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. 1978 Oct;75(10):4881–4885. doi: 10.1073/pnas.75.10.4881

Purification and characterization of branched chain α-keto acid dehydrogenase complex of bovine kidney

Flora H Pettit 1, Stephen J Yeaman 1, Lester J Reed 1
PMCID: PMC336225  PMID: 283398

Abstract

A branched chain α-keto acid dehydrogenase-dihydrolipoyl transacylase complex was purified to apparent homogeneity from bovine kidney mitochondria. As usually isolated, the complex (s20,w = 40 S) contained little, if any, dihydrolipoyl dehydrogenase. When saturated with the latter enzyme the complex had a specific activity of about 12 μmol of α-ketoisovalerate oxidized per min per mg of protein at 30° with NAD+ as electron acceptor. In addition to α-ketoisovalerate, the complex also oxidized α-ketoisocaproate, α-keto-β-methylvalerate, α-ketobutyrate, and pyruvate. The ratios of the specific activities were 2.0:1.5:1.0:1.0:0.4, and the apparent Km values were 40, 50, 37, 56, and 1000 μM. The complex was separated into its component enzymes. The branched chain α-keto acid dehydrogenase (6 S) consists of two different subunits with estimated molecular weights of 46,000 and 35,000. The dihydrolipoyl transacylase (20 S) contains apparently identical subunits of molecular weight about 52,000. In the electron microscope, the transacylase has the appearance of a cube, and the molecules of branched chain α-keto acid dehydrogenase appear to be distributed on the surface of the cube. In contrast to the pyruvate dehydrogenase complex of bovine kidney, the branched chain α-keto acid dehydrogenase complex apparently is not regulated by phosphorylation-dephosphorylation. Its activity, however, is subject to modulation by end-product inhibition.

Keywords: multienzyme complex, subunit composition, structural organization

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Selected References

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

  1. Collins J. H., Reed L. J. Acyl group and electron pair relay system: a network of interacting lipoyl moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes from Escherichia coli. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4223–4227. doi: 10.1073/pnas.74.10.4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Danner D. J., Lemmon S. K., Elsas L. J., 2nd Substrate specificity and stabilization by thiamine pyrophosphate of rat liver branched chain alpha-ketoacid dehydrogenase. Biochem Med. 1978 Feb;19(1):27–38. doi: 10.1016/0006-2944(78)90004-2. [DOI] [PubMed] [Google Scholar]
  4. Danson M. J., Perham R. N. Evidence for two lipoic acid residues per lipoate acetyltransferase chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Biochem J. 1976 Dec 1;159(3):677–682. doi: 10.1042/bj1590677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Goldberg A. L., Chang T. W. Regulation and significance of amino acid metabolism in skeletal muscle. Fed Proc. 1978 Jul;37(9):2301–2307. [PubMed] [Google Scholar]
  6. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  7. Linn T. C., Pelley J. W., Pettit F. H., Hucho F., Randall D. D., Reed L. J. -Keto acid dehydrogenase complexes. XV. Purification and properties of the component enzymes of the pyruvate dehydrogenase complexes from bovine kidney and heart. Arch Biochem Biophys. 1972 Feb;148(2):327–342. doi: 10.1016/0003-9861(72)90151-8. [DOI] [PubMed] [Google Scholar]
  8. Linn T. C. Studies on the apparent instability of bovine kidney alpha-ketoglutarate dehydrogenase complex. Arch Biochem Biophys. 1974 Apr 2;161(2):505–514. doi: 10.1016/0003-9861(74)90333-6. [DOI] [PubMed] [Google Scholar]
  9. Namba Y., Yoshizawa K., Ejima A., Hayashi T., Kaneda T. Coenzyme A- and nicotinamide adenine dinucleotide-dependent branched chain alpha-keto acid dehydrogenase. I. Purification and properties of the enzyme from Bacillus subtilis. J Biol Chem. 1969 Aug 25;244(16):4437–4447. [PubMed] [Google Scholar]
  10. Oliver R. M. Negative stain electron microscopy of protein macromolecules. Methods Enzymol. 1973;27:616–672. doi: 10.1016/s0076-6879(73)27029-5. [DOI] [PubMed] [Google Scholar]
  11. Parker P. J., Randle P. J. Branched chain 2-oxo-acid dehydrogenase complex of rat liver. FEBS Lett. 1978 Jun 1;90(1):183–186. doi: 10.1016/0014-5793(78)80325-1. [DOI] [PubMed] [Google Scholar]
  12. Parker P. J., Randle P. J. Partial purification and properties of branched-chain 2-oxo acid dehydrogenase of ox liver. Biochem J. 1978 Jun 1;171(3):751–757. doi: 10.1042/bj1710751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Roberts C. M., Sokatch J. R. Branched chain amino acids as activators of branched chain ketoacid dehydrogenase. Biochem Biophys Res Commun. 1978 Jun 14;82(3):828–833. doi: 10.1016/0006-291x(78)90857-4. [DOI] [PubMed] [Google Scholar]
  14. Rüdiger H. W., Langenbeck U., Goedde H. W. Oxidation of branched chain -ketoacids in Streptococcus faecalis and it's dependence on lipoic acid. Hoppe Seylers Z Physiol Chem. 1972 Jun;353(6):875–882. doi: 10.1515/bchm2.1972.353.1.875. [DOI] [PubMed] [Google Scholar]
  15. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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