Summary
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 α-ketoglutarate, NADPH and NH sup+inf4 are 1.2 mM, 9.7 µM 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 µM) 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 µM) yet N-ethylmaleimide does not.
In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.
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Abbreviations
- EDTA:
-
Ethylenediamine Tetraacetic Acid
- Tris:
-
Tris(hydroxymethyl)aminomethane
- Bis-tris:
-
bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane
- TRITON X-100:
-
iso-octylphenoxypoly-ethoxyethanol
- pHMB:
-
p-Hydroxymercuribenzoic acid
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Pamula, F., Wheldrake, J.F. Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum . Mol Cell Biochem 105, 85–92 (1991). https://doi.org/10.1007/BF00230377
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DOI: https://doi.org/10.1007/BF00230377