The Relationship Between Calcium Binding To Troponin C And Thin Filament Regulation Of Muscle Contraction (isometric Tension, Myofibrillar Atpase)

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Degree Name

Doctor of Philosophy (Ph.D.)




Endogenous troponin C (TnC) was extracted from rabbit fast skeletal muscle myofibrils and intact fibers by incubation with EDTA. Ca('2+)-induced myofibrillar ATPase and tension in fibers were reduced after extraction with EDTA but subsequent incubation with exogenous TnC restored Ca('2+) dependent activity to both preparations. Metal occupancy of the C-terminal Ca('2+)-Mg('2+) sites prevented the extraction of TnC consistent with previous suggestions that these sites function structurally. A fluorescent analogue of TnC (TnC(,DANZ)) which increases fluorescence intensity when Ca('2+) binds to the N-terminal Ca('2+)-specific sites was incorporated into TnC depleted fibers and myofibrils. Fluorescence and tension signals were recorded simultaneously from TnC(,DANZ) reconstituted fibers. The steady-state Ca('2+) sensitivity of the fluorescence change was found to be greater than the tension change. A similar relationship was found for simultaneously measured fluorescence and myofibrillar ATPase responses. Direct binding measurements demonstrated that Ca('2+) binding to either of the two Ca('2+)-specific sites produce the TnC(,DANZ) fluorescence change. The binding of Ca('2+) to the Ca('2+)-specific sites which was inferred from the fluorescence change was consistently greater than the resulting activity change. These results demonstrate that the change in activity correlates with Ca('2+) bound at more than one Ca('23+)-specific site. Models for multiple binding within individual TnC molecules and among TnC molecules along the thin filament are shown to be consistent with the data.


Chemistry, Biochemistry

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