Characterization of a novel alkaline phosphatase activity which co-purifies with a phosphorylase (phosphoprotein) phosphatase of Mr = 35,000 cardiac muscle.

In a previous communication (Li, H.-C., Hsiao, K.-J., and Chan, W. W. S. (1978) Eur. J. Biochem 84,215-225) we reported the purification of a divalent cation-independent, nonspecific phosphoprotein phosphatase (phosphatase S, M, = 35,000) from canine cardiac muscle to apparent homogeneity. It was found that the homogeneous enzyme preparation exhibited significant activity toward p-nitrophenyl phosphate. Since the enzymic activity had an optimum pH around 8.5, it was termed alkaline phosphatase S. Further studies demonstrated that the major alkaline phosphatase activity in the soluble fraction of the canine heart homogenate was co-purified with the phosphoprotein phosphatase S throughout the purification procedure. The alkaline and the phosphoprotein phosphatase activities were found to co-migrate on polyacrylamide gel electrophoresis, ion exchange, and gel filtration chromatographies and sucrose density gradient ultracentrifugation. These two activities, however, exhibited distinct thermostability, pH activity profile, and metal ion specificity, and could be partially separated by hydrophobic interaction chromatography. Discussions concerning whether these two activities reside in the same enzyme protein or in two different yet very similar polypeptide chains have been presented. The properties of alkaline phosphatase S appear to be different from other known mammalian alkaline phosphatases, and it may represent a new isozyme species of the hydrolytic enzyme. In contrast to other well studied alkaline phosphatases of membrane origin purified from various tissues, the cardiac muscle alkaline phosphatase S was of cytosolic origin and required the simultaneous presence of Mg2+ and a sulfhydryl compound for activity. The close association of alkaline phosphatase S activity with phosphoprotein phosphatase S suggests that it may play a role in the regulation of protein phosphorylation-dephosphorylation reaction.