Vacuolar H+-ATPase of adrenal secretory granules. Rapid partial purification and reconstitution into proteoliposomes

A procedure has been developed for the rapid purification and reconstitution into phospholipid vesicles of the proton-translocating ATPase of bovine adrenal chromaffin-granule membranes. It involves fractionation of the membranes with Triton X-114, resolubilization of the ATPase with n-octyl glucoside, addition of purified lipids and removal of detergent by gel filtration. The entire process can be completed within 2 h. H+ translocation was detected by the ATP-dependent quenching of the fluorescence of a permeant weak base. The effect of varying the lipid composition of the vesicles on ATP hydrolysis and H+ translocation by the reconstituted enzyme was examined. ATPase activity was maximally increased about 4-fold by added lipid, but was relatively insensitive to its composition, whereas vesicle acidification was absolutely dependent on the addition of phospholipids and cholesterol.