Sheu K F, Lai J C, Blass J P
J Neurochem. 1983 May;40(5):1366-72. doi: 10.1111/j.1471-4159.1983.tb13578.x.
The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.
通过测量从牛肾中纯化并经Mg.ATP磷酸化而失活的纯化磷酸化（即无活性）丙酮酸脱氢酶复合物（PDHC）的激活速率，来测定大鼠脑线粒体和匀浆中丙酮酸脱氢酶磷酸（PDHb）磷酸酶的活性。纯化线粒体中的PDHb磷酸酶活性相对于其底物磷酸化PDHC呈现出饱和动力学。其最适pH在7.0至7.4之间，依赖于Mg和Ca，并受NaF和K - 磷酸盐抑制。这些特性与来自牛心的高度纯化酶的特性一致。在亚细胞分级分离时，PDHb磷酸酶与线粒体标记酶（延胡索酸酶和PDHC）共纯化，并与胞质标记酶（乳酸脱氢酶）和膜标记酶（乙酰胆碱酯酶）分离，表明它与其底物一样位于线粒体中。PDHb磷酸酶在纯化线粒体和匀浆中具有相似的动力学特性：依赖于Mg和Ca，不依赖于二氯乙酸，并受NaF和K - 磷酸盐抑制。这些结果与大鼠脑制剂中仅存在一种类型的PDHb磷酸酶一致。它们支持在脑匀浆中测量该酶活性的有效性。
