Uno I, Matsumoto K, Adachi K, Ishikawa T
J Biol Chem. 1984 Jan 25;259(2):1288-93.
The active NAD-dependent glutamate dehydrogenase of wild type yeast cells fractionated by DEAE-Sephacel chromatography was inactivated in vitro by the addition of either the cAMP-dependent or cAMP-independent protein kinases obtained from wild type cells. cAMP-dependent inhibition of glutamate dehydrogenase activity was not observed in the crude extract of bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase. The cAMP-dependent protein kinase of CYR3 mutant cells, which has a high K alpha value for cAMP in the phosphorylation reaction, required a high cAMP concentration for the inactivation of NAD-dependent glutamate dehydrogenase. An increased inactivation of partially purified active NAD-dependent glutamate dehydrogenase (Mr = 450,000) was observed to correlate with increased phosphorylation of a protein subunit (Mr = 100,000) of glutamate dehydrogenase. The phosphorylated protein was labeled by an NADH analog, 5'-p-fluorosulfonyl[14C]benzoyladenosine. Activation and dephosphorylation of inactive NAD-dependent glutamate dehydrogenase fractions were observed in vitro by treatment with bovine alkaline phosphatase or crude yeast cell extracts. These results suggested that the conversion of the active form of NAD-dependent glutamate dehydrogenase to an inactive form is regulated by phosphorylation through cAMP-dependent and cAMP-independent protein kinases.
通过DEAE-葡聚糖凝胶色谱法分离的野生型酵母细胞的活性NAD依赖性谷氨酸脱氢酶，在体外因添加从野生型细胞获得的cAMP依赖性或cAMP非依赖性蛋白激酶而失活。在缺乏cAMP依赖性蛋白激酶调节亚基的bcy1突变细胞的粗提物中未观察到cAMP对谷氨酸脱氢酶活性的依赖性抑制。CYR3突变细胞的cAMP依赖性蛋白激酶在磷酸化反应中对cAMP具有高Kα值，需要高浓度的cAMP才能使NAD依赖性谷氨酸脱氢酶失活。观察到部分纯化的活性NAD依赖性谷氨酸脱氢酶（Mr = 450,000）失活增加与谷氨酸脱氢酶的一个蛋白亚基（Mr = 100,000）磷酸化增加相关。磷酸化蛋白被NADH类似物5'-对氟磺酰基[14C]苯甲酰腺苷标记。通过用牛碱性磷酸酶或粗酵母细胞提取物处理，在体外观察到无活性NAD依赖性谷氨酸脱氢酶组分的激活和去磷酸化。这些结果表明，NAD依赖性谷氨酸脱氢酶的活性形式向无活性形式的转变受cAMP依赖性和cAMP非依赖性蛋白激酶磷酸化的调节。
