Abstract
As an initial step in our study of nitrogen metabolism in the coral/algal symbiosis we have purified glutamate dehydrogenase (EC 1.4.1.4) to homogeneity from polyp tissue of the staghorn coral Acropora formosa collected from Magnetic Island (North Queensland) in 1985–1986. The purified enzyme had a specific activity of 78 U mg-1. The native enzyme had a relative molecular weight, M r, of 360 000 (±20 000), and appears to be a hexamer with subunits of M r=56000 (±3 000). Like the enzyme from other coelenterates, the coral glutamate dehydrogenase (GDH) was absolutely specific with respect to the coenzyme substrate (NADP+/NADPH), and was insensitive to allosteric regulation by nucleotides; unlike other coelenterate GDHs, the coral enzyme was absorlutely specific for ammonium as amino group donor in the reductive amination reaction, and major differences in kinetic properties were apparent. Linear Michaelis-Menten kinetics were observed for the substrates a-ketoglutarate, NADPH and NADP+, the K m values being 0.93, 0.11 and 0.03 mM, respectively. However glutamate dehydrogenase displayed biphasic kinetics with respect to l-glutamate and ammonium, indicating two apparent K m values (18 and 81 mM for l-glutamate and 9.2 and 416 mM for ammonium). The enzyme also exhibits Scatchard plots, Hill coefficients and cooperativity indices characteristic of enzymes displaying negative cooperativity.
Similar content being viewed by others
Literature cited
Alex, S. and J. E. Bell: Dual nucleotide specificity of bovine glutamate dehydrogenase. The role of negative cooperativity. Biochem. J. 191, 299–304 (1980)
Barnes, D. J. and C. J. Crossland: Urease activity in the staghorn coral Acropora acuminata. Comp. Biochem. Physiol. 55B, 371–376 (1976)
Bishop, S. H., A. Klotz, L. L. Drolet, D. H. Smullin and R. J. Hoffman: NADP-specific glutamate dehydrogenase in Metridium senile. Comp. Biochem. Physiol. 61B, 185–187 (1978)
Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantitites of protein utilising the principle of protein-dye binding. Analyt. Biochem. 72, 248–254 (1976)
Crossland, C. J. and D. J. Barnes: Nitrate assimilation enzymes from two hard corals, Acropora acuminata and Goniastrea australensis. Comp. Biochem. Physiol. 57B, 151–157 (1977)
Hoffman, R. J., S. H. Bishop and C. Sassaman: Glutamate dehydrogenases from coelenterates NADP-specific. J. exp. Zool. 203, 165–170 (1978)
Koshland, D. E., Jr., G. Nemethy and D. Filmer: Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry, N.Y. 5, 365–385 (1966)
Kumar, S. and D. J. D. Nicholas: NAD+-and NADP+-dependent glutamate dehydrogenases in Nitrbacter agilis. J. gen. Microbiol. 130, 967–973 (1984)
Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature, Lond. 227, 680–685 (1970)
LeJohn, H. B. and S. Jackson: Allosteric interactios of a regulatory NAD-specific glutamate dehydrogenase from Blastocladiella. A molecular model for the enzyme. J. biol. Chem. 243, 3447–3457 (1968)
Levitzki, A. and D. E. Koshland: Negative cooperativity in regulatory enzymes. Proc. natn. Acad. Sci. U.S.A. 62, 1121–1128 (1969)
Male, K. B. and K. B. Storey: Kinetic characterisation of NADP-specific glutamate dehydrogenase from the sea anemone Anthopleura xanthogrammica, control of amino acid biosynthesis during osmotic stress. Comp. Biochem. Physiol. 76B, 823–829 (1983)
Maulik, P. and S. Ghosh: NADPH/NADH-dependent cold-labile glutamate dehydrogenase in Azospirillum brasilense. Purification and properties. Eur. J. Biochem. 155, 595–602 (1986)
Neet, K. E.: Cooperativity in enzyme functions: equilibrium and kinetic aspects. Meth. Enzym. 64, 139–193 (1980)
Smith, E. L., B. M. Austen, K. M. Blumenthal and J. F. Nyc: Glutamate dehydrogenases. In: The enzymes, 3rd ed. Vol. 11. pp 293–367. Ed. by P. D. Boyer. New York: Academic Press 1975
Taylor, D. J.: Nutrition of algal-invertebrate symbiosis. II. Effects of exogenous nitrogen sources on growth, photosynthesis and the rate of excretion by algal symbionts in vivo and in vitro. Proc. R. Soc. (Ser. B) 201,401–412 (1978)
Author information
Authors and Affiliations
Additional information
Communicated by G. F. Humphrey, Sydney
Rights and permissions
About this article
Cite this article
Catmull, J., Yellowlees, D. & Miller, D.J. NADP+-dependent glutamate dehydrogenase from Acropora formosa: purification and properties. Mar. Biol. 95, 559–563 (1987). https://doi.org/10.1007/BF00393099
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00393099