Role of cellular retinaldehyde-binding protein and interphotoreceptor retinoid-binding protein in retinoid transport and metabolism in the mammalian retina

Although many aspects of the mammalian visual cycle have been extensively studied, much of our understanding of the mechanisms of retinoid transport and metabolism is still incomplete. The work presented in this dissertation sheds some light on the following aspects of the visual cycle: (1) the physicochemical properties of the native bovine and recombinant human cellular-retinaldehyde binding protein (CRALBP) and (2) the role of the interphotoreceptor retinoid-binding protein (IRBP) in the polarized release of 11-cis retinal (RAL) from cultured retinal pigment epithelium (RPE).; Circular dichroism (CD) demonstrated that native bovine and recombinant human CRALBP are structurally and physically similar. They both bind 11-cis RAL, 11-cis retinol (ROL) and 9-cis RAL, behave similarly under heat-denaturing conditions, have the same secondary structural characteristics and are composed of a mixture of {dollar}alpha{dollar}-helix and {dollar}beta{dollar}-sheet. CD also confirmed that CRALBP's binding pocket is specific for cis retinoids.; Fetal bovine RPE were grown on porous supports and their apical surfaces were incubated with medium containing either apo IRBP, apo CRALBP, apo retinol-binding protein (RBP), bovine serum albumin or no binding protein. ({dollar}sp3{dollar}H) all-trans ROL was delivered to the basal surface of the RPE by RBP either in the presence or absence of basal apo IRBP. High-performance liquid chromatography demonstrated that equivalent, low levels of ({dollar}sp3{dollar}H) 11-cis RAL were detected in both the apical and basal media when apo IRBP was absent from both compartments, and may represent a constitutive, baseline release. ({dollar}sp3{dollar}H) 11-cis RAL was optimally released into the apical medium only when apo IRBP was present apically. The decrease in apical ({dollar}sp3{dollar}H) 11-cis RAL in the presence of binding proteins other than apo IRBP, or when incubated with medium alone, was concomitant with a buildup of intracellular {9B} ({dollar}sp3{dollar}H) all-trans retinyl palmitate and basal ({dollar}sp3{dollar}H) all-trans ROL. The presence of apo IRBP in the basal medium did not enhance the level of ({dollar}sp3{dollar}H) 11-cis RAL released into this compartment. Together these results suggest that the RPE is polarized for 11-cis RAL release and that this process may require an IRBP apical membrane receptor.