Phosphoprotein Phosphatases from Rat Cerebral Cortex

• Published in: The Journal of biological chemistry Vol. 247; no. 10; pp. 3269 - 3277
• Main Authors: Maeno, Hiroo, Greengard, Paul
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 25.05.1972
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)45241-1

The subcellular distribution of phosphoprotein phosphatases which release orthophosphate from phosphoprotein was studied in rat cerebral cortex. In contrast to several other tissues examined, more than 50% of the total protein phosphatase activity in rat cerebral cortex was found in the particulate fractions; the activity was especially high in the crude mitochondrial fraction. Further subfractionation of the crude mitochondrial fraction by sucrose density gradient centrifugation showed that, among the membrane fractions, the specific activity of protein phosphatase was highest in the fractions rich in synaptic membranes and lowest in the mitochondria. A considerable amount of the enzyme activity in synaptic and microsomal membranes existed in a latent form which could be partially unmasked by treatment with Triton X-100. The specific activity of the protein phosphatase of the cell sap and of the synaptoplasm was considerably higher than that of the membrane fractions. Column chromatography on DEAE-cellulose resolved the protein phosphatase activity of the cell sap into three distinct protein phosphatases, which were clearly distinguished from membrane enzyme by differences in substrate specificity and metal ion requirements. The soluble protein phosphatases, but not the enzyme released from the synaptic plasma membrane fraction by Triton X-100 treatment, were specifically activated by manganese chloride. Endogenous membrane protein was found to be the best among several phosphorylated proteins examined as substrates for membrane-bound protein phosphatase. Both membrane-bound and soluble protein phosphatase exhibited pH optima in the neutral range. Protein phosphatase catalyzed the stoichiometric release of orthophosphate from a phosphoserine residue of protamine. This was the only amino acid residue in protamine and histones which appeared to be phosphorylated by an adenosine 3',5'-monophosphate-dependent protein kinase purified from bovine brain.