Studies on conformational transitions of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. II. Conformational characteristics of stabilized reaction intermediates as revealed by fluorescent and paramagnetic probes.
Various reaction intermediates of sarcoplasmic reticulum Ca2+,Mg2+-ATPase were stabilized and accumulated by modifying a specific SH group or by using nucleotide analogs. Conformational changes of the Ca2+,Mg2+-ATPase during the catalytic cycle were studied in the stabilized intermediates by the use of fluorescent and spin probes, which were introduced at specific SH groups of ATPase, namely one highly reactive but functionally nonessential (SHN) and one essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609-617]. The fluorescence intensity of N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide attached to SHD decreased by 2.5% upon addition of 10 microM AMP-P(NH)P provided that Ca2+ was also present. The AMP-P(NH)P-induced fluorescence change could also be detected by using other fluorescent probes such as N-[p-(2-benzimidazolyl)phenyl]maleimide and N-(1-anilinonaphthyl-4)maleimide. Moreover, labeling at SHN gave similar results. When SHN was labeled with N-[p-(2-benzimidazolyl)phenyl]maleimide, the fluorescence intensity also decreased by 2.5% upon addition of ATP only in the presence of Ca2+, where E-P formation took place. A conformational difference between ECa1-P X ADP and ECa1-P was suggested from saturation transfer ESR measurement of spin-labeled ATPase by using ADP beta S as an ADP analog to cause accumulation of ECa1-P X ADP beta S complex. Possible structural similarities among some of the intermediates are discussed based on these findings.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004355:Drug Stability; D004533:Egtazic Acid / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013113:Spin Labels / Q000494:pharmacology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D005456:Fluorescent Dyes; D013113:Spin Labels; D013439:Sulfhydryl Reagents; D004533:Egtazic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases