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. 1992 Dec 1;288(Pt 2):369–373. doi: 10.1042/bj2880369

Regulation of pyruvate dehydrogenase kinase activity from pig kidney cortex.

T Pawelczyk 1, M S Olson 1
PMCID: PMC1132021  PMID: 1463442

Abstract

The activity of pyruvate dehydrogenase (PDH) kinase in the purified PDH complex from pig kidney is sensitive to changes in ionic strength. The enzyme has optimum activity within a small range of ionic strength (0.03-0.05 M). An increase in ionic strength from 0.04 M to 0.2 M lowers the activity of PDH kinase by 32% and decreases the Km for ATP from 25 microM to 10 microM. At constant ionic strength (0.15 M) the enzyme has optimum activity over a broad pH range (7.2-8.0). The PDH kinase is stimulated 2.2-fold by 20 mM-K+, whereas Na+ even at high concentration (80 mM) has no effect on the enzyme activity. The stimulation of PDH kinase by K+ is not dependent on pH and ionic strength. PDH kinase is inhibited by HPO4(2-) in the presence of K+, whereas HPO4(2-) has no effect on the activity of this enzyme in the absence of K+. HPO4(2-) at concentrations of 2 and 10 mM inhibits PDH kinase by 28% and 55% respectively. The magnitude of this inhibition is not dependent on the ATP/ADP ratio. Inhibition by HPO4(2-) in the concentration range 0-10 mM is non-competitive with respect to ATP, and becomes mixed-type at concentrations over 10 mM. The Ki for HPO4(2-) is 10 mM. When HPO4(2-) is replaced by SO4(2-), the same effects on the activity of PDH kinase are observed. PDH kinase is also inhibited by Cl-. In the presence of 80 mM-Cl- the PDH kinase is inhibited by 40%. The inhibition by Cl- is not dependent on K+. In conclusion, we postulate that changes in phosphate concentrations may play a significant role in the regulation of PDH kinase activity in vivo.

