Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane |
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Authors: | M.P. Roisin J.P. Henry |
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Affiliation: | Service de Biochimie-Physique, Institut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, 75005 Paris France |
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Abstract: | Ghosts derived from bovine chromaffin granules have a 32Pi-ATP exchange activity which is associated with the H+ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (Km for ATP and Pi, ATP/Mg2+ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The 32Pi-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a 32Pi-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The α- and β-subunits of F1-ATPase were major components of the preparation. |
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Keywords: | Proton pump ATPase (Bovine adrenal gland) CCCP DCCD |
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