Abstract
An NADP+-specific isocitrate dehydrogenase has been purified and characterized from Rhizobium meliloti. The enzyme showed Mn++ or Mg++ requirement. The apparent Km values were 2.00×10-5 m and 1.51×10-5 m for dl-isocitrate and NADP+, respectively. The enzyme was inhibited by ATP, to a lesser extent by ADP and AMP. α-Ketoglutarate also inhibited the enzyme activity. Oxalacetate and glyoxylate together inhibited the enzyme activity. The inhibition was competitive. Studies with thiol inhibitors suggested that the enzyme contained a sulfhydryl group at or near the active site. The enzyme has an approximate molecular weight of 60 000. Fluorescence studies suggested that the enzyme contained tryptophan
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andrews, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel-filtration. — Biochem. J. 91: 222–233.
Barrera, C. R. and Jurtshuk, P. 1970. Characterization of the highly active isocitrate (NADP+) dehydrogenase of Azotobacter vinelandii. — Biochim. Biophys. Acta 220: 416–429.
Benemann, J. R. and Valentine, R. C. 1971. High-energy electrons in bacteria, p. 135–172. In A. H. Rose and J. F. Wilkinson, (eds.), Adv. Microb. Physiol., Vol. 5. — Academic Press, New York.
Chen, R. F. 1973. Extrinsic and intrinsic fluorescence of proteins, p. 467–540. In G. G. Guilbault, (ed.), Practical fluorescence, theory, methods and techniques. — Marcel Dekker Inc., New York.
Chung, A. E. and Franzen, J. S. 1969. Oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase from Azotobacter vinelandii. Isolation and characterization. —Biochemistry 8: 3175–3184.
Colman, R. F. 1968. Effect of modification of a methionyl residue on the kinetic and molecular properties of isocitrate dehydrogenase. — J. Biol. Chem. 243: 2454–2464.
Colowick, S. P. 1955. Separation of proteins by use of adsorbents, p. 98. In S. P. Colowick and N. O. Kaplan, (eds.), Methods in Enzymology, Vol. 1. — Academic Press, New York.
Davis, B. J. 1964. Disc electrophoresis. 11. Method and application to human serum proteins. — Ann. N.Y. Acad. Sci. 121: 404–427.
Hampton, M. L. and Hanson, R. S. 1969. Regulation of isocitrate dehydrogenase from Thiobacillus thiooxidans and Pseudomonas fluorescens. — Biochem. Biophys. Res. Commun. 36: 296–305.
Larsson, P.-O. and Mosbach, K. 1971. Preparation of a NAD(H)-polymer matrix showing coenzymic function of the bound pyridine nucleotide. — Biotechnol. Bioeng. 13: 393–398.
Lowe, C. R., Mosbach, K. and Dean, P. D. G. 1972. Some applications of insolubilised cofactors to the purification of pyridine nucleotide-dependent dehydrogenases. — Biochem. Biophys. Res. Commun. 48: 1004–1010.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. — J. Biol. Chem. 193: 265–275.
Marr, J. J. and Weber, M. M. 1968. Studies on the mechanism of purine nucleotide inhibition of a triphosphopyridine nucleotide-specific isocitrate dehydrogenase. — J. Biol. Chem. 243: 4973–4979.
Morris, C. J. O. R. 1964. Thin-layer chromatography of proteins on Sephadex G-100 and G-200. — J. Chromatogr. 16: 167–175.
Moustafa, E. and Leong, C. K. 1975. Effect of adenine nucleotides on NAD-depentent isocitrate dehydrogenases in rhizobia and bacteroids of legume root nodules. — Biochim. Biophys. Acta 391: 9–14.
Mumford, F. E., Carnahan, J. E. and Castle, J. E. 1959. Nitrogen fixation in a mutant of Azotobacter vinelandii. — J. Bacteriol. 77: 86–90.
Nambiar, P. T. C. and Shethna, Y. I. 1975. Isocitrate dehydrogenase in Rhizobium species. —Curr. Sci. 44: 385–386.
Reeves, H. C., Rabin, R., Wegener, W. S. and Ajil, S. J. 1971. Assays of enzymes of the tricarboxylic acid and glyoxylate cycles, p. 425–462. In J. R. Morris and D. W. Ribbons, (eds.), Methods in Microbiology, Vol. 6A. — Academic Press, New York.
Shiio, I. and Ozaki, H. 1968. Concerted inhibition of isocitrate dehydrogenase by glyoxylate plus oxalacetate. — J. Biochem. 64: 45–53.
Wong, P., Evans, H. J., Klucas, R. and Russell, S. 1971. Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids, p. 525–543. In T. A. Lie and E. G. Mulder, (eds.), Biological nitrogen fixation, Proc. Tech. Meeting on Biological Nitrogen Fixation of the Int. Biol. Programme (Section PP-N), Prague and Wageningen, 1970. —Plant & Soil, Special Volume, M. Nijhoff, The Hague.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Nambiar, P.T.C., Shethna, Y.I. Purification and properties of an NADP+-specific isocitrate dehydrogenase from Rhizobium meliloti . Antonie van Leeuwenhoek 42, 471–482 (1976). https://doi.org/10.1007/BF00410178
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00410178