Abstract
The α-oxoglutarate dehydrogenase multienzyme complex was purified from Acinetobacter lwoffi to a high degree of homogeneity as shown by gel electrophoresis and analytical ultracentrifugation. Sedimentation-velocity analyses gave s20,w values which increased with increasing protein concentration, suggesting dissociation of the complex in dilute solution. The maximum s20,w value thereby obtained and the value determined by active enzyme centrifugation were both in the range 28–29S. Electron micrographs of the complex indicated a molecular diameter of 20–22nm (200–220Å). The overall activity of the complex was inhibited by NADH, and kinetic studies indicated sites of action on the first and third enzyme components. AMP and ADP relieved this inhibition and also stimulated enzyme activity. Assays specific for the first enzyme component showed this to be the site of action of the adenylates. The activity of the complex varied with energy charge in a manner consistent with its role in energy metabolism.
Full text
PDF








Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Atkinson D. E. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry. 1968 Nov;7(11):4030–4034. doi: 10.1021/bi00851a033. [DOI] [PubMed] [Google Scholar]
- Cecil R., Weitzman P. D. The electroreduction of the disulphide bonds of insulin and other proteins. Biochem J. 1964 Oct;93(1):1–11. doi: 10.1042/bj0930001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chapman A. G., Fall L., Atkinson D. E. Adenylate energy charge in Escherichia coli during growth and starvation. J Bacteriol. 1971 Dec;108(3):1072–1086. doi: 10.1128/jb.108.3.1072-1086.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chrambach A., Reisfeld R. A., Wyckoff M., Zaccari J. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal Biochem. 1967 Jul;20(1):150–154. doi: 10.1016/0003-2697(67)90272-2. [DOI] [PubMed] [Google Scholar]
- Cohen R., Mire M. Analytical-band centrifugation of an active enzyme-substrate complex. 1. Principle and practice of the centrifugation. Eur J Biochem. 1971 Nov 11;23(2):267–275. doi: 10.1111/j.1432-1033.1971.tb01618.x. [DOI] [PubMed] [Google Scholar]
- Cohen R., Mire M. Analytical-band centrifugation of an active enzyme-substrate complex. 2. Determination of active units of various enzymes. Eur J Biochem. 1971 Nov 11;23(2):276–281. doi: 10.1111/j.1432-1033.1971.tb01619.x. [DOI] [PubMed] [Google Scholar]
- DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
- Dietrich J., Henning U. Regulation of pyruvate dehydrogenase complex synthesis in Escherichia coli K 12. Identification of the inducing metabolite. Eur J Biochem. 1970 Jun;14(2):258–269. doi: 10.1111/j.1432-1033.1970.tb00285.x. [DOI] [PubMed] [Google Scholar]
- ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
- Hansen H. G., Henning U. Regulation of pyruvate dehydrogenase activity in Escherichia coli K12. Biochim Biophys Acta. 1966 Aug 10;122(2):355–358. doi: 10.1016/0926-6593(66)90076-2. [DOI] [PubMed] [Google Scholar]
- Hansford R. G. The effect of adenine nucleotides upon the 2-oxoglutarate dehydrogenase of blowfly flight muscle. FEBS Lett. 1972 Mar 15;21(2):139–141. doi: 10.1016/0014-5793(72)80122-4. [DOI] [PubMed] [Google Scholar]
- Hirashima M., Hayakawa T., Koike M. Mammalian alpha-keto acid dehydrogenase complexes. II. An improved procedure for the preparation of 2-oxoglutarate dehydrogenase complex from pig heart muscle. J Biol Chem. 1967 Mar 10;242(5):902–907. [PubMed] [Google Scholar]
- KOIKE M., REED L. J., CARROLL W. R. alpha-Keto acid dehydrogenation complexes. I. Purification and properties of pyruvate and alpha-ketoglutarate dehydrogenation complexes of Escherichia coli. J Biol Chem. 1960 Jul;235:1924–1930. [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]
- MUKHERJEE B. B., MATTHEWS J., HORNEY D. L., REED L. J. RESOLUTION AND RECONSTITUTION OF THE ESCHERICHIA COLI ALPHA-KETOGLUTARATE DEHYDROGENASE COMPLEX. J Biol Chem. 1965 May;240:2268–2269. [PubMed] [Google Scholar]
- Orr M. D., Blakley R. L., Panagou D. Discontinuous buffer systems for analytical and preparative electrophoresis of enzymes on polyacrylamide gel. Anal Biochem. 1972 Jan;45(1):68–85. doi: 10.1016/0003-2697(72)90008-5. [DOI] [PubMed] [Google Scholar]
- Parker M. G., Weitzman P. D.J. Regulation of NADP-linked isocitrate dehydrogenase activity in Acinetobacter. FEBS Lett. 1970 May 1;7(4):324–326. doi: 10.1016/0014-5793(70)80195-8. [DOI] [PubMed] [Google Scholar]
- Reed L. J., Oliver R. M. The multienzyme alpha-keto acid dehydrogenase complexes. Brookhaven Symp Biol. 1968 Jun;21(2):397–412. [PubMed] [Google Scholar]
- Schwartz E. R., Old L. O., Reed L. J. Regulatory properties of pyruvate dehydrogenase from Escherichia coli. Biochem Biophys Res Commun. 1968 May 10;31(3):495–500. doi: 10.1016/0006-291x(68)90504-4. [DOI] [PubMed] [Google Scholar]
- Shen L. C., Atkinson D. E. Regulation of pyruvate dehydrogenase from Escherichia coli. Interactions of adenylate energy charge and other regulatory parameters. J Biol Chem. 1970 Nov 25;245(22):5974–5978. [PubMed] [Google Scholar]
- Tanaka N., Koike K., Hamada M., Otsuka K. I., Suematsu T., Koike M. Mammalian -keto acid dehydrogenase complexes. VII. Resolution and reconstitution of the pig heart 2-oxoglutarate dehydrogenase complex. J Biol Chem. 1972 Jun 25;247(12):4043–4049. [PubMed] [Google Scholar]
- Wedding R. T., Black M. K. Nucleotide activation of cauliflower alpha-ketoglutarate dehydrogenase. J Biol Chem. 1971 Mar 25;246(6):1638–1643. [PubMed] [Google Scholar]
- Weitzman P. D.J. Regulation of alpha-ketoglutarate dehydrogenase activity in Acinetobacter. FEBS Lett. 1972 May 15;22(3):323–326. doi: 10.1016/0014-5793(72)80261-8. [DOI] [PubMed] [Google Scholar]
- Weitzman P. D., Jones D. Regulation of citrate synthase and microbial taxonomy. Nature. 1968 Jul 20;219(5151):270–272. doi: 10.1038/219270a0. [DOI] [PubMed] [Google Scholar]