Abstract
Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Nicklas, W. J. 1988. Glutamate and glutamine in mammals: an overview. Pages 1–4in Kvamme, E. (ed.), Glutamine and Glutamate in Mammals, vol. 1 CRC Press, Boca Raton.
Tildon, J. T., and Zielke, H. R. 1988. Glutamine: an energy source for mammalian tissues. Pages 167–182in Kvamme, E. (ed.), Glutamine and Glutamate in Mammals, vol. 1. CRC Press, Boca Raton.
Syed, S.-E.H., and Engel, P. C. 1984. Ox liver glutamate dehydrogenase: the use of chemical modification to study the relationship between catalytic sites for different amino acid substrates and the question of kinetic non-equivalence of subunits. Biochem. J. 222:621–626.
Couée, I., and Tipton, K. F. 1989. Activation of glutamate dehydrogenase byl-leucine. Biochim. Biophys. Acta. 995:97–101.
Tipton, K. F., and Couée, I. 1988. Glutamate dehydrogenase. Pages 81–100in Kvamme, E. (ed.), Glutamine and Glutamate in Mammals, vol I. CRC Press, Boca Raton.
Nemat-Gorgani, M., and Dodd, G. 1977. The interaction of phospholipid membranes and detergents with glutamate dehydrogenase. II. Fluorescence and stopped-flow studies. Eur. J. Biochem. 74:139–147.
Nemat-Gorgani, M., and Dodd, G. 1977. The interaction of phospholipid membranes and detergents with glutamate dehydrogenase. I. Kinetic studies. Eur. J. Biochem. 74:129–137.
Couée, I. and Tipton, K. F. 1989. The effects of phospholipids on the activation of glutamate dehydrogenase byl-leucine. Biochem. J. 261:921–925.
Shemisa, O. A., and Fahien, L. A. 1971. Modification of glutamate dehydrogenase by various drugs which affect behavior. Mol. Pharmacol. 7:8–25.
Veronese, F. M., Bevilacqua, R., and Chaiken, I. M. 1979. Drug-protein interactions: evaluation of the binding of antipsychotic drugs to glutamate dehydrogenase by quantitative affinity chromatography. Mol. Pharmacol. 15:313–321.
Couée, I., and Tipton, K. F. 1990. The inhibition of glutamate dehydrogenase by some antipsychotic drugs. Biochem. Pharmacol. 39:827–832.
Couée, I., and Tipton, K. F. 1990. Inhibition of ox brain glutamate dehydrogenase by perphenazine. Biochem. Pharmacol. 39:1167–1174.
McCarthy, A. D., Walker, J. M., and Tipton, K. F. 1980. Purification of glutamate dehydrogenase from ox brain and liver. Biochem. J. 191:605–611.
McCarthy, A. D., and Tipton, K. F. 1983. Glutamate dehydrogenase. Pages 19–32,in Hertz, L, Kvamme, E. McGeer, E. G. and Schousboe, A. (eds.), Glutamine, Glutamate and GABA in the Central Nervous System. A. R. Liss, New York.
McCarthy, A. D., and Tipton, K. F. 1985. Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations. Biochem. J. 230:95–99.
Graham, L. D., Griffin, T. O., Beattie, R. E., McCarthy, A. D., and Tipton, K. F. 1985. Purification of liver glutamate dehydrogenase by affinity precipitation and studies on its denaturation. Biochim. Biophys. Acta 828:266–269.
Felipo, V., Miralles, V., Knecht, E., Hernandez-Yaso, J., and Grisolia, S. 1983. The precursor of rat liver mitochondrial glutamate dehydrogenase has enzymatic activity. Eur. J. Biochem. 133:641–644.
Ellman, G. L. 1959. Tissue sulphydryl groups. Arch. Biochem. Biophys. 82:70–77.
Olson, J. A., and Anfinsen, C. B. 1959. Crystallization and characterization ofl-glutamic dehydrogenase. J. Biol. Chem. 197:67–79.
Moon, K. and Smith, E. L. 1973. Sequence of bovine liver glutamate dehydrogenase. J. Biol. Chem. 248:3082–3088.
Riddles, P. W., Blakeley, R. L., and Zerner, B. 1983. Reassessment of Ellman's reagent. Meth. Enzymol. 91:9–60.
Habeeb, A. F. S. A. 1972. Reaction of protein sulfhydryl groups with Ellman's reagent. Meth. Enzymol. 25:457–464.
Gray, W. R. 1972. End-group analysis using dansyl chloride. Meth. Enzymol. 25:121–138.
Bitensky, M. W., Yielding, K. L., and Tomkins, G. M. 1965. The effect of allosteric modifiers on the rate of denaturation of glutamate dehydrogenase. J. Biol. Chem. 240:1077–1082.
Woods, K. R., and Wang, K.-T. 1967. Separation of dansylamino acids by polyamide layer chromatography. Biochim. Biophys. Acta. 133:369–370.
Niederwieser, A. 1972. Thin-layer chromatography of amino acids and derivatives. Meth. Enzymol. 25:60–99.
Perham, R. N. 1978. Techniques for determining the amino-acid composition and sequence of proteins. Pages 1–52in Kornberg, H. L., Metcalfe, J. C., Northcote, D. H., Pogson, C. I. and Tipton, K. F. (eds.), Techniques in the Life Sciences. B1/1. Elsevier, Amsterdam.
Moon, K., Piszkiewicz, D., and Smith, E. L. 1973. Amino acid sequence of chicken liver glutamate dehydrogenase. J. Biol. Chem. 248:3093–3103.
Julliard, J. H. and Smith, E. L. 1979. Partial amino acid sequence of the glutamate dehydrogenase of human liver and a revision of the sequence of the bovine enzyme. J. Biol. Chem. 254:3427–3438.
Smith, E. L., Austen, B. M., Blumenthal, K. M., and Nyc, J. F. 1975. Glutamate dehydrogenases. Pages 293–367in Boyer, P. D. (ed.), The Enzymes 3rd Ed. vol. 11. Academic Press, New York.
Amuro, N., Ooki, K., Ito, A., Goto, Y., and Okazaki, T. 1989. Nucleotide sequence of rat liver glutamate dehydrogenase cDNA. Nucleic Acid Res. 17:2356.
Mavrothalassitis, G., Tzimagiorgis, G., Mitsialis, A., Zannis, V., Plaitakis, A., Papamatheakis, J., and Moschonas, N. 1988. Isolation and characterization of cDNA clones encoding human liver glutamate dehydrogenase: Evidence for a small gene family. Proc. Nat. Acad. Sci. U.S.A. 85:3494–3498.
Ifflaender, U., and Sund, H. 1972. Association behaviour of rat liver glutamate dehydrogenase. FEBS Lett. 20:287–290.
Suva, R. H., and Abeles, R. H. 1978. Studies on the mechanism of action of plasma amine oxidase. Biochemistry 17:3538–3545.
Ferdinand, W., Stein, W. H., and Moore, S. 1965. An unusual disulfide bond in streptococcal proteinase. J. Biol. Chem. 240:1150–1155.
Marsh, E. N., and Leadlay, P. F. 1988. Methylmalonyl-CoA mutase fromPropionibacterium shermanii: evidence for the presence of two masked cysteinyl residues. Biochem. J. 260:339–343.
Author information
Authors and Affiliations
Additional information
Special issue dedicated to Dr. Santiago Grisolia.
Rights and permissions
About this article
Cite this article
Couée, I., Tipton, K.F. The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities. Neurochem Res 16, 773–780 (1991). https://doi.org/10.1007/BF00965686
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00965686