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The mechanism of the scallop myosin ATPase.

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thesis
posted on 19.11.2015, 09:08 by Andrew Paul. Jackson
Molluscan adductor muscles display thick filament regulation. A calcium binding site (regulatory domain) near the neck of the myosin molecule is responsible for controlling the ATPase activity, hence the rate of contraction. In the absence of calcium, the ATPase activity is highly suppressed. When calcium binds to the regulatory domain the inhibition is relieved allowing contraction to occur. Steady-state measurements are insufficient to characterise the ATPase activity in detail because of the dominant contribution from unregulated myosin molecules. Therefore spectoscopic techniques, allied to transient kinetic analysis were used to determine the effect of calcium on the various steps of the HMM ATPase mechanism. ATP, ADP and calcium binding to HMM caused small, but measurable, enhancements (upto 8%) in the proteins tryptophan fluorescence. Stopped-flow fluorescence spectroscopy allowed the kinetics of binding and dissociation to be elucidated. Calcium bound to, and dissociated from, HMM rapidly (108 M-1 S-1 and 25 S-1 respectively). When calcium was bound to the regulatory domain the affinity of the active site for nucleotide was reduced, an effect seen as an increase in the rate constant for nucleotide dissociation. Fluorescent ATP analogues, based on formycin were synthesised. These nucleotides displayed large fluorescence enhancements on binding to HMM (upto 500%). Turnover of FTP by HMM was suppressed 100-fold by the removal of calcium, as determined by transient kinetic measurements. The large fluorescence enhancements seen on binding of various formycin nucleotides allowd the effect of calcium on the association and dissociation processes to be examined in great detail. Binding was found to be a complex, multistep process in which the presence of calcium increased the rate of interconversion of the various HMM/nucleotide complexes by several orders of magnitude.

History

Date of award

01/01/1988

Author affiliation

Biochemistry

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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