Abstract
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.
Highlights
Two forms of protein phosphatase which dephospho- light chain subunit of myosin
A monoclonal antibody and polyclonal antiserum directed against an M, = 38,000 protein phosphatase from this tissue reacted with theM,= 38,000 subunit of PT-1 and PT-2
Identification of Cardiac Myosin and Troponin Phosphatases-Myosin and troponin are associated with the myofibrils which separate from soluble material during low speed centrifugation
Summary
Identification of Cardiac Myosin and Troponin Phosphatases-Myosin and troponin are associated with the myofibrils which separate from soluble material during low speed centrifugation. Native gel analysis of PT-1 revealed a single major Coomassie Blue staining band which corresponded to a large peak of light chain phosphatase activity (Fig. 4).The stained band near the top of the gel appears to be a contaminant in the preparation The amount of this band varies between different preparations and correlates with the presence of a high molecular weight contaminant observed on SDS gels of some preparations. PT-C was able to effectively interact with the antiif the M , = 55,000 and 63,000 subunits of PT-1 and PT-2 serum as indicated by a loss of specific rabbit immunoglobuwere distinct proteins the purified preparations were analyzed lins from the wells when increasing amounts of PT-C were by immunochemical methods.In order to produce antibodies preincubated with the antiserum (Fig. 11).Four-fold lessPT-.
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