Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1970 Sep;119(3):553–564. doi: 10.1042/bj1190553

Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria

P B Garland 1,*, D W Yates 1, B A Haddock 1
PMCID: PMC1179387  PMID: 5500316

Abstract

1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.–) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with εmM 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg2+ and corresponded to a previously described `external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg2+. Of these two `internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.–), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do the two internal acyl-CoA synthetases.

Full text

PDF

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. CHANCE B., WILLIAMS G. R. The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem. 1956;17:65–134. doi: 10.1002/9780470122624.ch2. [DOI] [PubMed] [Google Scholar]
  2. Galzigna L., Rossi C. R., Sartorelli L., Gibson D. M. A guanosine triphosphate-dependent acyl coenzyme A synthetase from rat liver mitochondria. J Biol Chem. 1967 May 10;242(9):2111–2115. [PubMed] [Google Scholar]
  3. Garland P. B., Shepherd D., Yates D. W. Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate. Biochem J. 1965 Nov;97(2):587–594. doi: 10.1042/bj0970587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. HEYTLER P. G. uncoupling of oxidative phosphorylation by carbonyl cyanide phenylhydrazones. I. Some characteristics of m-Cl-CCP action on mitochondria and chloroplasts. Biochemistry. 1963 Mar-Apr;2:357–361. doi: 10.1021/bi00902a031. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. MAHLER H. R., WAKIL S. J., BOCK R. M. Studies on fatty acid oxidation. I. Enzymatic activation of fatty acids. J Biol Chem. 1953 Sep;204(1):453–468. [PubMed] [Google Scholar]
  7. Nicholls DG RAND P. B. Th control of isocitrate oxidation by rat liver mitochondria. Biochem J. 1969 Sep;114(2):215–225. doi: 10.1042/bj1140215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Rossi C. R., Galzigna L., Alexandre A., Gibson D. M. Oxidation of long chain fatty acids by rat liver mitochondria. J Biol Chem. 1967 May 10;242(9):2102–2110. [PubMed] [Google Scholar]
  9. Tubbs P. K., Garland P. B. Variations in tissue contents of coenzyme A thio esters and possible metabolic implications. Biochem J. 1964 Dec;93(3):550–557. doi: 10.1042/bj0930550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Yates D. W., Shepherd D., Garland P. B. Organization of fatty-acid activation in rat liver mitochondria. Nature. 1966 Mar 19;209(5029):1213–1215. doi: 10.1038/2091213a0. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES