CHEMICAL MODIFICATION OF CA2+-DEPENDENT ATPASE OF SARCOPLASMIC-RETICULUM FROM SKELETAL-MUSCLE .1. BINDING OF N-ETHYLMALEIMIDE TO SARCOPLASMIC-RETICULUM - EVIDENCE FOR SULFHYDRYL GROUPS IN ACTIVE-SITE OF ATPASE AND FOR CONFORMATIONAL-CHANGES INDUCED BY ADENOSINE-TRIPHOSPHATE AND DIPHOSPHATE

The time course of binding of N-ethylmaleimide (NEM) to the [rabbit] SR [sarcoplasmic reticulum] was measured at pH 7.5 in the presence of absence of ATP or ADP. Both in the presence and absence of nucleotide, the ATPase [EC 3.6.1.3] activity decreased linearly with increase in the amount of NEM bound to the fragmented SR, and was inhibited almost completely by the binding of 2 mol of NEM/105 g of the SR protein. The amount of NEM incorporated into the ATPase (MW = 105,000) was measured by SDS disc-gel electrophoresis. The ATPase activity was inhibited almost completely by the binding of 2 mol of NEM/mol of ATPase. The rate of binding of NEM to SR decreased by 30-40% in the presence of either ATP or ADP. The concentrations of both ATP and ADP for half-saturation were 0.1-0.2 mM. The effect of nucleotide on the rate of binding of NEM was not changed by the presence of Ca2+ and Mg2+O. Similar effects were also observed even when the SR membranes were solubilized with Triton X-100. One or 2 SH groups are probably located in the active site of the SR ATPase. Addition of ATP and ADP may induce conformational changes.