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Title: Kinetics and regulation of the chromaffin granule proton pump
Author: Webster, Lorna Christina
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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The ATPase from bovine adrenal secretory granules (chromaffin granules) was purified and reconstituted into proteoliposomes. The kinetics of ATP-dependent proton translocation were studied and a fluorimetric assay to allow accurate estimations of initial rates was developed. Rate analysis showed that the ATPase is allosterically regulated by MgADP: modelling studies predicted that it exists in two conformational states, with different activities and affinities for MgATP. Both conformations states have three binding sites for MgATP which bind MgADp competitively. MgADP also binds to a single regulatory site on the inactive conformational state. Other nucleotide diphosphates show the same properties as MgADP but have lower affinities. It was shown that the covalently-reacting inhibitor N-ethylmaleimide (NEM) inactivates the ATPase in a pseudo-first order reaction. This inactivation was inhibited by the presence of micromolar concentrations of MgADP and other nucleotide diphosphates in a way that correlated with their regulatory features in proton translocation. The kinetics of protection were consistent with the two states of the enzyme being inactivated at different rates and also confirmed the existence of different types of binding site for MgADP. The three 72kDa (catalytic) subunits had the same rate constant for alkylation by NEM, suggesting that the enzyme is symmetrical. Direct binding analysis confirmed the results of both the kinetics of proton translocation and protection from NEM. MgADP was shown to bind to the enzyme with a submicromolar dissociation constant and with a stoichiometry confirming the existence of a single regulatory site. Overall the results suggest that MgADP and other nucleotide diphosphates may be important physiological regulators of this enzyme.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available