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. 1974 Jun;140(3):531–538. doi: 10.1042/bj1400531

Degradation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in vivo and in vitro*

F J Ballard 1,2,3, M F Hopgood 1,2,3, Lea Reshef 1,2,3, R W Hanson 1,2,3
PMCID: PMC1168032  PMID: 4447628

Abstract

1. Phosphoenolpyruvate carboxykinase (GTP) in the cytosol fraction of liver was labelled in young rats by the injection of [3H]leucine and then isolated with specific antibody. Antibody-antigen precipitates from `pulse'-labelled animals and from animals in which the content of radioactive enzyme had been decreased by a period of degradation were separated by electrophoresis on sodium dodecyl sulphate–polyacrylamide gels. No radioactive breakdown products were found. 2. 3H-labelled phosphoenolpyruvate carboxykinase (GTP) was purified from rat liver and used to measure degradation in vitro. There was first a loss of catalytic activity, then a disappearance of immunological activity and finally a loss of solubility before any evidence of proteolytic cleavage. Proteolytic-cleavage fragments, when found, were also insoluble. 3. An analysis of the subcellular location of enzyme inactivation showed that phosphoenolpyruvate carboxykinase (GTP) was stable when incubated with liver cytosol fraction and was inactivated most rapidly by the microsomal fraction. 4. We propose that denaturation of the enzyme is the rate-limiting step in degradation in vivo, and precedes proteolytic cleavage when the enzyme is incubated with liver preparations in vitro.

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Selected References

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

  1. Auricchio F., Mollica L., Liguori A. Inactivation of tyrosine aminotransferase in neutral homogenates and rat liver slices. Biochem J. 1972 Oct;129(5):1131–1138. doi: 10.1042/bj1291131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballard F. J., Hanson R. W. Purification of phosphoenolpyruvate carboxykinase from the cytosol fraction of rat liver and the immunochemical demonstration of differences between this enzyme and the mitochondrial phosphoenolpyruvate carboxykinase. J Biol Chem. 1969 Oct 25;244(20):5625–5630. [PubMed] [Google Scholar]
  3. Ballard F. J., Hopgood M. F. Phosphopyruvate carboxylase induction by L-tryptophan. Effects on synthesis and degradation of the enzyme. Biochem J. 1973 Oct;136(2):259–264. doi: 10.1042/bj1360259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohley P., Kirschke H., Langner J., Ansorge S., Hanson H. Intrazellulärer Proteinabbau. 3. Intrazelluläre Verteilung des Zytosolproteinabbaues bei neutralem p. Acta Biol Med Ger. 1971;27(2):229–243. [PubMed] [Google Scholar]
  5. Bowers W. E., Finkenstaedt J. T., de Duve C. Lysosomes in lymphoid tissue. I. The measurement of hydrolytic activities in whole homogenates. J Cell Biol. 1967 Feb;32(2):325–337. doi: 10.1083/jcb.32.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang H. C., Lane M. D. The enzymatic carboxylation of phosphoenolpyruvate. II. Purification and properties of liver mitochondrial phosphoenolpyruvate carboxykinase. J Biol Chem. 1966 May 25;241(10):2413–2420. [PubMed] [Google Scholar]
  7. Davies M., Lloyd J. B., Beck F. The effect of Trypan Blue, suramin and aurothiomalate on the breakdown of 125 I-labelled albumin within rat liver lysosomes. Biochem J. 1971 Jan;121(1):21–26. doi: 10.1042/bj1210021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dice J. F., Dehlinger P. J., Schimke R. T. Studies on the correlation between size and relative degradation rate of soluble proteins. J Biol Chem. 1973 Jun 25;248(12):4220–4228. [PubMed] [Google Scholar]
  9. Dice J. F., Schimke R. T. Turnover and exchange of ribosomal proteins from rat liver. J Biol Chem. 1972 Jan 10;247(1):98–111. [PubMed] [Google Scholar]
  10. Haider M., Segal H. L. Some characteristics of the alanine aminotransferase- and arginase-inactivating system of lysosomes. Arch Biochem Biophys. 1972 Jan;148(1):228–237. doi: 10.1016/0003-9861(72)90136-1. [DOI] [PubMed] [Google Scholar]
  11. Hopgood M. F., Ballard F. J. Synthesis and degradation of phosphoenolpyruvate carboxylase in rat liver and adipose tissue. Changes during a starvation-re-feeding cycle. Biochem J. 1973 Jun;134(2):445–453. doi: 10.1042/bj1340445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kominami E., Kobayashi K., Kominami S., Katunuma N. Properties of a specific protease for pyridoxal enzymes and its biological role. J Biol Chem. 1972 Nov 10;247(21):6848–6855. [PubMed] [Google Scholar]
  13. Li J. B., Knox W. E. Inactivation of tryptophan oxygenase in vivo and in vitro. J Biol Chem. 1972 Dec 10;247(23):7550–7555. doi: 10.2172/4660691. [DOI] [PubMed] [Google Scholar]
  14. Mego J. L. The effect of pH on cathepsin activities in mouse liver heterolysosomes. Biochem J. 1971 May;122(4):445–452. doi: 10.1042/bj1220445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Philippidis H., Hanson R. W., Reshef L., Hopgood M. F., Ballard F. J. The initial synthesis of proteins during development. Phosphoenolpyruvate carboxylase in rat liver at birth. Biochem J. 1972 Mar;126(5):1127–1134. doi: 10.1042/bj1261127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. SAWANT P. L., SHIBKO, KUMTA U. S., TAPPEL A. L. ISOLATION OF RAT-LIVER LYSOSOMES AND THEIR GENERAL PROPERTIES. Biochim Biophys Acta. 1964 Apr 6;85:82–92. doi: 10.1016/0926-6569(64)90169-5. [DOI] [PubMed] [Google Scholar]
  17. Schimke R. T., Doyle D. Control of enzyme levels in animal tissues. Annu Rev Biochem. 1970;39:929–976. doi: 10.1146/annurev.bi.39.070170.004433. [DOI] [PubMed] [Google Scholar]
  18. Ward S., Wilson D. L., Gilliam J. J. Methods for fractionation and scintillation counting of radioisotope-labeled polyacrylamide gels. Anal Biochem. 1970 Nov;38(1):90–97. doi: 10.1016/0003-2697(70)90158-2. [DOI] [PubMed] [Google Scholar]
  19. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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