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Selected References

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  1. Adler S., Shoubridge E., Radda G. K. Estimation of cellular pH gradients with 31P-NMR in intact rabbit renal tubular cells. Am J Physiol. 1984 Sep;247(3 Pt 1):C188–C196. doi: 10.1152/ajpcell.1984.247.3.C188. [DOI] [PubMed] [Google Scholar]
  2. Akerboom T. P., Bookelman H., Zuurendonk P. F., van der Meer R., Tager J. M. Intramitochondrial and extramitochondrial concentrations of adenine nucleotides and inorganic phosphate in isolated hepatocytes from fasted rats. Eur J Biochem. 1978 Mar 15;84(2):413–420. doi: 10.1111/j.1432-1033.1978.tb12182.x. [DOI] [PubMed] [Google Scholar]
  3. Assimacopoulos-Jeannet F., McCormack J. G., Jeanrenaud B. Effect of phenylephrine on pyruvate dehydrogenase activity in rat hepatocytes and its interaction with insulin and glucagon. FEBS Lett. 1983 Aug 8;159(1-2):83–88. doi: 10.1016/0014-5793(83)80421-9. [DOI] [PubMed] [Google Scholar]
  4. Atkinson P. G., Butterworth P. J. Control of phosphate transport in liver. Biochem Soc Trans. 1990 Aug;18(4):625–625. doi: 10.1042/bst0180625. [DOI] [PubMed] [Google Scholar]
  5. Balaban R. S. Regulation of oxidative phosphorylation in the mammalian cell. Am J Physiol. 1990 Mar;258(3 Pt 1):C377–C389. doi: 10.1152/ajpcell.1990.258.3.C377. [DOI] [PubMed] [Google Scholar]
  6. Beck F., Bauer R., Bauer U., Mason J., Dörge A., Rick R., Thurau K. Electron microprobe analysis of intracellular elements in the rat kidney. Kidney Int. 1980 Jun;17(6):756–763. doi: 10.1038/ki.1980.88. [DOI] [PubMed] [Google Scholar]
  7. Berthon B., Binet A., Mauger J. P., Claret M. Cytosolic free Ca2+ in isolated rat hepatocytes as measured by quin2. Effects of noradrenaline and vasopressin. FEBS Lett. 1984 Feb 13;167(1):19–24. doi: 10.1016/0014-5793(84)80824-8. [DOI] [PubMed] [Google Scholar]
  8. Bünger R., Soboll S. Cytosolic adenylates and adenosine release in perfused working heart. Comparison of whole tissue with cytosolic non-aqueous fractionation analyses. Eur J Biochem. 1986 Aug 15;159(1):203–213. doi: 10.1111/j.1432-1033.1986.tb09854.x. [DOI] [PubMed] [Google Scholar]
  9. Cate R. L., Roche T. E. A unifying mechanism for stimulation of mammalian pyruvate dehydrogenase(a) kinase by reduced nicotinamide adenine dinucleotide, dihydrolipoamide, acetyl coenzyme A, or pyruvate. J Biol Chem. 1978 Jan 25;253(2):496–503. [PubMed] [Google Scholar]
  10. Chiang P. K., Sacktor B. Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase. J Biol Chem. 1975 May 10;250(9):3399–3408. [PubMed] [Google Scholar]
  11. Cooper R. H., Randle P. J., Denton R. M. Regulation of heart muscle pyruvate dehydrogenase kinase. Biochem J. 1974 Dec;143(3):625–641. doi: 10.1042/bj1430625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coty W. A., Pedersen P. L. Phosphate transport in rat liver mitochondria. Kinetics and energy requirements. J Biol Chem. 1974 Apr 25;249(8):2593–2598. [PubMed] [Google Scholar]
  13. Dennis S. C., Padmas A., DeBuysere M. S., Olson M. S. Studies on the regulation of pyruvate dehydrogenase in the isolated perfused rat heart. J Biol Chem. 1979 Feb 25;254(4):1252–1258. [PubMed] [Google Scholar]
  14. Denton R. M., Coore H. G., Martin B. R., Randle P. J. Insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue. Nat New Biol. 1971 May 26;231(21):115–116. doi: 10.1038/newbio231115a0. [DOI] [PubMed] [Google Scholar]
  15. Denton R. M., McCormack J. G., Marshall S. E. Persistence of the effect of insulin on pyruvate dehydrogenase activity in rat white and brown adipose tissue during the preparation and subsequent incubation of mitochondria. Biochem J. 1984 Jan 15;217(2):441–452. doi: 10.1042/bj2170441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Denton R. M., Midgley P. J., Rutter G. A., Thomas A. P., McCormack J. G. Studies into the mechanism whereby insulin activates pyruvate dehydrogenase complex in adipose tissue. Ann N Y Acad Sci. 1989;573:285–296. doi: 10.1111/j.1749-6632.1989.tb15005.x. [DOI] [PubMed] [Google Scholar]
  17. Hansford R. G. Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria. J Biol Chem. 1976 Sep 25;251(18):5483–5489. [PubMed] [Google Scholar]
  18. Hems D. A., McCormack J. G., Denton R. M. Activation of pyruvate dehydrogenase in the perfused rat liver by vasopressin. Biochem J. 1978 Nov 15;176(2):627–629. doi: 10.1042/bj1760627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hennig G., Löffler G., Wieland O. H. Active and inactive forms of pyruvatedehydrogenase in skeletal muscle as related to the metabolic and functional state of the muscle cell. FEBS Lett. 1975 Nov 15;59(2):142–145. doi: 10.1016/0014-5793(75)80361-9. [DOI] [PubMed] [Google Scholar]
