Shapiro R A, Morehouse R F, Curthoys N P
Biochem J. 1982 Dec 1;207(3):561-6. doi: 10.1042/bj2070561.
A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.
一种与膜相关的磷酸依赖性谷氨酰胺酶源自超声破碎的线粒体，并在纯化后基本不含γ-谷氨酰转肽酶活性。磷酸盐浓度的增加会导致膜结合谷氨酰胺酶呈S形激活。磷酸盐对两种亲和标记物L-2-氨基-4-氧代-5-氯戊酸和6-重氮-5-氧代-L-正亮氨酸使谷氨酰胺酶失活的速率也有类似影响，这与之前对溶解和纯化后的酶的观察结果一致。因此，两种形式的谷氨酰胺酶在磷酸盐诱导下经历相似的构象变化。开发了一种灵敏的放射性测定法，并用于确定谷氨酸对膜相关谷氨酰胺酶抑制作用的动力学。当磷酸盐浓度从5 mM增加到100 mM时，谷氨酰胺的Km值从36 mM降至4 mM。在高浓度和低浓度磷酸盐条件下，谷氨酸都是谷氨酰胺的竞争性抑制剂。然而，随着磷酸盐浓度的增加，谷氨酸的Ki值从5 mM增加到52 mM。因此，谷氨酰胺和谷氨酸与谷氨酰胺酶上的同一位点相互作用，但该位点的特异性由可用的磷酸盐浓度决定。
