STUDIES ON THE DISTRIBUTION AND METABOLISM OF STEROLS AND OTHER ISOPRENOIDS IN THE BOVINE RETINA
FLIESLER, STEVEN JAY
Doctor of Philosophy
The sterol composition of bovine retinas and rod outer segment (ROS) membranes was examined. Cholesterol accounted for >98% of the total sterol and (TURN)2% of the dry weight of both the retina and ROS membranes. Minor amounts of components having the chromatographic properties of 5(alpha)-cholestan-3(beta)-ol and 5(alpha)-cholest-7-en-3(beta)-ol were detected in whole retinas, and a "cholestanol-like" component was also detected, in minor amounts, in ROS membranes. Calculations indicated that the molar ratio of cholesterol:rhodopsin in ROS membranes was (TURN)5. Using literature values for the phospholipid content of bovine ROS membranes, the calculated cholesterol:phospholipid molar ratio was (TURN)0.06 and cholesterol only represented (TURN)5-7 mole % of the total ROS membrane lipid. The metabolism of ('3)H-labelled mevalonic acid was studied in vitro with "intact" retinas and with the 10,000 x g supernatant (S(,10)) fraction. The major nonsaponifiable products obtained from incubations of "intact" retinas had the chromatographic properties of squalene and lanosterol; only minor amounts of label were incorporated into material having the chromatographic properties of C(,27) monohydroxy sterols. The retinas also converted mevalonic acid to isoprenoid acids; no label was incorporated into retina n-fatty acids. In the incubations with the S(,10) fraction, <1% of the label incorporated into nonsaponifiable lipids was precipitable with digitonin. The major nonsaponifiable lipids had the chromatographic properties of squalene and various open-chain isoprenoid alcohols, but did not correspond to the alcohols of the allyl pyrophosphates which are known intermediates in the biosynthesis of squalene. Material having the chromatographic properites of C(,30) and C(,27) monohydroxy sterols was detected in small amounts in these incubations, but cholesterol represented only a minor fraction of this material. Isoprenoid acids (primarily C(,20) isomers, with lesser amounts of C(,15) isomers) were formed as the major products of these incubations. In addition to material having the properties of the all-trans isomers of farnesoic acid and geranylgeranoic acid, the biosynthesis of compounds having the chromatographic properites of the cis,cis- and cis,trans- (or trans,cis-) isomers of farnesoic acid was noted. Also, relatively large amounts of a C(,20) isomer of geranylgeranoic acid (apparently having at least one cis double bond) were recovered from such incubations. These isoprenoid acids were not detected as endogenous components of the retina. In an in vivo experiment, intraocularly-injected ('14)C-labelled mevalonic acid was taken up and metabolized to nonsaponifiable lipids by several ocular tissues (predominantly by the retina) over a period of up to 2 hours. In each tissue, the major labelled nonsaponifiable lipid was squalene, with lesser amounts of material having the chromatographic properties of lanosterol. Only a few per cent of the retina nonsaponifiable products behaved chromatographically like C(,27) monohydroxy sterols. It was inferred from these results that the bovine retina has a very limited capacity for de novo sterol biosynthesis and must rely on alternate sources of cholesterol for the biogenesis of ROS membranes.