Demonstration of two different reactive sulfhydryl groups in the ATP-binding sites of Ca2+-ATPase of sarcoplasmic reticulum by disulfides of thioinosine triphosphates.
1. The disulfide of thioinosine triphosphate, (SnoPPP)2, is a substrate of the Ca2+-pump and the Ca2+-ATPase of sarcoplasmic reticulum (Km = 400 microM). 2. 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 dissociation constant of 130 microM for a low-affinity binding site. ATP protected Ca2+-ATPase against the inactivation under these conditions with a dissociation constant of 140 microM. 3. Kinetic analysis of the inactivations of Ca2+-ATPase by (SnoPP[CH2]P)2 in the absence of Ca2+ and Mg2+ but the presence of K+ and EGTA led to the appearance of two nucleotide binding sites with two different inactivation velocities. Inactivation rate constants k2 were found for the rapid inactivating part (k2' = 1.44 X 10(-2) s-1) and the slow inactivating part (k2" = 1.15 X 10(-3) s-1). From the protective effect of ATP under these conditions a high-affinity (Kd = 48.78 microM) and a low-affinity ATP binding site (Kd = 114 microM) were apparent. 4. 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. 5. (SnoPPP)2-inactivated Ca2+-ATPase was reactivated by incubation with dithiothreitol. 6. 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. 7. It is concluded that (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 sulfhydryl group within the active site.
D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004220:Disulfides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds; D013873:Thionucleotides / Q000494:pharmacology*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D013873:Thionucleotides; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D011188:Potassium; D002118:Calcium; D004229:Dithiothreitol