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Purification and properties of NADP‐dependent glutamate dehydrogenase from Sphaerostilbe repens
Author(s) -
Balton Bernard,
Msatef Yamina
Publication year - 1983
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1983.tb04227.x
Subject(s) - glutamate dehydrogenase , chemistry , polyacrylamide gel electrophoresis , ammonium , chromatography , substrate (aquarium) , gel electrophoresis , reductive amination , enzyme , biochemistry , glutamate receptor , biology , organic chemistry , catalysis , ecology , receptor
NADP‐dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from Sphaerostilbe repens was purified to homogeneity by using ammonium sullate fractionation hydroxyapatite and DEAE‐cellulose column chromatography and, finally, preparative polyacrylamide gel electrophoresis. The turnover number of the enzyme for the amination reaction was about 66000 mol substrate transformed min ‐1 (molecule of GDH) ‐1 . Molecular weight of the native enzyme was estimated to be 280000 dalton by polyacrylamide gradient gel electrophoresis. The same technique in the presence of sodium dodecyl sulfatc gave a single protein band that corresponded to the subunit molecular weight of 48000 dalton. Thus, it is concluded that NADP‐GDH is composed of six identical polypeptidic chains. The pH optimums were 6.9 and 8.4 for the forward and reverse reactions respectively. The NADP‐GDH lost practically none of its activity for ten days at 4°C and for 15 h at room temperature, but was inactivated by higher temperatures. Thiol compounds such as 2‐mercaptoethanol and dithiolhrcitol protected the enzyme from rapid inactivation. The Michaelis constants for GDH were 0.64, 0.049. 0.043 and 5.5 m M for α‐ketoglutaratc. NADPH, NADP and glutamate, respectively. The enzyme had a negative cooperativity for ammonium (Hill number of 0.66), and its K m value increased from 2.6 to 21.2 m M when the ammonium concentration exceeded 16 m M . The deamination reaction was highly sensitive to inhibition by ammonium, while the amination reaction was only slightly inhibited by glutamate. These results, considered together with the K m values, indicate that the NADP‐GDH in Sphaerostilbe repens is primarily concerned with glutamate biosynthesis.