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. 1987 Jan;84(2):378–382. doi: 10.1073/pnas.84.2.378

Malonyl-CoA binding site and the overt carnitine palmitoyltransferase activity reside on the opposite sides of the outer mitochondrial membrane.

M S Murthy, S V Pande
PMCID: PMC304210  PMID: 3540964

Abstract

The overt carnitine palmitoyltransferase (palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) activity of intact mitochondria from rat heart and liver was found to be resistant to the action of proteases such as Nagarse (subtilisin, EC 3.4.21.14). Nagarse under the same conditions, however, greatly decreased the malonyl-CoA inhibition of carnitine palmitoyltransferase activity, the high-affinity binding of malonyl-CoA to mitochondria, and the ability of malonyl-CoA to shift to the right the sigmoid activity curve of carnitine palmitoyltransferase observed with variations in palmitoyl-CoA concentration. No noticeable effect of Nagarse pretreatment was observed on the binding of octanoyl-CoA to mitochondria. Subfractionation of liver mitochondria using a combination of swelling, shrinking, and density gradient centrifugation yielded a membrane fraction in which the specific activities of the outer membrane marker enzymes were enriched greater than or equal to 16-fold together with a near-parallel enrichment of malonyl-CoA-inhibitable carnitine palmitoyltransferase activity. The percent recovery of this carnitine palmitoyltransferase in the outer membrane vesicles also matched that of the known outer membrane markers. The carnitine palmitoyltransferase activity of these out-side-out vesicles became susceptible to added Nagarse only on their cosonication. These findings show that whereas the malonyl-CoA binding site relevant to the inhibition of carnitine palmitoyltransferase is situated on the outer side of the outer membrane, the overt carnitine palmitoyltransferase activity resides on the inner side of the outer membrane.

