Solubilization and reconstitution of the adenosine 5'-triphosphate dependent noradrenaline uptake system of bovine chromaffin granule membrane.

The ATP-dependent catecholamine uptake system of chromaffin granule membrane has been solubilized and reconstituted in phospholipidic vesicles. The activity of the vesicles implies that both the ATP-dependent H+-translocase and the noradrenaline carrier have been successfully reconstituted. The membrane was solubilized by sodium cholate in presence of asolectin and the asolectin to cholate ratio appeared to be critical. Omission of asolectin resulted in reconstitution of vesicles with an active H+-pump and an inactive transport system. The detergent was removed from the solubilized membranes by filtration on Sephadex G-50 and it has been verified that the residual detergent of the reconstituted preparation was below the concentration inhibitory to the ghost H+-pump. The pH-dependence, Km for ATP and Km for noradrenaline of the reconstituted vesicles were similar to those of the ghosts, but their specific activity and reserpine-resistance were somehow variable. Vesicle activity was limited by transporter reconstitution, thus suggesting that reconstitution of the complete system might be used as an assay for the transporter. The noradrenaline carrier is not physically linked to dopamine beta-hydroxylase and bears no wheat germ agglutinin binding sites.