Partial purification and characterization of heat stable protein phosphatase inhibitor-2 from rabbit reticulocytes.
Previous studies indicated that the species of type 1 and type 2 protein phosphatases (PP-1, PP-2) in rabbit reticulocytes are similar to those of rabbit skeletal muscle and rabbit liver. Reticulocyte PP-1 was found to be selectively inhibited by the heat stable protein phosphatase inhibitor-2 (I-2) from rabbit skeletal muscle. Of interest was the observation that muscle I-2 appeared to regulate protein synthesis in reticulocyte lysates by inhibiting an eIF-2 alpha phosphatase with type 1 properties. In this study we have characterized reticulocyte inhibitor-2 (I-2) and find that its properties are similar to those of skeletal muscle I-2. (i) Both I-2 species are stable to boiling and to acid treatment, and have similar chromatographic profiles on DEAE-cellulose and on Blue Sepharose CL-6B. (ii) The two I-2 species migrate electrophoretically as 26-28,000 dalton polypeptides in SDS-acrylamide gels. (iii) Both skeletal muscle I-2 and reticulocyte I-2 selectively inhibit isolated reticulocyte PP-1 and endogenous PP-1 in the lysate. (iv) Reticulocyte I-2 co-chromatographs with PP-1 on DEAE-cellulose, and over 90% of lysate I-2 can be isolated from this partially purified PP-1. (v) Both inhibitor-2 species are active in the unphosphorylated state, but upon addition to lysates, both are phosphorylated by endogenous cAMP-independent protein kinase(s). In addition a preliminary analysis using a polyclonal antibody against muscle inhibitor-1 confirmed biochemical analyses which indicate that lysates are deficient in inhibitor-1.
D000818:Animals; D004791:Enzyme Inhibitors / Q000097:blood*; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000097:blood*; D011506:Proteins; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D000097847:Protein Phosphatase Inhibitory Proteins
D004791:Enzyme Inhibitors; D010749:Phosphoprotein Phosphatases; D011506:Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins