Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1986 Apr 15;235(2):429–434. doi: 10.1042/bj2350429

Rat tissue concentrations of branched-chain 2-oxo acid dehydrogenase complex. Re-evaluation by immunoassay and bioassay.

P A Patston, J Espinal, J M Shaw, P J Randle
PMCID: PMC1146704  PMID: 3741400

Abstract

A rabbit polyclonal antibody to purified ox kidney branched-chain oxo acid dehydrogenase complex was shown by a variety of techniques to be an antibody to the E2 (acyltransferase) component. Rocket immunoelectrophoresis showed that the antibody does not discriminate between phosphorylated (inactive) or dephosphorylated (active) complex, and the same technique is used to assay total branched-chain complex (sum of active and inactive forms) in rat liver and heart mitochondrial extracts. The values obtained in normal rats fed on normal diet were comparable with those obtained by spectrophotometric assay of the holocomplex reaction after conversion of inactive complex into active complex. The values obtained in liver mitochondria from rats fed on 0%-casein diet or starved for 48 h were comparable with those in rats fed on normal diet, whereas earlier studies using spectrophotometric assay had shown substantial decreases in rats fed on 0%-casein diet or starved for 48 h. It has been shown that conversion of inactive complex into active complex requires prolonged incubation (120 min) in the presence of ketoleucine (4-methyl-2-oxopentanoate; to inhibit branched-chain oxo acid dehydrogenase kinase) to effect complete conversion in mitochondria from rats fed on 0%-casein diet, or starved for 48 h, or made diabetic with alloxan. By this technique, total activity of the complex in rat liver mitochondria was unaffected by diet or diabetes. The effects of diet and diabetes to decrease the activity of branched-chain complex in rat liver are therefore apparently mediated wholly through inactivation of the complex by phosphorylation.

Full text

PDF
429

Images in this article

431Fig. 1.
on p.431
432Fig. 2.
on p.432

Selected References

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

  1. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  2. Cook K. G., Bradford A. P., Yeaman S. J., Aitken A., Fearnley I. M., Walker J. E. Regulation of bovine kidney branched-chain 2-oxoacid dehydrogenase complex by reversible phosphorylation. Eur J Biochem. 1984 Dec 17;145(3):587–591. doi: 10.1111/j.1432-1033.1984.tb08597.x. [DOI] [PubMed] [Google Scholar]
  3. Cook K. G., Bradford A. P., Yeaman S. J. Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex. Biochem J. 1985 Feb 1;225(3):731–735. doi: 10.1042/bj2250731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Damuni Z., Merryfield M. L., Humphreys J. S., Reed L. J. Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4335–4338. doi: 10.1073/pnas.81.14.4335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Espinal J., Patston P. A., Fatania H. R., Lau K. S., Randle P. J. Purification and properties of a protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex. Biochem J. 1985 Jan 15;225(2):509–516. doi: 10.1042/bj2250509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fatania H. R., Lau K. S., Randle P. J. Activation of phosphorylated branched chain 2-oxoacid dehydrogenase complex. FEBS Lett. 1982 Oct 4;147(1):35–39. doi: 10.1016/0014-5793(82)81006-5. [DOI] [PubMed] [Google Scholar]
  7. Fatania H. R., Lau K. S., Randle P. J. Inactivation of purified ox kidney branched-chain 2-oxoacid dehydrogenase complex by phosphorylation. FEBS Lett. 1981 Sep 28;132(2):285–288. doi: 10.1016/0014-5793(81)81180-5. [DOI] [PubMed] [Google Scholar]
  8. Foden S., Randle P. J. Calcium metabolism in rat hepatocytes. Biochem J. 1978 Mar 15;170(3):615–625. doi: 10.1042/bj1700615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gillim S. E., Paxton R., Cook G. A., Harris R. A. Activity state of the branched chain alpha-ketoacid dehydrogenase complex in heart, liver, and kidney of normal, fasted, diabetic, and protein-starved rats. Biochem Biophys Res Commun. 1983 Feb 28;111(1):74–81. doi: 10.1016/s0006-291x(83)80119-3. [DOI] [PubMed] [Google Scholar]
  10. Heffelfinger S. C., Sewell E. T., Danner D. J. Antibodies to bovine liver branched-chain 2-oxo acid dehydrogenase cross-react with this enzyme complex from other tissues and species. Biochem J. 1983 Aug 1;213(2):339–344. doi: 10.1042/bj2130339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lau K. S., Fatania H. R., Randle P. J. Inactivation of rat liver and kidney branched chain 2-oxoacid dehydrogenase complex by adenosine triphosphate. FEBS Lett. 1981 Apr 6;126(1):66–70. doi: 10.1016/0014-5793(81)81034-4. [DOI] [PubMed] [Google Scholar]
  12. Odessey R. Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity. Biochem J. 1982 Apr 15;204(1):353–356. doi: 10.1042/bj2040353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Parker P. J., Randle P. J. Inactivation of rat heart branched-chain 2-oxoacid dehydrogenase complex by adenosine triphosphate. FEBS Lett. 1978 Nov 1;95(1):153–156. doi: 10.1016/0014-5793(78)80072-6. [DOI] [PubMed] [Google Scholar]
  14. Patston P. A., Espinal J., Randle P. J. Effects of diet and of alloxan-diabetes on the activity of branched-chain 2-oxo acid dehydrogenase complex and of activator protein in rat tissues. Biochem J. 1984 Sep 15;222(3):711–719. doi: 10.1042/bj2220711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pettit F. H., Yeaman S. J., Reed L. J. Purification and characterization of branched chain alpha-keto acid dehydrogenase complex of bovine kidney. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4881–4885. doi: 10.1073/pnas.75.10.4881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Randle P. J., England P. J., Denton R. M. Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart. Biochem J. 1970 May;117(4):677–695. doi: 10.1042/bj1170677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wagenmakers A. J., Schepens J. T., Veerkamp J. H. Effect of starvation and exercise on actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues. Biochem J. 1984 Nov 1;223(3):815–821. doi: 10.1042/bj2230815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wagenmakers A. J., Schepens J. T., Veldhuizen J. A., Veerkamp J. H. The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues. Biochem J. 1984 May 15;220(1):273–281. doi: 10.1042/bj2200273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yeaman S. J., Cook K. G., Boyd R. W., Lawson R. Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex. FEBS Lett. 1984 Jun 25;172(1):38–42. doi: 10.1016/0014-5793(84)80868-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES