Demonstration of Two Different Reactive Sulfhydryl Groups in the ATP-Binding Sites of Ca2+-ATPase of Sarcoplasmic Reticulum by Disulfides of Thioinosine Triphosphates

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Abstract
The disulfide of thioinosine triphosphate, (SnoPPP)2, is a substrate of the Ca2+-pump and the Ca2+-ATPase of [rabbit] sarcoplasmic reticulum (Km = 400 .mu.M). Inactivation of Ca2+-ATPase by the .beta.,.gamma.-methylene diphosphonate analogue of the disulfide of thioinosine triphosphate, (SnoPP[CH2]P)2, in the presence of (Ca2+ + Mg2+ + K+) is preceeded by a dissociable enzyme inhibitor complex with a Kd of 130 .mu.M for a low-affinity binding site. ATP protected Ca2+-ATPase against the inactivation under these conditions with a Kd of 140 .mu.M. Kinetic analysis of the inactivations of Ca2+-ATPase by (SnoPP[CH2]P)2 is the absence of Ca2+ and Mg2+, but the presence of K+ and EGTA [ethylene glycol bis(2-amino ethyl ether)-N,N''-tetraacetic acid] led to the appearance of 2 nucleotide binding sites with 2 different inactivation velocities. Inactivation rate constants K2 were found for the rapid inactivating part .**GRAPHIC**. = 1.44 .times. 10-2 s-1) and the slow inactivating part .**GRAPHIC**. = 1.15 .times. 10-3 s-1). From the protective effect of ATP under these conditions a high-affinity (Kd = 48.78 .mu.M) and a low-affinity ATP binding site (Kd = 114 .mu.M) were apparent. The affinity of the analogues to the enzyme is decreased in the sequence: (SnoPPP)2 > (SnoPP[NH]P)2 > (SnoPP[CH2]P)2 > (SnoP)2. (SnoPPP)2-inactivated Ca2+-ATPase was reactivated by incubation with dithiothreitol. Inactivation of Ca2+-ATPase by [.gamma.-32P](SnoPPP)2 in the presence of (Mg2+ + K+ + Ca2+) or (EGTA + K+) was accompanied by the incorporation of hydroxylamine-insensitive radioactivity into the acid-precipitable protein. The enzyme-bound [.gamma.-32P]SnoPPP was cleaved by dithiothreitol. (SnoPPP)2 and its non-hydrolyzable analogues (SnoPP[NH]P)2 and (SnoPP[CH2]P)2 act as ATP affinity labels and form mixed disulfides with a SH group within the active site.