Inhibitors of protein phosphatase‐1

Protein phosphatase inhibitor‐1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP‐dependent protein kinase on a threonyI residue; this phosphorylation was necessary for inhibition of protein phosphatase‐1. Bovine adipose tissue inhibitor‐1 was compared directly with rabbit skeletal muscle inhibitor‐1 and with a 32000‐ M r , dopamine‐ and cAMP‐regulated phosphoprotein from bovine brain (DARPP‐32), also an inhibitor of protein phosphatase‐1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor‐1 was found to be very similar and possibly identical to DARPP‐32 and was clearly distinct from skeletal muscle inhibitor‐1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus‐aureus ‐V8‐protease‐generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two‐dimensional thin‐layer electrophoresis/chromatography; elution on reverse‐phase HPLC; chymotryptic peptide maps as analysed by reverse‐phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase‐1 (DARPP‐32), which is distinct from that of skeletal muscle (inhibitor‐1).