Uptake and isomerization of all-trans retinol by isolated bovine retinal pigment epithelial cells: further clues to the visual cycle.

The site and substrate for all-trans to 11-cis isomerization in the visual cycle have remained obscure for several decades. Only recently studies on a subcellular level have begun to shed some light on these phenomena. We have addressed this system on a cellular level by utilizing intact isolated bovine retinal pigment epithelial cells, maintained during short-term incubation in vitro. Supplementation with labeled all-trans retinol incorporated in a lipid vesicle carrier, in a range of 1-6 nmol per 10(6) cells, resulted in a rapid uptake of retinol. The majority of the internalized retinol was processed prior to mixing with endogenous retinoid pools and most of it was converted into all-trans retinylester. Up to 10% of the incorporated label was isomerized yielding 11-cis retinol, 11-cis retinaldehyde and 11-cis retinylester. The kinetics of the 11-cis retinoid formation indicated that 11-cis retinol is the first isomerization product. The level of 11-cis retinol apparently 'triggered' further processing into other 11-cis retinoids. An updated model with discussion topics is presented for the retinoid pathway relevant to the visual cycle.

A rapid versatile microassay for cellular retinol-binding protein using Lipidex-1000 microcolumns.

A new, rapid and versatile microassay for cellular retinol-binding protein has been developed based on separation of bound and free ligand by means of Lipidex-1000, a hydrophobic Sephadex derivative. This requires quantitative manipulation of retinol in aqueous solution. The tendency of retinol to adhere to glass and plastic surfaces was overcome by addition of the detergent Ammonyx LO, which yields a micellar dispersion. Detergent concentrations up to 10 mM did not interfere with binding of retinol to Lipidex-1000 or binding protein. The binding capacity of Lipidex-1000 was found to exceed 400 nmol of retinol per ml of gel. Retinal pigment epithelium (RPE) cells were used as a source for cRBP (cellular retinol-binding protein). The binding protein is saturated with ligand by incubation for 60 min at room temperature at concentrations of free retinol over 180 nM. Separation of protein-bound retinol from free retinol is achieved via Lipidex-1000: protein-bound (specific and nonspecific) retinol is not retained and is eluted by buffer with the protein fraction. Free retinol is retained by Lipidex and is subsequently recovered by elution with methanol. Total recovery of ligand approaches 100%. Analysis time is about 4 hr for a maximum of ca. 50 samples. Nonspecific protein binding can be determined equally effectively either by incubation with 3 mM PCMBS or by addition of a 100-fold molar excess of nonlabeled retinol.(ABSTRACT TRUNCATED AT 250 WORDS)