CONTROL OF SUBSTRATE FLOW AT A BRANCH IN THE VISUAL CYCLE

Photoisomerization of rhodopsin's chromophore, 11-cis-retinaldehyde, a nd subsequent regeneration of the 11-cis configuration are accomplishe d in vertebrates by a series of reactions known as the visual cycle. A t one point in the cycle, 11-cis-retinol can either be enzymatically o xidized to 11-cis-retinaldehyde and exported for visual pigment regene ration or be enzymatically esterified and stored. Partition of substra te at this branch was examined in this study and found to be influence d by cellular retinaldehyde-binding protein (CRALBP), a retinoid-bindi ng protein found in retina. Esterification was reduced to about 10% an d oxidation stimulated 2-3-fold in the presence of this protein. Other experiments confirmed that ''free'' 11-cis-retinol was esterified mor e rapidly than 11-cis-retinol complexed with CRALBP and that CRALBP 11 -cis-retinol was not an inhibitor of the esterification. Following oxi dation of CRALBP 11-cis-retinol, the reaction product, 11-cis-retinald ehyde, was found associated with the binding protein. 11-cis-Retinalde hyde is not available for reaction with carbonyl reagents when the ret inoid is bound to CRALBP. However, enzymatic oxidation of CRALBP.11-ci s-retinol in the presence of O-ethylhydroxylamine produced ca. 30% ret inaldehyde 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 com petition for substrate between two important visual cycle enzymes, and suggest that CRALBP may act as a substrate-routing protein in vivo. T he mechanism for the opposite effect of CRALBP on oxidation and esteri fication is not understood.