Reduction of disulfide bonds in sarcoplasmic reticulum Ca(2+)-ATPase by dithiothreitol causes inhibition of phosphoenzyme isomerization in catalytic cycle. This reduction requires binding of both purine nucleotide and Ca2+ to enzyme.
Sarcoplasmic reticulum vesicles were treated with 6 mM dithiothreitol in the presence of 2 mM ATP and 0.1 mM Ca2+ at 25 degrees C and pH 7.0 for various periods. The Ca(2+)-ATPase was inhibited almost completely in 100 min. The content of the sulfhydryl group, which was measured in 2.5% SDS with 5,5'-dithiobis(2-nitrobenzoic acid), increased during the treatment. Extrapolation of the plot of the Ca(2+)-ATPase activity versus the increment of the sulfhydryl group showed that reduction of two disulfide bonds per phosphorylation site leads to a complete inhibition of the enzyme. This reduction required binding of both Ca2+ (with a high affinity) and a purine nucleotide to the enzyme. Adenosine 5'-(beta,gamma-methylene) triphosphate (a nonhydrolyzable ATP analog) was also effective for the reduction, while phosphorylation of the enzyme with acetyl phosphate or P(i) did not trigger the reduction. These results indicate that formation of a substrate-enzyme-calcium complex is responsible for the dithiothreitol-induced disulfide bond reduction. The partial reactions of the Ca(2+)-ATPase were examined with the vesicles that had been treated with dithiothreitol in the presence of ATP and Ca2+ for 100-200 min. Neither isomerization of the unphosphorylated enzyme from the low Ca2+ affinity form to the high Ca2+ affinity form nor phosphorylation of the enzyme with ATP or P(i) was inhibited. In contrast, isomerization of the phosphoenzyme intermediate from the ADP-sensitive form to the ADP-insensitive form was strongly inhibited. These results show that the observed inhibition of the Ca(2+)-ATPase is due to a selective blockage of the phosphoenzyme isomerization.
Daiho
T
T
Department of Biochemistry, Asahikawa Medical College, Japan.
Kanazawa
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D007536:Isomerism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D011817:Rabbits; D012265:Ribonucleotides / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D004220:Disulfides; D010750:Phosphoproteins; D012265:Ribonucleotides; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004229:Dithiothreitol