Phosphoprotein phosphatase inhibitor-2. Identification as a species of molecular weight 31,000 in rabbit muscle, liver, and other tissues.

Abstract

Specific antibodies, raised to purified rabbit skeletal muscle inhibitor-2, were used to analyze for the presence of inhibitor-2 in extracts of rabbit skeletal, cardiac, and diaphragm muscles, liver, kidney, brain, and lung. Direct analyses of the extracts by "Western blotting" revealed several immunoreactive species, apparent molecular weights in the range 26,000-136,000, as well as species with the electrophoretic mobility of inhibitor-2, apparent molecular weight 31,000. When supernatants from boiled extracts were similarly analyzed, most of the immunoreactive material was lost and the species corresponding to inhibitor-2 became prominent. Liver and muscle were studied in more detail; immunoprecipitates from either boiled or unboiled extracts were analyzed by Western blotting. The dominant polypeptide now was the species of apparent molecular weight 31,000, corresponding to inhibitor-2. Higher molecular weight species (115,000 in muscle and 136,000 in liver) were also detectable. The amount of inhibitor-2 detected in immunoprecipitates was not greatly different whether unboiled or boiled tissue extracts were used. In addition, extraction of the precipitates by boiling released material that inhibited purified type 1 protein phosphatase. The results suggest that inhibitor-2 is widely distributed in rabbit tissues and is found predominantly as a form of apparent molecular weight 31,000. In particular, the study provides direct demonstration of a species in rabbit liver with similar properties to rabbit muscle inhibitor-2.