Chemical Modification of the Ca2+-Dependent ATPase of Sarcoplasmic Reticulum from Skeletal Muscle

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Abstract
SR vesicles were allowed to react for 15 min at pH 8.0 and 0˚C with an impermeant reagent, TBS, fixing the Ca2+-dependent ATPase [EC 3.6.1.3] in any one of the three enzymatic states:MgE, MgEATPand EP The TNP-SR was digested with trypsin, and three major subfragments of the ATPase (I, II, and III) with molecular weights of about 50,000, 32,000, and 22,000 daltons were separated by SDS-polyacrylamide gel electrophoresis. The amounts of TNP incorporated into these subfragments were measured after eluting them from gels with 1% SDS. When SR was incubated with TBS in the enzymatic state MgE, about 1, 0.5, and 0.5 mol of TNP were incorporated for each mole of subfragments I, II, and III, respectively. About 0.5, 0.5 and 0 mol of TNP were incorporated for each mole of subfragments I, II, and III, respectively, when the enzymatic state was fixed at on the other hand, about 1.5, 1, and 1 mol of TNP were incorporated for each mole of subfragments I, II, and III, respectively, in state EP. SR was further purified by sucrose density gradient centrifugation, and was digested with trypsin. The SDS-gel electrophoretograms of tryptic digests changed markedly with change in the enzymatic state of the ATPase, MgE, MgEATP or EP. Based on these results, together with those on the molecular mechanism of coupling of the ATPase reaction with Ca2+ transport, a diagrammatic model explaining the functional movements of the ATPase molecule coupled with elementary steps of the ATPase reaction is proposed.