Structural characterization of the ATP-hydrolyzing portion of the coated vesicle proton pump

Abstract

The ATP-hydrolyzing portion of the proton pump from clathrin-coated vesicles (isolated from calf brain) was solubilized with three nondenaturing detergents (cholate, ocytl glucoside, and Triton X-100). The hydrodynamic properties of the solubilized (Mg2+)-ATPase were then determined by sedimentation analysis in H2O and D2O and gel filtration on Sepharose 4B. The coated vesicle (Mg2+)-ATPase migrated under all conditions as a single peak of activity. In cholate, the sedimentation coefficient (s20,w), Stokes radius (a), and partial specific volume (.hivin.vc) were 8.25 (.+-. 0.20) S, 68 (.+-. 2) .ANG., and 0.71 (.+-. 0.03) cm3/g, respectively. In octyl glucoside and Triton X-100 these values were respectively 7.90 (.+-. 0.20) and 7.45 (.+-. 0.20) S, 68 (.+-. 3) and 101 (.+-. 5) .ANG., and 0.74 (.+-. 0.03) and 0.75 (.+-. 0.03) cm3/g. Application of the Svedberg equation to these data gave a molecular weight for the protein-detergent complex of 217000 .+-. 21000 (cholate), 234000 .+-. 26000 (octyl glucoside), and 337000 .+-. 40000 (Triton X-100). Assuming the protein binds one micelle of detergent, these values correspond to a protein molecular weight of 215000 .+-. 21000 (cholate), 226000 .+-. 26000 (octyl glucoside), and 247000 .+-. 40000 (Triton X-100). The cholate-solubilized, gradient-purified (Mg2+)-ATPase, when combined with a 100000 g pellet fraction, could be reconstituted by dialysis into phospholipid vesicles which displayed ATP-dependent proton uptake. The sensitivity of this acidification and the (Mg2+)-ATPase activity of the detergent-solubilized protein to 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and N-ethylmaleimide (NEM) and their resistance to ouabain are consistent with this protein complex corresponding to the ATP-hydrolyzing portion of the coated vesicle proton pump.