Cardiac contractile protein phosphatases. Purification of two enzyme forms and their characterization with subunit-specific antibodies.
Two forms of protein phosphatase which dephosphorylate cardiac myosin or myosin light chains and the inhibitory subunit of cardiac troponin were purified from bovine cardiac muscle. The enzymes were composed of subunits of Mr = 63,000, 55,000, and 38,000 in a 1:1:1 molar ratio (PT-1) or Mr = 63,000 and 38,000 in a 1:1 molar ratio (PT-2). Native gel electrophoresis and sucrose gradient sedimentation indicated that activity toward all three substrates was due to a single enzyme species. A monoclonal antibody and polyclonal antiserum directed against an Mr = 38,000 protein phosphatase from this tissue specifically reacted with the Mr = 38,000 subunit of PT-1 and PT-2. The specificity of antibodies for the Mr = 38,000 subunit indicated that it was distinct from the other subunits. The Mr = 63,000 subunits of PT-1 and PT-2 were identical based on mobility on sodium dodecyl sulfate gels and one-dimensional peptide maps. Specificity of antiserum against the Mr = 55,000 subunit of PT-1 showed that this subunit was a distinct protein and not derived from the Mr = 63,000 subunit by proteolysis. PT-2 but not PT-1 could interact with antiserum against the Mr = 38,000 catalytic subunit in competitive immunoassays indicating that the presence of the Mr = 55,000 subunit may alter or mask antigenic site(s). Analysis of the enzymatic properties of PT-1 and PT-2 showed that PT-2 had higher activity with myosin, myosin light chains, and phosphorylase while PT-1 had higher activity with troponin. The results indicate that the presence of the Mr = 55,000 subunit may alter the enzymatic properties of the catalytic subunit.
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009200:Myocardial Contraction; D009206:Myocardium / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000276:immunology / Q000302:isolation & purification*; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; C052279:troponin phosphatase; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase