AFFINITY-CHROMATOGRAPHY OF REGULATORY SUBUNITS OF PROTEIN PHOSPHATASE-1

In this study we demonstrate that recombinant rabbit muscle protein ph osphatase-1 immobilized on CH-Sepharose is an efficient affinity chrom atography support for the isolation of subunits of phosphatase-1. The support was used to isolate the glycogen binding subunit of phosphatas e-1 from muscle and nonmuscle rat tissues. Examination of the affinity -purified material from rat heart and liver showed that the major comp onent was a 160-kDa polypeptide on SDS-PAGE. The identity of the purif ied liver 160-kDa polypeptide as the glycogen binding subunit was conf irmed by the demonstration that it is capable of binding to glycogen, and is phosphorylated by the catalytic subunit of PKA. The novel obser vation was made that the phosphorylation was dependent on the presence of glycogen. Examination of the material from heart, lung, liver, kid ney, and brain showed a similar phenomenon. Our studies show that this subunit is widely distributed in tissues. The affinity support was al so efficient in the isolation of the NIPP-1 (nuclear inhibitor of prot ein phosphatase-1) proteins from calf thymus. Examination of heat-trea ted extracts of rat liver led to the isolation of a novel 19-kDa inhib itory protein which could also be phosphorylated by PKA and may repres ent the rat liver homolog of calf thymus NIPP-1. (C) 1996 Academic Pre ss, Inc.