Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1981 May;67(5):1463–1471. doi: 10.1172/JCI110176

Pyruvate Dehydrogenase Complex Activity in Normal and Deficient Fibroblasts

Kwan-Fu Rex Sheu 1, Chii-Whei C Hu 1, Merton F Utter 1
PMCID: PMC370714  PMID: 6262377

Abstract

Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37°C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-14C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of 14CO2 production on the presence of thiamin-PP, coenzyme A (CoA), Mg++, and NAD+. Also, it has been shown that acetyl-CoA and 14CO2 are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg++ and Ca++, or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity.

Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency, and that the present assay is sufficient to detect the heterozygous carriers of the deficiency. Application of the same procedures to fibroblasts obtained from 16 individuals who were believed to have normal PDC activities showed a range from about 2-2.5 nmol/min per mg protein for adults to 5-6 nmol/min per mg protein for cells from infants.

Full text

PDF
1463

Selected References

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

  1. Blass J. P., Avigan J., Uhlendorf B. W. A defect in pyruvate decarboxylase in a child with an intermittent movement disorder. J Clin Invest. 1970 Mar;49(3):423–432. doi: 10.1172/JCI106251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blass J. P., Cederbaum S. D., Kark R. A., Rodriguez-Budelli M. Pyruvate dehydrogenase deficiency in 35 patients. Monogr Hum Genet. 1978;9:12–15. doi: 10.1159/000401603. [DOI] [PubMed] [Google Scholar]
  3. Blass J. P. Disorders of pyruvate metabolism. Neurology. 1979 Mar;29(3):280–286. doi: 10.1212/wnl.29.3.280. [DOI] [PubMed] [Google Scholar]
  4. Blass J. P., Kark A. P., Engel W. K. Clinical studies of a patient with pyruvate decarboxylase deficiency. Arch Neurol. 1971 Nov;25(5):449–460. doi: 10.1001/archneur.1971.00490050083007. [DOI] [PubMed] [Google Scholar]
  5. Blass J. P., Kark R. A., Menon N. K. Low activities of the pyruvate and oxoglutarate dehydrogenase complexes in five patients with Friedreich's ataxia. N Engl J Med. 1976 Jul 8;295(2):62–67. doi: 10.1056/NEJM197607082950202. [DOI] [PubMed] [Google Scholar]
  6. Blass J. P., Lonsdale D., Uhlendorf B. W., Hom E. Intermittent ataxia with pyruvate-decarboxylase deficiency. Lancet. 1971 Jun 19;1(7712):1302–1302. doi: 10.1016/s0140-6736(71)91822-8. [DOI] [PubMed] [Google Scholar]
  7. Bremer J. Pyruvate dehydrogenase, substrate specificity and product inhibition. Eur J Biochem. 1969 Apr;8(4):535–540. doi: 10.1111/j.1432-1033.1969.tb00559.x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Cederbaum S. D., Blass J. P., Minkoff N., Brown W. J., Cotton M. E., Harris S. H. Sensitivity to carbohydrate in a patient with familial intermittent lactic acidosis and pyruvate dehydrogenase deficiency. Pediatr Res. 1976 Aug;10(8):713–720. doi: 10.1203/00006450-197608000-00002. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. DeVivo D. C., Haymond M. W., Obert K. A., Nelson J. S., Pagliara A. S. Defective activation of the pyruvate dehydrogenase complex in subacute necrotizing encephalomyelopathy (Leigh disease). Ann Neurol. 1979 Dec;6(6):483–494. doi: 10.1002/ana.410060605. [DOI] [PubMed] [Google Scholar]
  12. Dennis S. C., DeBuysere M., Scholz R., Olson M. S. Studies on the relationship between ketogenesis and pyruvate oxidation in isolated rat liver mitochondria. J Biol Chem. 1978 Apr 10;253(7):2229–2237. [PubMed] [Google Scholar]
  13. Denton R. M., Randle P. J., Bridges B. J., Cooper R. H., Kerbey A. L., Pask H. T., Severson D. L., Stansbie D., Whitehouse S. Regulation of mammalian pyruvate dehydrogenase. Mol Cell Biochem. 1975 Oct 31;9(1):27–53. doi: 10.1007/BF01731731. [DOI] [PubMed] [Google Scholar]
  14. Falk R. E., Cederbaum S. D., Blass J. P., Gibson G. E., Kark R. A., Carrel R. E. Ketonic diet in the management of pyruvate dehydrogenase deficiency. Pediatrics. 1976 Nov;58(5):713–721. [PubMed] [Google Scholar]
  15. Farrell D. F., Clark A. F., Scott C. R., Wennberg R. P. Absence of pyruvate decarboxylase activity in man: a cause of congenital lactic acidosis. Science. 1975 Mar 21;187(4181):1082–1084. doi: 10.1126/science.803713. [DOI] [PubMed] [Google Scholar]
  16. Haworth J. C., Perry T. L., Blass J. P., Hansen S., Urquhart N. Lactic acidosis in three sibs due to defects in both pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. Pediatrics. 1976 Oct;58(4):564–572. [PubMed] [Google Scholar]
  17. 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]
  18. Hutson N. J., Kerbey A. L., Randle P. J., Sugden P. H. Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat. Biochem J. 1978 Aug 1;173(2):669–680. doi: 10.1042/bj1730669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kark R. A., Rodriguez-Budelli M. Pyruvate dehydrogenase deficiency in spinocerebellar degenerations. Neurology. 1979 Jan;29(1):126–131. doi: 10.1212/wnl.29.1.126. [DOI] [PubMed] [Google Scholar]
  20. Koster J. F., Slee R. G., Fernandes J. Lactic acidosis due to a defect in the pyruvate dehydrogenase complex: a possible brain pyruvate dehydrogenase phosphatase deficiency. Monogr Hum Genet. 1978;9:7–11. doi: 10.1159/000401601. [DOI] [PubMed] [Google Scholar]
  21. Kuroda Y., Kline J. J., Sweetman L., Nyhan W. L., Groshong T. D. Abnormal pyruvate and alpha-ketoglutarate dehydrogenase complexes in a patient with lactic acidemia. Pediatr Res. 1979 Aug;13(8):928–931. doi: 10.1203/00006450-197908000-00011. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Leiter A. B., Weinberg M., Isohashi F., Utter M. F. Relationshiop between phosphorylation and activity of pyruvate dehydrogenase in rat liver mitochondria and the absence of such a relationship for pyruvate carboxylase. J Biol Chem. 1978 Apr 25;253(8):2716–2723. [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Linn T. C., Pettit F. H., Reed L. J. Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc Natl Acad Sci U S A. 1969 Jan;62(1):234–241. doi: 10.1073/pnas.62.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lynen A., Sedlaczek E., Wieland O. H. Partial purification and characterization of a pyruvate dehydrogenase-complex-inactivating enzyme from rat liver. Biochem J. 1978 Feb 1;169(2):321–328. doi: 10.1042/bj1690321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Löffler G., Bard S., Wieland O. H. Control of pyruvate dehydrogenase interconversion by palmitoyl-coenzyme A as related to adenine nucleotide translocation in isolated fat cell mitochondria. FEBS Lett. 1975 Dec 15;60(2):269–274. doi: 10.1016/0014-5793(75)80729-0. [DOI] [PubMed] [Google Scholar]
  29. Ohlen J., Siess E. A., Löffler G., Wieland O. H. The effect of insulin on pyruvate dehydrogenase interconversion in heart muscle of alloxan-diabetic rats. Diabetologia. 1978 Feb;14(2):135–139. doi: 10.1007/BF01263452. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Pettit F. H., Roche T. E., Reed L. J. Function of calcium ions in pyruvate dehydrogenase phosphatase activity. Biochem Biophys Res Commun. 1972 Oct 17;49(2):563–571. doi: 10.1016/0006-291x(72)90448-2. [DOI] [PubMed] [Google Scholar]
