1887

Abstract

The purification and some properties of NADP-dependent glutamate dehydrogenase (GDH) and glutamine synthetase (GS) from the facultatively anaerobic Gram-negative bacterium were investigated. The enzymes were purified to homogeneity using a procedure which involved affinity chromatography on Blue Sepharose CL-6B as the major purification step. The recoveries in the purification of GDH and GS were 28% and 64%, respectively. The specific activity of purified GDH was 183 nkat (mg protein)(deaminating reaction). GDH was composed of subunits of molecular mass 47 kDa and the native enzyme was either a tetramer or hexamer. The apparent values for L-glutamate, NADP, 2-oxoglutarate, NADPH and ammonia were 1.5 m, 5.9 μ, 0.47m, 12.5 μ and 14 m, respectively. The specific activity of purified GS was 1125 nkat (mg protein)(transferase reaction). The molecular mass of native GS was 570 kDa; it was composed of 12 subunits of molecular mass 50.1 kDa. The apparent values for -glutamine and hydroxylamine in the transferase reaction were 2.1 and 2.4 mM, respectively; those of ammonia, -glutamate and ATP in the biosynthetic reaction were 0.03, 1 and 0.17 m, respectively. After the adenylylation of GS, the for -glutamine and L-glutamate increased and reached the values of 8.0 and 27 m, respectively. The effects of the changes in GS activity on the ammonia metabolism of are discussed.

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1992-08-01
2021-10-24
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