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.

Chronic low‐frequency stimulation elicits in rabbit fast‐twitch muscle a partial inactivation of the sarcoplasmic reticulum (SR) Ca2+‐ATPase and Ca2+‐uptake activities. Inactive Ca2+‐ATPase was enriched in a light microsomal fraction by sucrose density gradient centrifugation after calcium oxalate loading in the presence of ATP. This fraction showed a reduced specific activity and phosphoprotein formation of the Ca2+‐transport ATPase. These results suggest that the inactivation of the Ca2+‐ATPase as induced by increased contractile activity, is confined to a specific SR vesicle population.

Changes in the contractile properties and phosphorylation of myosin light chain 2 (LC2) in extensor digitorum longus (EDL) muscles of rats were studied for the 7-day period following denervation. Post-tetanic potentiation (PTP) of isometric twitch tension, a property of fast-twitch muscles, was recorded 10 sec after a l-sec tetanic stimulation (200 Hz) and the extent of LC2 phosphorylation was de-

The precise localization of a membrane-bound, arginine-specific mono-ADP-ribosyltransferase (mADP-RT) was assessed in rabbit skeletal muscle by studying membrane fractions isolated by successive sucrose density gradient centrifugations. mADP-RT activity was IO-fold enriched in sarcolemma1 and T-tubular membranes. The catalytic activity, determined in preparations with mainly right-side-out vesicles, was found to be on the cytoplasmic face. As revealed by SDS-PAGE and autoradiography endogenous mADP-RT activity labeled several proteins in the range between 15 kDa and 250 kDa. T-tubules contained the highest number of ["P]ADP-ribose-labeled proteins.

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