Inactivation of sarcoplasmic-reticulum Ca(2+)-ATPase in low-frequency-stimulated muscle results from a modification of the active site.
Molecular changes underlying the partial inactivation of the sarcoplasmic-reticulum (SR) Ca(2+-) ATPase in low-frequency-stimulated fast-twitch muscle were investigated in the present study. The specific Ca(2+)-ATPase activity, as well as the ATP- and acetyl phosphate-driven Ca2+ uptakes by the SR, were reduced by approx. 30% in 4-day-stimulated muscle. Phosphoprotein formation of the enzyme in the presence of ATP or Pi was also decreased to the same extent. Measurements of ATP binding revealed a 30% decrease in binding to the enzyme. These changes were accompanied by similar decreases in the ligand-induced (ATP, ADP, Pi) intrinsic tryptophan fluorescence. A decreased binding of fluorescein isothiocyanate (FITC) corresponded to the lower ATP binding and phosphorylation of the enzyme. Moreover, Pi-induced changes in fluorescence of the FITC-labelled enzyme did not differ between SR from stimulated and contralateral muscles, indicating that Ca(2+)- ATPase molecules which did not bind FITC were responsible for the decreased Pi-dependent phosphorylation, and therefore represented the inactive form of the enzyme. No differences existed between the Ca(2+)-induced changes in the intrinsic fluorescence of SR from stimulated and contralateral muscles which fit their similar Ca(2+)-binding characteristics. Taking the proposed architecture of the Ca2(+)-ATPase into consideration, our results suggest that the inactivation relates to a circumscribed structural alteration of the enzyme in sections of the active site consisting of the nucleotide-binding and phosphorylation domains.
Matsushita
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Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Pette
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent; D004558:Electric Stimulation; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009132:Muscles / Q000201:enzymology* / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D002413:Cations, Divalent; D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium