Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum.
NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent Kms for alpha-ketoglutarate, NADPH and NH4+ are 1.2 mM, 9.7 microM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 microM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn2+ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 microM) yet N-ethylmaleimide does not. In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.
Pamula
F
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School of Biological Sciences, Flinders University of South Australia, Bedford Park.
Wheldrake
J F
JF
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification*; D018086:Glutamate Dehydrogenase (NADP+); D006160:Guanosine Triphosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D008970:Molecular Weight; D009024:Morphogenesis
D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D005969:Glutamate Dehydrogenase; D018086:Glutamate Dehydrogenase (NADP+); D005033:Ethylmaleimide