Abstract
1. A spectrophotometric direct-reading assay for measurements of beta-oxidation by intact mitochondria is described. The procedure relies on the ability of ferricyanide to trap reducing equivalents generated by the acyl-CoA dehydrogenases (EC 1.3.99.3). The reduction of ferricyanide was recorded by using a dual-wavelength spectrophotometer. 2. Oxaloacetate or acetoacetate was used to stimulate the rate of beta-oxidation by rotenone-blocked mitochondria. Although both were effective with rat liver mitochondria, oxaloacetate gave about 75% more stimulation. With heart or kidney mitochondria, only oxaloacetate gave marked stimulation. Acetoacetate had no stimulatory effect with heart mitochondria, but a small stimulatory effect on the rate of beta-oxidation by kidney mitochondria. 3. The stoicheiometry of beta-oxidation-dependent reduction of ferricyanide was examined, and good correlations were found between experimental and theoretical amounts of ferricyanide reduced. 4. Ferricyanide appears as efficient a final electron acceptor as O2. With ferricyanide the rate of beta-oxidation by heart mitochondria can be measured without interference from the oxidation of tricarboxylic acid-cycle intermediates.
Full text
PDF












Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bremer J., Davis E. J. Phosphorylation coupled to acyl-coenzyme A dehydrogenase-linked oxidation of fatty acids by liver and heart mitochondria. Biochim Biophys Acta. 1972 Sep 20;275(3):298–301. doi: 10.1016/0005-2728(72)90210-1. [DOI] [PubMed] [Google Scholar]
- Bremer J., Wojtczak A. B. Factors controlling the rate of fatty acid -oxidation in rat liver mitochondria. Biochim Biophys Acta. 1972 Dec 8;280(4):515–530. doi: 10.1016/0005-2760(72)90131-2. [DOI] [PubMed] [Google Scholar]
- CLELAND W. W. Computer programmes for processing enzyme kinetic data. Nature. 1963 May 4;198:463–465. doi: 10.1038/198463a0. [DOI] [PubMed] [Google Scholar]
- Christophersen B. O., Bremer J. Erucic acid--an inhibitor of fatty acid oxidation in the heart. Biochim Biophys Acta. 1972 Dec 8;280(4):506–514. doi: 10.1016/0005-2760(72)90130-0. [DOI] [PubMed] [Google Scholar]
- Christophersen B. O., Christiansen R. Z. Studies on the mechanism of the inhibitory effects of erucylcarnitine in rat heart mitochondria. Biochim Biophys Acta. 1975 Jun 23;388(3):402–412. doi: 10.1016/0005-2760(75)90099-5. [DOI] [PubMed] [Google Scholar]
- Connelly J. L., Danner D. J., Bowden J. A. Branched chain alpha-keto acid metabolism. I. Isolation, purification, and partial characterization of bovine liver alpha-ketoisocaproic:alpha-keto-beta-methylvaleric acid dehydrogenase. J Biol Chem. 1968 Mar 25;243(6):1198–1203. [PubMed] [Google Scholar]
- Davis E. J. The effect of pyruvate on cyclic oxidations by heart sarcosomes. Biochim Biophys Acta. 1967 Jul 5;143(1):26–36. doi: 10.1016/0005-2728(67)90106-5. [DOI] [PubMed] [Google Scholar]
- Flatmark T., Pedersen J. I. Brown adipose tissue mitochondria. Biochim Biophys Acta. 1975 Mar 31;416(1):53–103. doi: 10.1016/0304-4173(75)90013-0. [DOI] [PubMed] [Google Scholar]
- Garland P. B., Chance B., Ernster L., Lee C. P., Wong D. Flavoproteins of mitochondrial fatty acid oxidation. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1696–1702. doi: 10.1073/pnas.58.4.1696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HALL L. M. Preparation of crystalline lithium acetoacetate. Anal Biochem. 1962 Jan;3:75–80. doi: 10.1016/0003-2697(62)90046-5. [DOI] [PubMed] [Google Scholar]
- Kean E. A. Improved method for isolation of hypoglycins A and B from fruit of Blighia sapida. J Pharm Pharmacol. 1974 Aug;26(8):639–640. doi: 10.1111/j.2042-7158.1974.tb10678.x. [DOI] [PubMed] [Google Scholar]
- Klingenberg M. Localization of the glycerol-phosphate dehydrogenase in the outer phase of the mitochondrial inner membrane. Eur J Biochem. 1970 Apr;13(2):247–252. doi: 10.1111/j.1432-1033.1970.tb00924.x. [DOI] [PubMed] [Google Scholar]
- LEHNINGER A. L., SUDDUTH H. C., WISE J. B. D-beta-Hydroxybutyric dehydrogenase of muitochondria. J Biol Chem. 1960 Aug;235:2450–2455. [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- MYERS D. K., SLATER E. C. The enzymic hydrolysis of adenosine triphosphate by liver mitochondria. I. Activities at different pH values. Biochem J. 1957 Dec;67(4):558–572. doi: 10.1042/bj0670558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mitchell P., Moyle J. Translocation of some anions cations and acids in rat liver mitochondria. Eur J Biochem. 1969 Jun;9(2):149–155. doi: 10.1111/j.1432-1033.1969.tb00588.x. [DOI] [PubMed] [Google Scholar]
- Osmundsen H., Sherratt H. S. A novel mechanism for inhibition of beta-oxidation by methylenecyclopropylacetyl-CoA, a metabolite of hypoglycin. FEBS Lett. 1975 Jul 15;55(1):38–41. doi: 10.1016/0014-5793(75)80951-3. [DOI] [PubMed] [Google Scholar]
- Podack E. R., Seubert W. On the mechanism of malonyl-CoA independent fatty acid synthesis. II. Isolation, properties and subcellular location of trans-2,3-hexenoyl-CoA and trans-2,3-decenoyl-CoA reductase. Biochim Biophys Acta. 1972 Oct 5;280(2):235–247. [PubMed] [Google Scholar]
- Quastel J. H., Wheatley A. H. Anaerobic oxidations. On ferricyanide as a reagent for the manometric investigation of dehydrogenase systems. Biochem J. 1938 May;32(5):936–943. doi: 10.1042/bj0320936. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stanley K. K., Tubbs P. K. The role of intermediates in mitochondrial fatty acid oxidation. Biochem J. 1975 Jul;150(1):77–88. doi: 10.1042/bj1500077. [DOI] [PMC free article] [PubMed] [Google Scholar]
- WALTER P., LARDY H. A. EFFECT OF ANTIMYCIN A ON OXIDATIVE PHOSPHORYLATION WITH FERRICYANIDE AS ELECTRON ACCEPTOR. Biochemistry. 1964 Jun;3:812–816. doi: 10.1021/bi00894a015. [DOI] [PubMed] [Google Scholar]
- Williamson D. H., Bates M. W., Page M. A., Krebs H. A. Activities of enzymes involved in acetoacetate utilization in adult mammalian tissues. Biochem J. 1971 Jan;121(1):41–47. doi: 10.1042/bj1210041. [DOI] [PMC free article] [PubMed] [Google Scholar]
