Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Jul;81(14):4335–4338. doi: 10.1073/pnas.81.14.4335

Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney.

Z Damuni, M L Merryfield, J S Humphreys, L J Reed
PMCID: PMC345583  PMID: 6589597

Abstract

Branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of approximately 460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of approximately 230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations. BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 microM, respectively. These inhibitions were reversed completely by 2 mM Mg2+. GTP was replaceable by guanosine 5'-(beta, gamma-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 micrograms/ml. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 microM. These inhibitions were not reversed by 2 mM Mg2+. BCKDH phosphatase activity was stimulated 1.5- to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 micrograms/ml.

Full text

PDF
4335

Selected References

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

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Fatania H. R., Patston P. A., Randle P. J. Dephosphorylation and reactivation of phosphorylated purified ox-kidney branched-chain dehydrogenase complex by co-purified phosphatase. FEBS Lett. 1983 Jul 25;158(2):234–238. doi: 10.1016/0014-5793(83)80585-7. [DOI] [PubMed] [Google Scholar]
  5. Harris R. A., Paxton R., DePaoli-Roach A. A. Inhibition of branched chain alpha-ketoacid dehydrogenase kinase activity by alpha-chloroisocaproate. J Biol Chem. 1982 Dec 10;257(23):13915–13918. [PubMed] [Google Scholar]
  6. Harris R. A., Paxton R., Parker R. A. Activation of the branched-chain alpha-ketoacid dehydrogenase complex by a broad specificity protein phosphatase. Biochem Biophys Res Commun. 1982 Aug 31;107(4):1497–1503. doi: 10.1016/s0006-291x(82)80168-x. [DOI] [PubMed] [Google Scholar]
  7. Hughes W. A., Halestrap A. P. The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation. Biochem J. 1981 May 15;196(2):459–469. doi: 10.1042/bj1960459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lau K. S., Fatania H. R., Randle P. J. Regulation of the branched chain 2-oxoacid dehydrogenase kinase reaction. FEBS Lett. 1982 Jul 19;144(1):57–62. doi: 10.1016/0014-5793(82)80568-1. [DOI] [PubMed] [Google Scholar]
  9. Lawson R., Cook K. G., Yeaman S. J. Rapid purification of bovine kidney branched-chain 2-oxoacid dehydrogenase complex containing endogenous kinase activity. FEBS Lett. 1983 Jun 27;157(1):54–58. doi: 10.1016/0014-5793(83)81115-6. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Odessey R. Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase. Biochem J. 1980 Oct 15;192(1):155–163. doi: 10.1042/bj1920155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Parker P. J., Randle P. J. Active and inactive forms of branched-chain 2-oxoacid dehydrogenase complex in rat heart and skeletal muscle. FEBS Lett. 1980 Apr 7;112(2):186–190. doi: 10.1016/0014-5793(80)80176-1. [DOI] [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. Patel T. B., Olson M. S. Evidence for the regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex by a phosphorylation/dephosphorylation mechanism. Biochemistry. 1982 Aug 31;21(18):4259–4265. doi: 10.1021/bi00261a012. [DOI] [PubMed] [Google Scholar]
  15. Paxton R., Harris R. A. Isolation of rabbit liver branched chain alpha-ketoacid dehydrogenase and regulation by phosphorylation. J Biol Chem. 1982 Dec 10;257(23):14433–14439. [PubMed] [Google Scholar]
  16. Pettit F. H., Reed L. J. Pyruvate dehydrogenase complex from bovine kidney and heart. Methods Enzymol. 1982;89(Pt 500):376–386. doi: 10.1016/s0076-6879(82)89067-8. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Stepp L. R., Pettit F. H., Yeaman S. J., Reed L. J. Purification and properties of pyruvate dehydrogenase kinase from bovine kidney. J Biol Chem. 1983 Aug 10;258(15):9454–9458. [PubMed] [Google Scholar]
  19. Teague W. M., Pettit F. H., Wu T. L., Silberman S. R., Reed L. J. Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney. Biochemistry. 1982 Oct 26;21(22):5585–5592. doi: 10.1021/bi00265a031. [DOI] [PubMed] [Google Scholar]
  20. Watanabe H., Green G. D., Shaw E. A comparison of the behavior of chymotrypsin and cathepsin B towards peptidyl diazomethyl ketones. Biochem Biophys Res Commun. 1979 Aug 28;89(4):1354–1360. doi: 10.1016/0006-291x(79)92158-2. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES