Structural characterization of cardiac protein phosphatase with a monoclonal antibody. Evidence that the Mr = 38,000 phosphatase is the catalytic subunit of the native enzyme(s).
The native structures of protein phosphatases have not been clearly established. Several tissues contain high molecular weight enzymes which are converted to active species of Mr approximately 35,000 by denaturing treatments or partial proteolysis. We have used a monoclonal antibody directed against purified bovine cardiac Mr = 38,000 protein phosphatase to determine whether this species is the native catalytic subunit or a proteolytic product of a larger polypeptide. Monoclonal antibody was obtained from a cloned hybrid cell line produced by the fusion of Sp2 myeloma cells with spleen cells from a mouse immunized with phosphatase coupled to hemocyanin. This antibody was specific for the Mr = 38,000 phosphatase as determined by immunoblot analysis of purified enzyme or cardiac tissue extracts after native or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single immunoreactive protein of Mr = 38,000 was present in cardiac tissue extracts including extracts prepared from freeze-clamped rat heart rapidly denatured in hot sodium dodecyl sulfate buffer. Precipitation of cardiac extract with 80% ethanol did not alter the Mr of the phosphatase nor did it liberate new immunoreactive material not observed in the extract. Ethanol precipitation caused the dissociation of both phosphatase activity and immunoreactivity from a high Mr form to a form of Mr between 30,000 and 40,000. An immunoreactive protein of Mr = 38,000 was identified in several bovine and rat tissues as well as tissues from rabbits, mice and chickens and human HT-29 cells. From these data we conclude that the Mr = 38,000 cardiac phosphatase is a native catalytic subunit of higher molecular complexes which are dissociated by ethanol precipitation. A very similar, or identical, protein is present in several tissues and species suggesting that this catalytic subunit is a ubiquitous enzyme important in many dephosphorylation reactions.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002417:Cattle; D011232:Chemical Precipitation; D002645:Chickens; D000431:Ethanol / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000276:immunology; D051381:Rats
D000911:Antibodies, Monoclonal; D000431:Ethanol; D010749:Phosphoprotein Phosphatases