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. 1986 Jun 1;236(2):527–533. doi: 10.1042/bj2360527

Rat liver pyruvate carboxylase. Purification, detection and quantification of apo and holo forms by immuno-blotting and by an enzyme-linked immunosorbent assay.

F Ahmad, P M Ahmad, A Mendez
PMCID: PMC1146872  PMID: 3753465

Abstract

A simple scheme for the purification of pyruvate carboxylase from rat liver mitochondria is described. It is rapid and provides high-purity pyruvate carboxylase with excellent yield and reproducibility. The final enzyme preparations appear to be homogeneous by the following criteria: elution behaviour on molecular-sizing matrix, SDS/polyacrylamide-gel electrophoresis, Ouchterlony double-diffusion analysis and Western blotting. Detection and quantification of nanogram amounts of pyruvate carboxylase (apo and holo forms) in total tissue homogenates by immuno-blotting and by enzyme-linked immunosorbent assay are described. The data provided suggest that under normal physiological conditions (both in vivo and in vitro) essentially all the pyruvate carboxylase molecules are biotinylated.

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Selected References

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

  1. Ahmad F., Ahmad P. M. Studies on rat mammary gland acetyl-CoA carboxylase. Ann N Y Acad Sci. 1985;447:189–201. doi: 10.1111/j.1749-6632.1985.tb18437.x. [DOI] [PubMed] [Google Scholar]
  2. Ahmad P. M., Feltman D. S., Ahmad F. Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding. Biochem J. 1982 Apr 1;203(1):45–50. doi: 10.1042/bj2030045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ahmad P. M., Russell T. R., Ahmad F. Increase in fatty acid synthetase content of 3T3-L cells undergoing spontaneous and chemically induced differentiation to adipocytes. Biochem J. 1979 Aug 15;182(2):509–514. doi: 10.1042/bj1820509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ashman L. K., Keech D. B., Wallace J. C., Nielsen J. Sheep kidney pyruvate carboxylase. Studies on its activation by acetyl coenzyme A and characteristics of its acetyl coenzyme A independent reaction. J Biol Chem. 1972 Sep 25;247(18):5818–5824. [PubMed] [Google Scholar]
  5. Atkin B. M., Utter M. F., Weinberg M. B. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase activity in leukocytes and fibroblasts from a patient with pyruvate carboxylase deficiency. Pediatr Res. 1979 Jan;13(1):38–43. doi: 10.1203/00006450-197901000-00009. [DOI] [PubMed] [Google Scholar]
  6. Attwood P. V., Keech D. B. Pyruvate carboxylase. Curr Top Cell Regul. 1984;23:1–55. doi: 10.1016/b978-0-12-152823-2.50005-2. [DOI] [PubMed] [Google Scholar]
  7. Ballard F. J., Hanson R. W. The citrate cleavage pathway and lipogenesis in rat adipose tissue: replenishment of oxaloacetate. J Lipid Res. 1967 Mar;8(2):73–79. [PubMed] [Google Scholar]
  8. Barden R. E., Taylor B. L., Isoashi F., Frey W. H., Zander G., Lee J. C., Utter M. F. Structural properties of pyruvate carboxylases from chicken liver and other sources. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4308–4312. doi: 10.1073/pnas.72.11.4308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cohen N. D., Beegen H., Utter M. F., Wrigley N. G. A re-examination of the electron microscopic appearance of pyruvate carboxylase from chicken liver. J Biol Chem. 1979 Mar 10;254(5):1740–1747. [PubMed] [Google Scholar]
  10. Freytag S. O., Utter M. F. Induction of pyruvate carboxylase apoenzyme and holoenzyme in 3T3-L1 cells during differentiation. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1321–1325. doi: 10.1073/pnas.77.3.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gershoni J. M., Palade G. E. Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal Biochem. 1982 Aug;124(2):396–405. doi: 10.1016/0003-2697(82)90056-2. [DOI] [PubMed] [Google Scholar]
  12. Goss N. H., Dyer P. Y., Keech D. B., Wallace J. C. An electron microscopic study of pyruvate carboxylase. J Biol Chem. 1979 Mar 10;254(5):1734–1739. [PubMed] [Google Scholar]
  13. Gravel R. A., Robinson B. H. Biotin-dependent carboxylase deficiencies (propionyl-CoA and pyruvate carboxylases). Ann N Y Acad Sci. 1985;447:225–234. doi: 10.1111/j.1749-6632.1985.tb18441.x. [DOI] [PubMed] [Google Scholar]
  14. Green N. M., Toms E. J. The properties of subunits of avidin coupled to sepharose. Biochem J. 1973 Aug;133(4):687–700. doi: 10.1042/bj1330687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henrikson K. P., Allen S. H., Maloy W. L. An avidin monomer affinity column for the purification of biotin-containing enzymes. Anal Biochem. 1979 Apr 15;94(2):366–370. doi: 10.1016/0003-2697(79)90374-9. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Lau E. P., Cochran B. C., Munson L., Fall R. R. Bovine kidney 3-methylcrotonyl-CoA and propionyl-CoA carboxylases: each enzyme contains nonidentical subunits. Proc Natl Acad Sci U S A. 1979 Jan;76(1):214–218. doi: 10.1073/pnas.76.1.214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu T. Y., Chang Y. H. Hydrolysis of proteins with p-toluenesulfonic acid. Determination of tryptophan. J Biol Chem. 1971 May 10;246(9):2842–2848. [PubMed] [Google Scholar]
  20. Mackall J. C., Lane M. D. Role of pyruvate carboxylase in fatty acid synthesis: alterations during preadipocyte differentiation. Biochem Biophys Res Commun. 1977 Dec 7;79(3):720–725. doi: 10.1016/0006-291x(77)91171-8. [DOI] [PubMed] [Google Scholar]
  21. McClure W. R., Lardy H. A., Kneifel H. P. Rat liver pyruvate carboxylase. I. Preparation, properties, and cation specificity. J Biol Chem. 1971 Jun 10;246(11):3569–3578. [PubMed] [Google Scholar]
  22. Nyhan W. L. Clinical problems relating to biotin. Ann N Y Acad Sci. 1985;447:222–224. doi: 10.1111/j.1749-6632.1985.tb18440.x. [DOI] [PubMed] [Google Scholar]
  23. Rubin C. S., Hirsch A., Fung C., Rosen O. M. Development of hormone receptors and hormonal responsiveness in vitro. Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells. J Biol Chem. 1978 Oct 25;253(20):7570–7578. [PubMed] [Google Scholar]
  24. Russell T. R., Ho R. Conversion of 3T3 fibroblasts into adipose cells: triggering of differentiation by prostaglandin F2alpha and 1-methyl-3-isobutyl xanthine. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4516–4520. doi: 10.1073/pnas.73.12.4516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. SCRUTTON M. C., UTTER M. F. PYRUVATE CARBOXYLASE. 3. SOME PHYSICAL AND CHEMICAL PROPERTIES OF THE HIGHLY PURIFIED ENZYME. J Biol Chem. 1965 Jan;240:1–9. [PubMed] [Google Scholar]
  26. Scrutton M. C. Pyruvate carboxylase from chicken liver: effects of sulfate and other anions on catalytic activity and structural parameters. Arch Biochem Biophys. 1972 Jun;150(2):636–647. doi: 10.1016/0003-9861(72)90084-7. [DOI] [PubMed] [Google Scholar]
  27. Seufert D., Herlemann E. M., Albrecht E., Seubert W. On the mechanism of gluconeogenesis and its regulation. VII. Purification and properties of pyruvate carboxylase from rat liver. Hoppe Seylers Z Physiol Chem. 1971 Mar;352(3):459–478. doi: 10.1515/bchm2.1971.352.1.459. [DOI] [PubMed] [Google Scholar]
  28. Sweetman L., Burri B. J., Nyhan W. L. Biotin holocarboxylase synthetase deficiency. Ann N Y Acad Sci. 1985;447:288–296. doi: 10.1111/j.1749-6632.1985.tb18446.x. [DOI] [PubMed] [Google Scholar]
  29. Towbin H., Gordon J. Immunoblotting and dot immunobinding--current status and outlook. J Immunol Methods. 1984 Sep 4;72(2):313–340. doi: 10.1016/0022-1759(84)90001-2. [DOI] [PubMed] [Google Scholar]
  30. Utter M. F., Barden R. E., Taylor B. L. Pyruvate carboxylase: an evaluation of the relationships between structure and mechanism and between structure and catalytic activity. Adv Enzymol Relat Areas Mol Biol. 1975;42:1–72. doi: 10.1002/9780470122877.ch1. [DOI] [PubMed] [Google Scholar]
  31. Weinberg M. B., Utter M. F. Effect of streptozotocin-induced diabetes mellitus on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase. Biochem J. 1980 Jun 15;188(3):601–608. doi: 10.1042/bj1880601. [DOI] [PMC free article] [PubMed] [Google Scholar]

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