  20. Hucho F., Randall D. D., Roche T. E., Burgett M. W., Pelley J. W., Reed L. J. -Keto acid dehydrogenase complexes. XVII. Kinetic and regulatory properties of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase from bovine kidney and heart. Arch Biochem Biophys. 1972 Jul;151(1):328–340. doi: 10.1016/0003-9861(72)90504-8. [DOI] [PubMed] [Google Scholar]
  21. Humphrey S. M., Buckman J. E., Holliss D. G. Subcellular distribution of energy metabolites in the pre-ischaemic and post-ischaemic perfused working rat heart. Eur J Biochem. 1990 Aug 17;191(3):755–759. doi: 10.1111/j.1432-1033.1990.tb19184.x. [DOI] [PubMed] [Google Scholar]
  22. Hutson N. J., Randle P. J. Enhanced activity of pyruvate dehydrogenase kinase in rat heart mitochondria in alloxan-diabetes or starvation. FEBS Lett. 1978 Aug 1;92(1):73–76. doi: 10.1016/0014-5793(78)80724-8. [DOI] [PubMed] [Google Scholar]
  23. Iles R. A., Stevens A. N., Griffiths J. R., Morris P. G. Phosphorylation status of liver by 31P-n.m.r. spectroscopy, and its implications for metabolic control. A comparison of 31P-n.m.r. spectroscopy (in vivo and in vitro) with chemical and enzymic determinations of ATP, ADP and Pi. Biochem J. 1985 Jul 1;229(1):141–151. doi: 10.1042/bj2290141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kashiwagura T., Deutsch C. J., Taylor J., Erecińska M., Wilson D. F. Dependence of gluconeogenesis, urea synthesis, and energy metabolism of hepatocytes on intracellular pH. J Biol Chem. 1984 Jan 10;259(1):237–243. [PubMed] [Google Scholar]
  25. Katz L. A., Swain J. A., Portman M. A., Balaban R. S. Intracellular pH and inorganic phosphate content of heart in vivo: a 31P-NMR study. Am J Physiol. 1988 Jul;255(1 Pt 2):H189–H196. doi: 10.1152/ajpheart.1988.255.1.H189. [DOI] [PubMed] [Google Scholar]
  26. Kerbey A. L., Radcliffe P. M., Randle P. J., Sugden P. H. Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex. Biochem J. 1979 Aug 1;181(2):427–433. doi: 10.1042/bj1810427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Larner J. Insulin-signaling mechanisms. Lessons from the old testament of glycogen metabolism and the new testament of molecular biology. Diabetes. 1988 Mar;37(3):262–275. doi: 10.2337/diab.37.3.262. [DOI] [PubMed] [Google Scholar]
  28. Linn T. C., Pettit F. H., Hucho F., Reed L. J. Alpha-keto acid dehydrogenase complexes. XI. Comparative studies of regulatory properties of the pyruvate dehydrogenase complexes from kidney, heart, and liver mitochondria. Proc Natl Acad Sci U S A. 1969 Sep;64(1):227–234. doi: 10.1073/pnas.64.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mason J., Beck F., Dörge A., Rick R., Thurau K. Intracellular electrolyte composition following renal ischemia. Kidney Int. 1981 Jul;20(1):61–70. doi: 10.1038/ki.1981.105. [DOI] [PubMed] [Google Scholar]
  30. McCormack J. G. Studies on the activation of rat liver pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase by adrenaline and glucagon. Role of increases in intramitochondrial Ca2+ concentration. Biochem J. 1985 Nov 1;231(3):597–608. doi: 10.1042/bj2310597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pawelczyk T., Easom R. A., Olson M. S. Effect of ionic strength and pH on the activity of pyruvate dehydrogenase complex from pig kidney cortex. Arch Biochem Biophys. 1992 Apr;294(1):44–49. doi: 10.1016/0003-9861(92)90134-i. [DOI] [PubMed] [Google Scholar]
  32. Pawelczyk T., Easom R. A., Olson M. S. The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex. Biochem J. 1988 Aug 1;253(3):819–825. doi: 10.1042/bj2530819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pettit F. H., Pelley J. W., Reed L. J. Regulation of pyruvate dehydrogenase kinase and phosphatase by acetyl-CoA/CoA and NADH/NAD ratios. Biochem Biophys Res Commun. 1975 Jul 22;65(2):575–582. doi: 10.1016/s0006-291x(75)80185-9. [DOI] [PubMed] [Google Scholar]
  34. Pratt M. L., Roche T. E. Mechanism of pyruvate inhibition of kidney pyruvate dehydrogenasea kinase and synergistic inhibition by pyruvate and ADP. J Biol Chem. 1979 Aug 10;254(15):7191–7196. [PubMed] [Google Scholar]
  35. Rahmatullah M., Roche T. E. Modification of bovine kidney pyruvate dehydrogenase kinase activity by CoA esters and their mechanism of action. J Biol Chem. 1985 Aug 25;260(18):10146–10152. [PubMed] [Google Scholar]
