INTERACTIONS WITH RETINOL AND RETINOIDS OF BOVINE CELLULAR RETINOL-BINDING PROTEIN

The interactions with retinol and retinol analogs of bovine cellular r etinol-binding protein (CRBP) have been investigated, by means of fluo rescence titrations, to obtain more information on the structural feat ures of retinoids that may be required for their interaction with the binding protein. An approximately stoichiometric binding of retinol to bovine CRBP (K'(d) similar to 2 nM) has been found in direct binding assays. Although retinal exhibited relatively high binding affinity to bovine CRBP (K'(d) similar to 30 nM), a large excess of the retinoid could not compete with retinol for the carrier protein. On the assumpt ion that retinol and retinal interact with the same binding site, this result indicates that the above-mentioned apparent dissociation const ant for retinol . CRBP may be an overestimate and that its value may b e as low as 0.1 nM. The finding of an exceedingly tight binding of ret inol to CRBP provides further support for the possible role of CRBP-bo und retinol, rather than its uncomplexed labile form, as substrate of enzymes involved in the metabolism of the vitamin. The results of thes e and previous studies indicate that CRBP is particularly sensitive to modifications of the retinol hydroxyl end group. Axerophthene, a reti nol analog bearing a hydrogen atom in place of the hydroxyl end group, and beta-ionone exhibit rather low binding affinities for CRBP (K'(d) similar to 0.2 mu M and similar to 4 mu M, respectively), suggesting that the hydroxyl group and isoprene tail moieties contribute substant ially to the retinol-binding affinity and specificity. These findings are consistent with the indications emerging from the three-dimensiona l structure determination of retinol . CRBP [Cowan, S. W., Newcomer, M . E. and Jones, T. A. (1993) J. Mel. Biol. 230, 1225-1246]. Additional ly, the bulky end groups of fenretinide and N-ethyl retinamide replaci ng the retinol hydroxyl group have been found to prevent retinoid bind ing to CRBP. The primary structure of bovine CRBP has been determined and is highly similar to the structures of both human and rat CRBP (97 % and 95% identical, respectively).