Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers.
Intrinsic troponin C (TnC) was extracted from small bundles of rabbit psoas fibers and replaced with TnC labeled with dansylaziridine (5-dimethylaminonaphthalene-1-sulfonyl). The flourescence of incorporated dansylaziridine-labeled TnC was enhanced by the binding of Ca2+ to the Ca2+-specific (regulatory) sites of TnC and was measured simultaneously with force (Zot, H.G., Güth, K., and Potter, J.D. (1986) J. Biol. Chem. 261, 15883-15890). Various myosin cross-bridge states also altered the fluorescence of dansylaziridine-labeled TnC in the filament, with cycling cross-bridges having a greater effect than rigor cross-bridges; and in both cases, there was an additional effect of Ca2+. The paired fluorescence and tension data were used to calculate the apparent Ca2+ affinity of the regulatory sites in the thin filament and were shown to increase at least 10-fold during muscle activation presumably due to the interaction of cycling cross-bridges with the thin filament. The cross-bridge state responsible for this enhanced Ca2+ affinity was shown to be the myosin-ADP state present only when cross-bridges are cycling. The steepness of the pCa force curves (where pCa represents the -log of the free Ca2+ concentration) obtained in the presence of ATP at short and long sarcomere lengths was the same, suggesting that cooperative interactions between adjacent troponin-tropomyosin units may spread along much of the actin filament when cross-bridges are attached to it. In contrast to the cycling cross-bridges, rigor bridges only increased the Ca2+ affinity of the regulatory sites 2-fold. Taken together, the results presented here indicate a strong coupling between the Ca2+ regulatory sites and cross-bridge interactions with the thin filament.
Güth
K
K
Department of Pharmacology, University of Miami School of Medicine, Florida 33101.
Potter
J D
JD
D000818:Animals; D001388:Aziridines / Q000494:pharmacology*; D001389:Azirines / Q000494:pharmacology*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003619:Dansyl Compounds / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D009119:Muscle Contraction / Q000187:drug effects; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D001388:Aziridines; D001389:Azirines; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D014336:Troponin; D019209:Troponin C; C017173:dansylaziridine; D002118:Calcium