Fractionation of rhodopsin and other components in the rod outer segment membrane by ammonium sulfate salting-out

Abstract

Purified bovine rod outer segment membrane was solubilized in a mixture of 1.5% cholic acid/20% saturated ammonium sulfate and 0.05 M phosphate buffer (pH 7.5). The solubilized rod outer segment membrane was fractionated with ammonium sulfate and 70--90% of rhodopsin (A278/A498= 1.6--1.9) was recovered in the fraction of 50 to 60% saturation with ammonium sulfate, giving a highly concentrated solution of purified rhodopsin (A1CM 498 = 83). By the method of ammonium sulfate salting-out, the solubilized rod outer segment membrane was divided into several fractions without a loss of components. The components in each fraction were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rhodopsin and opsin amounted to 93% of the total protein in the membrane. Other proteins with molecular weights of 46 000, 52 000, 56 000, 70 000, 95 000, 105 000, 130 000 and 270 000 were also detected. Most of phospholipids in the rod outer segment membrane remained in the supernatant above 60% saturation with ammonium sulfate.