Structure and function of the bovine chromaffin granule proton pump

• Main Author: Perez-Castineira, Jose Roman
• Published: University of Edinburgh 1991
• Subjects: 611
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.660537

The secretory granules of the adrenal medulla, known as 'chromaffin granules', store catecholamines, proteins and nucleotides that afterwards will be secreted by exocytosis. The accumulation of the catecholamines within the granules occurs by a chemiosmotic mechanism involving a membrane-bound H<SUP>+</SUP>-translocating ATPase, coupled to a separate H<SUP>+</SUP>/catecholamine exchanger. This ATP belongs to a recently identified group of proteins called 'vacuolar' of V-type ATPases, that are distinct from the P-type H<SUP>+</SUP>-ATPases of fungalplasma membranes and F-type H<SUP>+4-ATPases of energy-transducing membranes. The aim of this thesis is the structural and functional characterization of the bovine chromaffin granule ATPase. First, a new and rapid method of purification and reconstitution of the enzyme was developed. The method can be accomplished in less than two hours, yielding a partially purified form of the enzyme incorporated into liposomes of defined composition. This permitted the measurement of the H</SUP>^+ -translocation activity of the enzyme by use of the fluorescent probe ACMA. The effect of lipid composition on ATP hydrolysis and H^+-translocationactivities was studied. Kinetic and regulatory studies were carried out on the reconstituted enzyme. MgATP, MgdATP, MgGTP and Mg/TP were all shown to be substrates for the enzyme and Mn^2+, Ca^2+, Co^2+ and Ni^2+ could substitute for Mg^2+. Kinetic parameters were calculated for all these cases. The inhibitory effects of ADP, GDP and IDP were also studied. The regulatory properties were consistent with the existence of several substrate and inhibitor binding sites and the kinetic data could be fitted to a model involving positive homotropic and negative heterotropic effects. Structural studies of the purified enzyme were performed. It was shown that one of the subunits (116 kDa) was a glycoprotein. A hydrophobic probe specifically labelled two subunits (116 and 16 kDa) suggesting that they form the part of the enzymic complex embedded in the granule membrane. The stoichiometry of the complex was calculated from quantitative aminoacid analysis of the subunits separated by electrophoresis in polyacrylamide gels. In order to do this a purer form of the enzyme was obtained by centrifugation through glycerol density gradients. This separated the active enzyme from a complex formed by polypeptides of molecular weights 116, 40, 19 and 16 kDa, suggesting that the enzyme contains two separable domains.