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) Ca2+-ATPase in low-frequency-stimulated fast-twitch muscle were investigated in the present study. The specific Ca2+-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 P(i) 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, P(i)) intrinsic tryptophan fluorescence. A decreased binding of fluorescein isothiocyanate (FITC) corresponded to the lower ATP binding and phosphorylation of the enzyme. Moreover, P(i)-induced changes in fluorescence of the FITC-labelled enzyme did not differ between SR from stimulated and contralateral muscles, indicating that Ca2+-ATPase molecules which did not bind FITC were responsible for the decreased P(i)-dependent phosphorylation, and therefore represented the inactive form of the enzyme. No differences existed between the Ca2+-induced changes in the intrinsic fluorescence of SR from stimulated and contralateral muscles which fit their similar Ca2+-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.