Phosphorylase phosphatase regulatory subunit. "Western" blotting with immunoglobulins against inhibitor-2 reveals a protein of Mr = 60,000.
Rabbit muscle phosphorylase phosphatase has been isolated in different laboratories as an inactive complex of Mr = 70,000, composed of separate catalytic (Mr = 38,000) and regulatory (Mr = 31,000) proteins. The regulatory protein is identical to one of two heatstable inhibitors called inhibitor-2 (I2). Antiserum raised in sheep against I2 by repeated immunization potently blocked inhibitory activity, whereas preimmune serum did not. Immunoglobulins which blocked inhibitory activity were purified by affinity chromatography with I2 as the immobilized ligand. Using a "Western" immunoblotting procedure, as little as 1-5 ng of pure I2, obtained by electroelution of the Mr = 31,000 band of I2 from a polyacrylamide gel segment, were detected. Immunoblotting of the immunogen revealed only a band at Mr = 31,000, indicating the absence of contaminating antigenic proteins. When extracts of skeletal muscle and other rabbit tissues were denatured directly in dodecyl sulfate for immunoblotting the most intensely stained band was present at Mr = 60,000, rather than at Mr = 31,000 as expected. A small amount of I2 and other bands were detected, in particular at Mr = 36,000 and 25,000. Subsequent to heat treatment of the tissue extracts, there was an enrichment of I2 content relative to the Mr = 60,000 band. The results indicate the existence of a Mr = 60,000 protein related to I2. Activation of phosphorylase phosphatase in a muscle extract by treatment with Co2+ plus trypsin exactly coincided with digestion of the Mr = 60,000 immunoreactive protein. Available data indicate that this protein may function as a regulatory subunit of phosphorylase phosphatase.
Gruppuso
P A
PA
Johnson
G L
GL
Constantinides
M
M
Brautigan
D L
DL
D000818:Animals; D002850:Chromatography, Gel; D003035:Cobalt / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007136:Immunoglobulins; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D010765:Phosphorylase Phosphatase / Q000032:analysis*; D011506:Proteins / Q000276:immunology*; D011817:Rabbits; D012756:Sheep; D014357:Trypsin / Q000378:metabolism; D000097847:Protein Phosphatase Inhibitory Proteins
D003035:Cobalt; D007136:Immunoglobulins; D046911:Macromolecular Substances; D010749:Phosphoprotein Phosphatases; D010765:Phosphorylase Phosphatase; D011506:Proteins; D014357:Trypsin; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins