Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Nov;173(21):6844–6848. doi: 10.1128/jb.173.21.6844-6848.1991

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

J C Hoyt 1, K E Johnson 1, H C Reeves 1
PMCID: PMC209036  PMID: 1938889

Abstract

Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.

Full text

PDF
6847

Images in this article

6846FIG. 1
on p.6846

Selected References

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

  1. Atomi H., Ueda M., Hikida M., Hishida T., Teranishi Y., Tanaka A. Peroxisomal isocitrate lyase of the n-alkane-assimilating yeast Candida tropicalis: gene analysis and characterization. J Biochem. 1990 Feb;107(2):262–266. doi: 10.1093/oxfordjournals.jbchem.a123036. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochim Biophys Acta. 1963 Jan 8;67:104–137. doi: 10.1016/0006-3002(63)91800-6. [DOI] [PubMed] [Google Scholar]
  4. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory. Biochim Biophys Acta. 1963 Feb 12;67:173–187. doi: 10.1016/0006-3002(63)91815-8. [DOI] [PubMed] [Google Scholar]
  5. CLELAND W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection. Biochim Biophys Acta. 1963 Feb 12;67:188–196. doi: 10.1016/0006-3002(63)91816-x. [DOI] [PubMed] [Google Scholar]
  6. Carpenter W. D., Beevers H. Distribution and Properties of Isocitritase in Plants. Plant Physiol. 1959 Jul;34(4):403–409. doi: 10.1104/pp.34.4.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chell R. M., Sundaram T. K., Wilkinson A. E. Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp. Biochem J. 1978 Jul 1;173(1):165–177. doi: 10.1042/bj1730165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Comai L., Dietrich R. A., Maslyar D. J., Baden C. S., Harada J. J. Coordinate expression of transcriptionally regulated isocitrate lyase and malate synthase genes in Brassica napus L. Plant Cell. 1989 Mar;1(3):293–300. doi: 10.1105/tpc.1.3.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garnak M., Reeves H. C. Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli. J Biol Chem. 1979 Aug 25;254(16):7915–7920. [PubMed] [Google Scholar]
  10. Hoyt J. C., Reeves H. C. In vivo phosphorylation of isocitrate lyase from Escherichia coli D5H3G7. Biochem Biophys Res Commun. 1988 Jun 16;153(2):875–880. doi: 10.1016/s0006-291x(88)81177-x. [DOI] [PubMed] [Google Scholar]
  11. Hoyt J. C., Robertson E. F., Berlyn K. A., Reeves H. C. Escherichia coli isocitrate lyase: properties and comparisons. Biochim Biophys Acta. 1988 Jul 14;966(1):30–35. doi: 10.1016/0304-4165(88)90125-0. [DOI] [PubMed] [Google Scholar]
  12. Kornberg H. L. The role and control of the glyoxylate cycle in Escherichia coli. Biochem J. 1966 Apr;99(1):1–11. doi: 10.1042/bj0990001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LaPorte D. C., Chung T. A single gene codes for the kinase and phosphatase which regulate isocitrate dehydrogenase. J Biol Chem. 1985 Dec 5;260(28):15291–15297. [PubMed] [Google Scholar]
  14. 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]
  15. McCarthy J. T., Charles A. M. Studies on isocitrate lyase from the facultative autotroph Thiobacillus novellus. Can J Microbiol. 1973 Apr;19(4):513–519. doi: 10.1139/m73-082. [DOI] [PubMed] [Google Scholar]
  16. OLSON J. A. The purification and properties of yeast isocitric lyase. J Biol Chem. 1959 Jan;234(1):5–10. [PubMed] [Google Scholar]
  17. Pinzauti G., Giachetti E., Vanni P. Isocitrate lyase of conifers (Pinus pinea). Int J Biochem. 1982;14(4):267–275. doi: 10.1016/0020-711x(82)90087-8. [DOI] [PubMed] [Google Scholar]
  18. Reeves H. C., O'Neil S., Weitzman P. D. Modulation of isocitrate dehydrogenase activity in Acinetobacter calcoaceticus by acetate. FEBS Lett. 1983 Nov 14;163(2):265–268. doi: 10.1016/0014-5793(83)80832-1. [DOI] [PubMed] [Google Scholar]
  19. Robertson E. F., Dannelly H. K., Malloy P. J., Reeves H. C. Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Anal Biochem. 1987 Dec;167(2):290–294. doi: 10.1016/0003-2697(87)90166-7. [DOI] [PubMed] [Google Scholar]
  20. Robertson E. F., Hoyt J. C., Reeves H. C. Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli. J Biol Chem. 1988 Feb 15;263(5):2477–2482. [PubMed] [Google Scholar]
  21. Rogers J. E., McFadden B. A. Isocitrate lyase from Pseudomonas indigofera: pH dependence of catalysis and binding of substrates and inhibitors. Arch Biochem Biophys. 1976 Jun;174(2):695–704. doi: 10.1016/0003-9861(76)90400-8. [DOI] [PubMed] [Google Scholar]
  22. SHIIO I., SHIIO T., MCFADDEN B. A. ISOCITRATE LYASE FROM PSEUDOMONAS INDIGOFERA. I. PREPARATION, AMINO ACID COMPOSITION AND MOLECULAR WEIGHT. Biochim Biophys Acta. 1965 Jan;96:114–122. doi: 10.1016/0005-2787(65)90615-5. [DOI] [PubMed] [Google Scholar]
  23. SHIIO I., SHIIO T., MCFADDEN B. A. ISOCITRATE LYASE FROM PSEUDOMONAS INDIGOFERA. II. ACTIVATION, INHIBITION AND STABILIZATION. Biochim Biophys Acta. 1965 Jan;96:123–133. doi: 10.1016/0005-2787(65)90616-7. [DOI] [PubMed] [Google Scholar]
  24. Turley R. B., Choe S. M., Trelease R. N. Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase. Biochim Biophys Acta. 1990 Jun 21;1049(2):223–226. doi: 10.1016/0167-4781(90)90045-4. [DOI] [PubMed] [Google Scholar]
  25. Vanni P., Giachetti E., Pinzauti G., McFadden B. A. Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol B. 1990;95(3):431–458. doi: 10.1016/0305-0491(90)90002-b. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES