Interaction of adenosine-5'-O-(3-thiotriphosphate) with Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum.
Interaction of adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) with Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. The nucleotide was slowly hydrolyzed by the ATPase at 30 degrees C at a rate of about 0.5% that of ATP hydrolysis. Whereas at 0 degrees C, ATP gamma S showed only a limited reactivity toward the ATPase in that a thiophosphorylated intermediate was formed and ADP was released, but hydrolysis of the intermediate to complete the catalytic cycle did not occur. A fairly stable analog of the E-P intermediate could thus be obtained. Presence of the thiophosphorylated intermediate was indicated by the [3H]ADP in equilibrium ATP gamma S exchange reaction and also by using [35S]ATP gamma S. When the ATPase was reacted with ATP gamma S at 0 degrees C in the presence of ferricyanide, EP-forming activity was rapidly lost. Free Ca2+ ions were required for this inactivation. Disulfide bond formation between a cysteinyl residue located near the substrate binding site and the enzyme-bound ATP gamma S or the thiophosphorylated intermediate was suggested by the fact that 2-mercaptoethanol reversed the inactivation. The reaction may prove to be a useful tool for affinity labeling of the active site of the ATPase.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005292:Ferricyanides / Q000494:pharmacology; D006868:Hydrolysis; D066298:In Vitro Techniques; D008623:Mercaptoethanol / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013696:Temperature; D013873:Thionucleotides / Q000378:metabolism*
D005292:Ferricyanides; D013873:Thionucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D008623:Mercaptoethanol; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; C028033:potassium ferricyanide