摘要
浑球红假单胞菌菌株601经超声击碎,粗提液通过Triton处理,硫酸铵沉淀,DE—52和DEAE—sephadex A—50柱层析及 Seqhadex G—200凝胶过滤等步骤,将谷氨酸合酶(GOGAT)分离纯化,在聚丙烯酰胺凝胶电泳上呈现一条带。GOGAT表观分子量约为138 kD。该酶最大光吸收在278,375,450 nm和475 nm处,表明GOGAT可能是一种黄素蛋白。纯化的GOGAT对其底物 Gln,α—酮戊二酸和NADPH的表观K_m值分别为830,150和6μmol/L。反应产物Gln和NADP,几种氨基酸对GOGAT活力有不同程度的抑制作用,Gln类似物DON对GOGAT活力有强烈的抑制作用。
Glutamate synthase [L-gutamate:NADP^+oxidoreductase (transaminating), E.C.1.4. 1. 13](GOGAT) of Rhodobacter sphaeroides strain 601was purified to electrophoretic homogeneityby Triton treatment, ammonium sulfate preci-pitation, DE-52 and DEAE-sephadex A--50chromatography and Sephadex G200 filtration.The specific activity of purified glutamate syn-thase was 2.018 U/mg protein, which represen-ted a 208-fold purification with a 15% yield(Table 1). The close agreement of the mole-cular weights between the native enzyme (ap-proximately 138 kD, Fig. 4) and the polypep-tide chain (139 kD, Fig. 5) showed clearly thatthe enzyme consisted of a single chain, R. spha-eroides glutamate synthase showed substrate spe-cificity to L-glutamine, a-Ketoglutarate, andNADPH for catalytic activity (Table. 2). NADHdid not support activity. Neither could NH_4C1or L-asparagine substitute for L-glutamine norcould oxalacetate or pyruvate substitute fora-Ketoglutarate. The UV-visible spectrumof the enzyme exhibited maxima at 278, 375,450 and 475 nm (Fig. 6)indicating that it may bea flavoprotein similar to that from other prokary-otes. The apparent optimum pH of the enzymewas between 8.3～8.9 (Fig. 7) and its K_mvalues for L-glutamine, a-Ketoglutarate andNADPH were 830, 150, 6 umol/L respectively(Fig. 8). Its products (L-glutamate andNADPH) and several ammino acids (Met, Leu,Asp and Asn) inhibited the activity of the enzy-me (Table. 3). An analogue of Gln, DON (6-diazo-5-oxo-L-norleucine) was found to be apotent inhibitor of the enzyme (Fig. 9).
关键词
假单胞菌
谷氨酸合酶
纯化
glutamate synthase
apparent K_m
Rhodobacter sphaeroides