  36. Reed L. J., Damuni Z., Merryfield M. L. Regulation of mammalian pyruvate and branched-chain alpha-keto acid dehydrogenase complexes by phosphorylation-dephosphorylation. Curr Top Cell Regul. 1985;27:41–49. doi: 10.1016/b978-0-12-152827-0.50011-6. [DOI] [PubMed] [Google Scholar]
  37. Robertson J. G., Barron L. L., Olson M. S. Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase. J Biol Chem. 1986 Jan 5;261(1):76–81. [PubMed] [Google Scholar]
  38. Robertson J. G., Barron L. L., Olson M. S. Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase. J Biol Chem. 1986 Jan 5;261(1):76–81. [PubMed] [Google Scholar]
  39. Roche T. E., Reed L. J. Function of the nonidentical subunits of mammalian pyruvate dehydrogenase. Biochem Biophys Res Commun. 1972 Aug 21;48(4):840–846. doi: 10.1016/0006-291x(72)90684-5. [DOI] [PubMed] [Google Scholar]
  40. Roche T. E., Reed L. J. Monovalent cation requirement for ADP inhibition of pyruvate dehydrogenase kinase. Biochem Biophys Res Commun. 1974 Aug 19;59(4):1341–1348. doi: 10.1016/0006-291x(74)90461-6. [DOI] [PubMed] [Google Scholar]
  41. Ross B., Freeman D., Chan L. Contributions of nuclear magnetic resonance to renal biochemistry. Kidney Int. 1986 Jan;29(1):131–141. doi: 10.1038/ki.1986.15. [DOI] [PubMed] [Google Scholar]
  42. Siess E. A., Kientsch-Engel R. I., Fahimi F. M., Wieland O. H. Possible role of Pi supply in mitochondrial actions of glucagon. Eur J Biochem. 1984 Jun 15;141(3):543–548. doi: 10.1111/j.1432-1033.1984.tb08227.x. [DOI] [PubMed] [Google Scholar]
  43. Siess E. A., Wieland O. H. Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells. Biochem J. 1976 Apr 15;156(1):91–102. doi: 10.1042/bj1560091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Soboll S., Scholz R., Heldt H. W. Subcellular metabolite concentrations. Dependence of mitochondrial and cytosolic ATP systems on the metabolic state of perfused rat liver. Eur J Biochem. 1978 Jun 15;87(2):377–390. doi: 10.1111/j.1432-1033.1978.tb12387.x. [DOI] [PubMed] [Google Scholar]
  45. Thomas A. P., Alexander J., Williamson J. R. Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2+ induced by vasopressin in isolated hepatocytes. J Biol Chem. 1984 May 10;259(9):5574–5584. [PubMed] [Google Scholar]
  46. Tullson P. C., Goldstein L. Effects of acute acid-base changes on rat renal pyruvate dehydrogenase. Renal pyruvate dehydrogenase during acid-base alterations. Enzyme. 1987;37(3):127–133. doi: 10.1159/000469249. [DOI] [PubMed] [Google Scholar]
  47. Veech R. L., Lawson J. W., Cornell N. W., Krebs H. A. Cytosolic phosphorylation potential. J Biol Chem. 1979 Jul 25;254(14):6538–6547. [PubMed] [Google Scholar]
  48. Wang W., Messner G., Oberleithner H., Lang F., Deetjen P. The effect of ouabain on intracellular activities of K+, Na+, Cl-, H+ and Ca2+ in proximal tubules of frog kidneys. Pflugers Arch. 1984 May;401(1):6–13. doi: 10.1007/BF00581526. [DOI] [PubMed] [Google Scholar]
  49. Wastila W. B., Stull J. T., Mayer S. E., Walsh D. A. Measurement of cyclic 3',5'-denosine monophosphate by the activation of skeletal muscle protein kinase. J Biol Chem. 1971 Apr 10;246(7):1996–2003. [PubMed] [Google Scholar]
  50. Weiss L., Löffler G., Schirmann A., Wieland O. Control of pyruvate dehydrogenase interconversion in adipose tissue by insulin. FEBS Lett. 1971 Jun 24;15(3):229–231. doi: 10.1016/0014-5793(71)80318-6. [DOI] [PubMed] [Google Scholar]
  51. Wieland O. H., Portenhauser R. Regulation of pyruvate-dehydrogenase interconversion in rat-liver mitochondria as related to the phosphorylation state of intramitochondrial adenine nucleotides. Eur J Biochem. 1974 Jun 15;45(2):577–588. doi: 10.1111/j.1432-1033.1974.tb03584.x. [DOI] [PubMed] [Google Scholar]
  52. Wieland O. H. The mammalian pyruvate dehydrogenase complex: structure and regulation. Rev Physiol Biochem Pharmacol. 1983;96:123–170. doi: 10.1007/BFb0031008. [DOI] [PubMed] [Google Scholar]
  53. Wieland O. H., Urumow T., Drexler P. Insulin, phospholipase, and the activation of the pyruvate dehydrogenase complex: an enigma. Ann N Y Acad Sci. 1989;573:274–284. doi: 10.1111/j.1749-6632.1989.tb15004.x. [DOI] [PubMed] [Google Scholar]
  54. Wolff S. D., Eng C., Balaban R. S. NMR studies of renal phosphate metabolites in vivo: effects of hydration and dehydration. Am J Physiol. 1988 Oct;255(4 Pt 2):F581–F589. doi: 10.1152/ajprenal.1988.255.4.F581. [DOI] [PubMed] [Google Scholar]

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