Glycerinated rabbit fast skeletal muscle fibers were chemically skinned with 1% Brij 35 and partially depleted of endogenous troponin C subunit (TnC) by exposure of the fibers to EDTA. The TnC-depleted fibers exhibited a decrease in maximal tension that was mostly restored by readdition of TnC or by the addition of the fluorescent 5-dimethylaminonaphthalene-1-sulfonyl aziridine analogue, TnC(Danz). TnC(Danz) is known to undergo an increase in fluorescence intensity when Ca2+ binds to the two low affinity Ca2+-specific regulatory sites of TnC. Steady-state fractional fluorescence and tension changes were measured simultaneously as a function of Ca2+. The Ca2+ sensitivity of the fluorescence curve was about 0.6 log unit greater than the tension curve. This difference in sensitivity could be explained if separate conformational states of TnC, brought about by Ca2+ binding to the Ca2+-specific sites, produce the fluorescence and tension changes. TnC-depleted fibers were also reconstituted with the fluorescent 2-[(4'-iodoacetamido)analino] naphthalene-6-sulfonic acid analogue, cardiac TnC(Iaans), which undergoes an increase in fluoresence intensity when Ca2+ binds to the single Ca2+-specific regulatory site. The steady-state fractional fluorescence and tension curves for fibers reconstituted with cardiac TnC(Iaans) had nearly the same Ca2+ sensitivity. The steady-state fractional fluorescence of myofibrils reconstituted with TnC(Danz) was found to have a greater sensitivity to Ca2+ than the simultaneously measured ATPase. In all cases paired fractional fluorescence and activity curves tended to have parallel dependence on Ca2+. These procedures make it possible to study the Ca2+ binding properties of the Ca2+-specific sites in intact myofibrils and skinned fibers; the results presented suggest that the Ca2+ affinity of the Ca2+-specific sites of troponin are reduced in the thin filament compared to that of troponin in solution.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1986|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology