Removal of amino group containing phospholipids from rhodopsin: XVII. Biochemical aspects of the visual process

Abstract

Previously we showed that phospholipase C treatment and hexane extraction of cattle rhodopsin in rod outer segment membrane preparations removed 95% of the phospholipids originally present in these preparations, reducing thereby the phosphatidyl ethanolamine and phosphatidyl serine contents from 30 and 9 to 0.2 and 1.6 mole per mole rhodopsin, respectively, while leaving the pigment completely intact. Now it is shown that, upon incubation of this rhodopsin preparation with phospholipase A 2 from Crotalus adamanteus, most of the remaining phosphatidyl serine was hydrolyzed to lysophosphatidyl serine and fatty acid. Repeated extraction of this preparation with aqueous bovine serum albumin solution completely removes the lyso compound. After this procedure 64% of the rhodopsin was still spectrally intact. After removal of some “free” retinaldehyde, originating from decomposed rhodopsin, by means of endogenous retinol dehydrogenase activity and added NADPH, the resulting rhodopsin had the same absorption spectrum, molar absorbance value, and photolytic properties as native rhodopsin. Quantitative analysis of the phospholipids present in the final product yielded values of 0.10 mole per mole rhodopsin for each of the two amino group containing phospholipids phosphatidyl ethanolamine and phosphatidyl serine. These findings prove that the “chromophoric” retinaldehyde cannot be linked to either phospholipid in native cattle rhodopsin.