CONTROL OF SUBSTRATE FLOW AT A BRANCH IN THE VISUAL CYCLE

Photoisomerization of rhodopsin's chromophore, 11-cis-retinaldehyde, and subsequent regeneration of the 11-cis configuration are accomplished in vertebrates by a series of reactions known as the visual cycle. At one point in the cycle, 11-cis-retinol can either be enzymatically oxidized to 11-cis-retinaldehyde and exported for visual pigment regeneration or be enzymatically esterified and stored. Partition of substrate at this branch was examined in this study and found to be influenced by cellular retinaldehyde-binding protein (CRALBP), a retinoid-binding protein found in retina. Esterification was reduced to about 10% and oxidation stimulated 2-3-fold in the presence of this protein. Other experiments confirmed that ''free'' 11-cis-retinol was esterified more rapidly than 11-cis-retinol complexed with CRALBP and that CRALBP 11-cis-retinol was not an inhibitor of the esterification. Following oxidation of CRALBP 11-cis-retinol, the reaction product, 11-cis-retinaldehyde, was found associated with the binding protein. 11-cis-Retinaldehyde is not available for reaction with carbonyl reagents when the retinoid is bound to CRALBP. However, enzymatic oxidation of CRALBP.11-cis-retinol in the presence of O-ethylhydroxylamine produced ca. 30% retinaldehyde O-ethyloxime and 70% free 11-cis-retinaldehyde, suggesting that about one-third of the retinol oxidized had dissociated from the binding protein. Neither oxidation nor esterification of CRALBP.11-cis-retinol was inhibited by including CRALBP 11-cis-retinaldehyde in the reaction mixture. The results indicate that CRALBP influences the competition for substrate between two important visual cycle enzymes, and suggest that CRALBP may act as a substrate-routing protein in vivo. The mechanism for the opposite effect of CRALBP on oxidation and esterification is not understood.