  32. Portenhauser R., Wieland O. H., Wenzel H. Regulation of pyruvate dehydrogenase in heart mitochondria. Hoppe Seylers Z Physiol Chem. 1977 Jun;358(6):647–658. doi: 10.1515/bchm2.1977.358.1.647. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Reynolds S. F., Blass J. A possible mechanism for selective cerebellar damage in partial pyruvate dehydrogenase deficiency. Neurology. 1976 Jul;26(7):625–628. doi: 10.1212/wnl.26.7.625. [DOI] [PubMed] [Google Scholar]
  35. Robinson B. H., Sherwood W. G. Pyruvate dehydrogenase phosphatase deficiency: a cause of congenital chronic lactic acidosis in infancy. Pediatr Res. 1975 Dec;9(12):935–939. doi: 10.1203/00006450-197512000-00015. [DOI] [PubMed] [Google Scholar]
  36. Robinson B. H., Taylor J., Sherwood W. G. Deficiency of dihydrolipoyl dehydrogenase (a component of the pyruvate and alpha-ketoglutarate dehydrogenase complexes): a cause of congenital chronic lactic acidosis in infancy. Pediatr Res. 1977 Dec;11(12):1198–1202. doi: 10.1203/00006450-197712000-00006. [DOI] [PubMed] [Google Scholar]
  37. Roche T. E., Cate R. L. Evidence for lipoic acid mediated NADH and acetyl-CoA stimulation of liver and kidney pyruvate dehydrogenase kinase. Biochem Biophys Res Commun. 1976 Oct 18;72(4):1375–1383. doi: 10.1016/s0006-291x(76)80166-0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Rodriguez-Budelli M., Kark P. Kinetic evidence for a structural abnormality of lipoamide dehydrogenase in two patients with Friedreich ataxia. Neurology. 1978 Dec;28(12):1283–1286. doi: 10.1212/wnl.28.12.1283. [DOI] [PubMed] [Google Scholar]
  41. Severson D. L., Denton R. M., Pask H. T., Randle P. J. Calcium and magnesium ions as effectors of adipose-tissue pyruvate dehydrogenase phosphate phosphatase. Biochem J. 1974 May;140(2):225–237. doi: 10.1042/bj1400225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Siess E. A., Wieland O. H. Purification and characterization of pyruvate-dehydrogenase phosphatase from pig-heart muscle. Eur J Biochem. 1972 Mar 15;26(1):96–105. doi: 10.1111/j.1432-1033.1972.tb01744.x. [DOI] [PubMed] [Google Scholar]
  44. Stansbie D., Denton R. M., Bridges B. J., Pask H. T., Randle P. J. Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet. Biochem J. 1976 Jan 15;154(1):225–236. doi: 10.1042/bj1540225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Strömme J. H., Borud O., Moe P. J. Fatal lactic acidosis in a newborn attributable to a congenital defect of pyruvate dehydrogenase. Pediatr Res. 1976 Jan;10(1):62–66. doi: 10.1203/00006450-197601000-00012. [DOI] [PubMed] [Google Scholar]
  46. Stumpf D. A., Parks J. K. Friedreich ataxia. II. Normal kinetics of lipoamide dehydrogenase. Neurology. 1979 Jun;29(6):820–826. doi: 10.1212/wnl.29.6.820. [DOI] [PubMed] [Google Scholar]
  47. Tsai C. S., Burgett M. W., Reed L. J. Alpha-keto acid dehydrogenase complexes. XX. A kinetic study of the pyruvate dehydrogenase complex from bovine kidney. J Biol Chem. 1973 Dec 25;248(24):8348–8352. [PubMed] [Google Scholar]
  48. Utter M. F., Sheu K. F. Biochemical mechanisms of biotin and thiamin action and relationships to genetic disease. Birth Defects Orig Artic Ser. 1980;16(1):289–304. [PubMed] [Google Scholar]
  49. Whitehouse S., Cooper R. H., Randle P. J. Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids. Biochem J. 1974 Sep;141(3):761–774. doi: 10.1042/bj1410761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Whitehouse S., Randle P. J. Activation of pyruvate dehydrogenase in perfused rat heart by dichloroacetate (Short Communication). Biochem J. 1973 Jun;134(2):651–653. doi: 10.1042/bj1340651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wick H., Schweizer K., Baumgartner R. Thiamine dependency in a patient with congenital lacticacidaemia due to pyruvate dehydrogenase deficiency. Agents Actions. 1977 Sep;7(3):405–410. doi: 10.1007/BF01969575. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES