Chronic low-frequency stimulation of rabbit fast-twitch muscle induces partial inactivation of the sarcoplasmic reticulum Ca2(+)-ATPase and changes in its tryptic cleavage.
Persistently increased contractile activity as induced by low-frequency stimulation in fast-twitch rabbit muscle elicits a partial inactivation of the sarcoplasmic reticulum Ca2(+)-ATPase function with regard to Ca2+ transport and ATP hydrolysis. Electron microscopy showed no differences in the frequency and structure of the two-dimensional Ca2(+)-ATPase crystals between microsomal fractions from normal and stimulated muscles. However, differences existed between the tryptic digestion of the Ca2(+)-ATPase in both the membrane-bound and solubilized enzyme at the first tryptic cleavage site, named T1 (Arg505). This followed from a delayed appearance of the A and B fragments of the Ca2(+)-ATPase in the electrostimulated muscle. No differences existed with regard to the second tryptic cleavage site, named T2 (Arg198). Confirming previous results, fluorescein isothiocyanate (FITC) binding to the enzyme of the chronically stimulated muscle was markedly reduced. The FITC-labeled fraction of the enzyme from both the normal and the stimulated muscle followed similar time courses of tryptic cleavage. The fraction of Ca2(+)-ATPase that did not bind TITC was identified by immunoblot analysis as the trypsin-resistant form. In view of the vicinity of T1, the FITC- and the ATP-binding sties, these results point to a modification of the enzyme in that region leading to an inactivation of about 50% of the sarcoplasmic reticulum Ca2(+)-ATPase molecules.
Dux
L
L
Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Green
H J
HJ
Pette
D
D
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004558:Electric Stimulation; D004789:Enzyme Activation; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D008297:Male; D009119:Muscle Contraction; D009132:Muscles / Q000201:enzymology* / Q000648:ultrastructure; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D013861:Thiocyanates / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
D005452:Fluoresceins; D013861:Thiocyanates; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium