Title

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

Date of Award

1986

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmacology

Abstract

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.

Keywords

Chemistry, Biochemistry

Link to Full Text

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