Date of Award

Spring 1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Howard D. White

Committee Member

Frank A. Lattanzio

Committee Member

Laura K. Moen

Committee Member

Paul H. Ratz

Abstract

Muscle contraction is thought to be accomplished by sliding of myosin filaments along actin filaments. Although actomyosin naturally uses ATP as an energy source, it can also use other nucleoside triphosphates (NTP) as substrates. In this work, the dependence of rate and equilibrium constants of the various steps in the muscle contraction mechanism upon nucleotide structure was investigated to unravel the dependence of the mechanical properties of the muscle upon changes in different biochemical steps of the mechanism.

The experiments measuring the dissociation of actomyosin by NTP (or mant-NTP) shows that both the rate constant of actomyosin-S1 dissociation and second order rate constant of NTP binding to acto-S1 are dependent upon NTP structure. The magnitude of some of the rate constants may vary as much as 20 fold with different nucleoside triphosphates.

The step sizes calculated from muscle fiber shortening experiments or in-vitro motility assays and pre-steady state measurements in solution give a constant value (4 ~ 6 nm), which is independent of the NTP structure (for ATP, CTP, mant-ATP and mant-CTP) and protein species (for acto-RS.HMM and acto-BV.myosin). This indicates that the geometric constraints of the myolattice and/or high concentration of actin and myosin in the fibers does not significantly affect the rate of NTP binding. The step size, as a structural parameter, is a more fundamental property of the actomyosin interaction.

The rate constant of the dissociation of nucleoside diphosphate (NDP) from actomyosin, the equilibrium constant of NDP to actomyosin (Ki), and the binding rate constant of NDP to actomyosin are all dependent upon the structure of NDP.

A large fluorescence decrease observed in actin binding to rabbit skeletal myosin-S1-mant-NDP is thought to measure the conformation change preceding mant-NDP release, the slowest step in this reaction.

The release of mant-ADP from acto-bovine cardiac myosin-S1, the rate-limiting step in this reaction, is also accompanied by fluorescence decrease. The dissociation of acto-S1-mant-NDP limited by different steps is a significant difference between skeletal and cardiac muscle contraction mechanisms.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/yeay-df39

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