotyohann’ L: W. and Neely J. R. (1978) Coenzyme A and carnitine distribution in normal and ischemic hearts. J. hi@/. Cfienr. 253, 43104318. LaNoue K. F., Watts J. A. and Koch C. D. (1981) Adenine nucleotide transport during cardiac ischemia. Am. J. Ph~siol. 241, H663-H671. Moore K. H., Radloff J. F., Hull F. E. and Sweeley C. C. (1980) Incomplete fatty acid oxidation by ischemic heart: /I-hydroxy fatty acid production. Am. J. Physiol. 239, H257--H265. Moore K. H., Koen A. E. and Hull F. E. (1982) a-hydroxy fatty acid production by ischemic rabbit heart: distribution and chemical states. 1. c/in. Inwsr. 69, 377-383. Neely J. R., Rovetto M. J. and Whitmer J. T. (1976) Rate-limiting steps of carbohydrate and fatty acid metabolism in ischemic’heart. Acta Med. .ycand. S.&l. 587,9-l 5. Owens K.. Kennett F. F. and Wealicki W. B. (1982) Effects of fatty acid intermediates on Ni ’ -K +-ATPase activity in cardiac sarcolemma. Am. J. Physiol. 242, H456H461.
inhibition
185
Pande S. V. and Blanchaer M. C. (1971) J. hi&. Chem. 246, 402m~4I I. Pullman M. E. (I 973) A convenient and versatile method for purification of CoA thiol esters. Anal??. Biochem. 54, 188~198. Rapport M. M. and Alonzo N. (1955) Photometric determination of fatty acid ester groups in phospholipids. J. biol. Chem. 217, 193.-197. Shrago E., Shug A., Elson C, Spennetta T. and Crosby C. (1974) Regulation of metabolite transport in rat and guinea pig liver mitochondria by long chain fatty acyI coenzyme A esters. Biol. Chem. 249, 5269-5214. Shug A., Lerner E., Elson C. and Shrago E. (1971) The inhibition of adenine nucleotide translocase activity by oleyl CoA and it’s reversal in rat liver mitochondria. Biochem. hiophys. Res. Commun. 43, 557-563. Shug A. L., Shrago E., Bittar N., Felts J. and Koke J. R. (1975) Acyl-CoA inhibition of adenine nucleotide translocation in ischemic myocardium Am. J. Phy~M. 228, 689-692. Sordahl D. A. and Schwartz A. (1967) Effects of dipyridmole on heart muscle mitochondria. M&c. fharmaco(. 3, 509-5 15. Wojtczak L. and Zaluska H. (1967) The inhibition of translocation of adenine nucleotides through mitochondrial membranes by oleate. Bioc~re~?z.hiophys. Res. C~)rn~~~~z. 28, 768 I