Glutamate‐Stimulated Phosphorylation of a Specific Protein in P2 Fractions of Rat Cerebral Cortex

Abstract

— Incubation of P₂ fractions from rat cerebral cortex with ³²P_i in the presence of l‐glutamate caused an increased phosphorylation of a protein with apparent molecular weight of 43,000 (P₄₃) as demonstrated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and autoradiography. This glutamate‐stimulated phosphorylation of P₄₃ was already detectable 10 s after the addition of glutamate and was dependent on the concentration of glutamate in the incubation medium. Other excitatory amino acids such as D‐glutamate, l‐aspartate, d,l‐cysteic acid, l‐cysteinesulfinic acid, and d,l‐α‐aminoadipic acid did not stimulate the phosphorylation of P₄₃. In contrast, α‐ketoglutarate and succinate stimulated the phosphorylation of this protein. Glutamate‐stimulated phosphorylation of P₄₃ seemed not to be mediated by either cAMP or cGMP and was inhibited by the presence of Ca²⁺ in the incubation medium. Experiments performed with metabolic inhibitors indicated that glutamate‐stimulated protein phosphorylation is localized in mitochondria. This conclusion is supported by the occurrence of glutamate‐stimulated phosphorylation of P₄₃ in mitochondrial fractions from several peripheral tissues. The present results are consistent with the hypothesis that P₄₃ is a component of the pyruvate dehydrogenase complex of mitochondria.