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Selected References

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  1. Bergstrom J. D., Reitz R. C. Studies on carnitine palmitoyl transferase: the similar nature of CPTi (inner form) and CPTo (outer form). Arch Biochem Biophys. 1980 Oct 1;204(1):71–79. doi: 10.1016/0003-9861(80)90008-9. [DOI] [PubMed] [Google Scholar]
  2. Bird M. I., Saggerson E. D. Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity. Biochem J. 1984 Sep 15;222(3):639–647. doi: 10.1042/bj2220639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brosnan J. T., Kopec B., Fritz I. B. The localization of carnitine palmitoyltransferase on the inner membrane of bovine liver mitochondria. J Biol Chem. 1973 Jun 10;248(11):4075–4082. [PubMed] [Google Scholar]
  4. Catravas G. N., Takenaga J., McHale C. G. Effect of chronic administration of morphine on monoamine oxidase activity in discrete regions of the brain of rats. Biochem Pharmacol. 1977 Feb 1;26(3):211–214. doi: 10.1016/0006-2952(77)90305-7. [DOI] [PubMed] [Google Scholar]
  5. Declercq P. E., Venincasa M. D., Mills S. E., Foster D. W., McGarry J. D. Interaction of malonyl-CoA and 2-tetradecylglycidyl-CoA with mitochondrial carnitine palmitoyltransferase I. J Biol Chem. 1985 Oct 15;260(23):12516–12522. [PubMed] [Google Scholar]
  6. FRITZ I. B., YUE K. T. LONG-CHAIN CARNITINE ACYLTRANSFERASE AND THE ROLE OF ACYLCARNITINE DERIVATIVES IN THE CATALYTIC INCREASE OF FATTY ACID OXIDATION INDUCED BY CARNITINE. J Lipid Res. 1963 Jul;4:279–288. [PubMed] [Google Scholar]
  7. Fiek C., Benz R., Roos N., Brdiczka D. Evidence for identity between the hexokinase-binding protein and the mitochondrial porin in the outer membrane of rat liver mitochondria. Biochim Biophys Acta. 1982 Jun 14;688(2):429–440. doi: 10.1016/0005-2736(82)90354-6. [DOI] [PubMed] [Google Scholar]
  8. Grantham B. D., Zammit V. A. Binding of [14C]malonyl-CoA to rat liver mitochondria after blocking of the active site of carnitine palmitoyltransferase I. Displacement of low-affinity binding by palmitoyl-CoA. Biochem J. 1986 Jan 15;233(2):589–593. doi: 10.1042/bj2330589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haddock B. A., Yates D. W., Garland P. B. The localization of some coenzyme A-dependent enzymes in rat liver mitochondria. Biochem J. 1970 Sep;119(3):565–573. doi: 10.1042/bj1190565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hoppel C. L., Tomec R. J. Carnitine palmityltransferase. Location of two enzymatic activities in rat liver mitochondria. J Biol Chem. 1972 Feb 10;247(3):832–841. [PubMed] [Google Scholar]
  11. Kiorpes T. C., Hoerr D., Ho W., Weaner L. E., Inman M. G., Tutwiler G. F. Identification of 2-tetradecylglycidyl coenzyme A as the active form of methyl 2-tetradecylglycidate (methyl palmoxirate) and its characterization as an irreversible, active site-directed inhibitor of carnitine palmitoyltransferase A in isolated rat liver mitochondria. J Biol Chem. 1984 Aug 10;259(15):9750–9755. [PubMed] [Google Scholar]
  12. Lund H., Bremer J. Carnitine acetyltransferase. Effect of malonyl-CoA, fasting and clofibrate feeding in mitochondria from different tissues. Biochim Biophys Acta. 1983 Jan 7;750(1):164–170. doi: 10.1016/0005-2760(83)90216-3. [DOI] [PubMed] [Google Scholar]
  13. Mannella C. A., Capolongo N., Berkowitz R. Correlation between outer-membrane lysis and susceptibility of mitochondria to inhibition by adriamycin and polyamines. Biochim Biophys Acta. 1986 Mar 12;848(3):312–316. doi: 10.1016/0005-2728(86)90205-7. [DOI] [PubMed] [Google Scholar]
  14. McGarry J. D., Leatherman G. F., Foster D. W. Carnitine palmitoyltransferase I. The site of inhibition of hepatic fatty acid oxidation by malonyl-CoA. J Biol Chem. 1978 Jun 25;253(12):4128–4136. [PubMed] [Google Scholar]
  15. McGarry J. D., Mannaerts G. P., Foster D. W. A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. J Clin Invest. 1977 Jul;60(1):265–270. doi: 10.1172/JCI108764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mills S. E., Foster D. W., McGarry J. D. Interaction of malonyl-CoA and related compounds with mitochondria from different rat tissues. Relationship between ligand binding and inhibition of carnitine palmitoyltransferase I. Biochem J. 1983 Jul 15;214(1):83–91. doi: 10.1042/bj2140083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Murthy M. S., Pande S. V. Mechanism of carnitine acylcarnitine translocase-catalyzed import of acylcarnitines into mitochondria. J Biol Chem. 1984 Jul 25;259(14):9082–9089. [PubMed] [Google Scholar]
  18. Murthy M. S., Pande S. V. Microcompartmentation of transported carnitine, acetylcarnitine and ADP occurs in the mitochondrial matrix. Implications for transport measurements and metabolism. Biochem J. 1985 Sep 15;230(3):657–663. doi: 10.1042/bj2300657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Norum K. R., Farstad M., Bremer J. The submitochondrial distribution of acid:CoA ligase (AMP) and palmityl-CoA:carnitine palmityltransferase in rat liver mitochondria. Biochem Biophys Res Commun. 1966 Sep 8;24(5):797–804. doi: 10.1016/0006-291x(66)90397-4. [DOI] [PubMed] [Google Scholar]
  20. Noy N., Zakim D. Substrate specificity of fatty-acyl-CoA ligase in liver microsomes. Biochim Biophys Acta. 1985 Feb 8;833(2):239–244. doi: 10.1016/0005-2760(85)90196-1. [DOI] [PubMed] [Google Scholar]
  21. Pande S. V., Blanchaer M. C. Preferential loss of ATP-dependent long-chain fatty acid activating enzyme in mitochondria prepared using Nagarse. Biochim Biophys Acta. 1970 Feb 10;202(1):43–48. doi: 10.1016/0005-2760(70)90216-x. [DOI] [PubMed] [Google Scholar]
  22. Pande S. V., Blanchaer M. C. Reversible inhibition of mitochondrial adenosine diphosphate phosphorylation by long chain acyl coenzyme A esters. J Biol Chem. 1971 Jan 25;246(2):402–411. [PubMed] [Google Scholar]
  23. Pande S. V., Goswami T., Parvin R. Protective role of adenine nucleotide translocase in O2-deficient hearts. Am J Physiol. 1984 Jul;247(1 Pt 2):H25–H34. doi: 10.1152/ajpheart.1984.247.1.H25. [DOI] [PubMed] [Google Scholar]
  24. Pande S. V., Murthy M. S., Noël H. Differential effects of phosphatidylcholine and cardiolipin on carnitine palmitoyltransferase activity. Biochim Biophys Acta. 1986 Jun 27;877(2):223–230. doi: 10.1016/0005-2760(86)90298-5. [DOI] [PubMed] [Google Scholar]
  25. Saggerson E. D. Carnitine acyltransferase activities in rat liver and heart measured with palmitoyl-CoA and octanoyl-CoA. Latency, effects of K+, bivalent metal ions and malonyl-CoA. Biochem J. 1982 Feb 15;202(2):397–405. doi: 10.1042/bj2020397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Saggerson E. D., Carpenter C. A. Malonyl CoA inhibition of carnitine acyltransferase activities: effects of thiol-group reagents. FEBS Lett. 1982 Jan 11;137(1):124–128. doi: 10.1016/0014-5793(82)80329-3. [DOI] [PubMed] [Google Scholar]
  27. Saggerson E. D., Carpenter C. A. The effect of malonyl-CoA on overt and latent carnitine acyltransferase activities in rat liver and adipocyte mitochondria. Biochem J. 1983 Feb 15;210(2):591–597. doi: 10.1042/bj2100591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vercesi A., Reynafarje B., Lehninger A. L. Stoichiometry of H+ ejection and Ca2+ uptake coupled to electron transport in rat heart mitochondria. J Biol Chem. 1978 Sep 25;253(18):6379–6385. [PubMed] [Google Scholar]
  29. Yates D. W., Garland P. B. The partial latency and intramitochondrial distribution of carnitine-palmitoyltransferase (e.c.2.3.1.-), and the CoASH and carnitine permeable space of rat liver mitochondria. Biochem Biophys Res Commun. 1966 May 25;23(4):460–465. doi: 10.1016/0006-291x(66)90750-9. [DOI] [PubMed] [Google Scholar]
  30. Zammit V. A., Corstorphine C. G. Altered release of carnitine palmitoyltransferase activity by digitonin from liver mitochondria of rats in different physiological states. Biochem J. 1985 Sep 1;230(2):389–394. doi: 10.1042/bj2300389. [DOI] [PMC free article] [PubMed] [Google Scholar]

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