DIFFERENTIAL INTERACTION OF LECITHIN RETINOL ACYLTRANSFERASE WITH CELLULAR RETINOL BINDING-PROTEINS

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in K(d) values (0.63 and 0.44-mu-M, respectively) similar to bose obtained for esterification of retinol-CRBP (0.20 and 0.78-mu-M, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32-mu-M, respectively). Because K(d) values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10(-10) M, these similar K(m) values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a K(I) (0.21-mu-M) lower than the K(m) for CRBP-retinol (0.78-mu-M). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP (II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol. Thus, LRAT was able to discriminate between the apo and holo forms of the two binding proteins. Ligand binding altered the physical properties of both CRBP and CRBP(II), as demonstrated by changes in elution position during size-exclusion and ion-exchange chromatography, perhaps providing the basis for differential recognition by LRAT. Previous work has shown retinoid binding proteins can restrict the metabolism of the bound retinoid to certain enzymes; here, specific interaction of a particular enzyme with apo and holo binding proteins modulated the extent of that metabolism.