The enzymatic cleavage of rhodopsin by the retinal pigment epithelium. I. Enzyme preparation, properties and kinetics: characterization of the glycopeptide product.

An enzyme system has been detected in the bovine retinal pigmented epithelium (RPE) which carries out the degradation of rhodopsin. The substrate for these studies was bovine rhodopsin labeled by in vitro techniques with [3H]-glucosamine or [3H]-mannose, the former being the derivative most extensively examined. Labeled rhodopsin was purified by adsorption and affinity chromatography followed by preparative isoelectric focusing. Cell-free preparations from bovine RPE cleaved rhodopsin to a single glycopeptide. The pH optimum for the reaction was about 3.5. The apparent Km for rhodopsin was about 5 microns. Rhodopsin was cleaved about 55% under saturating conditions, the limited extent of reaction due primarily to its partial inactivation for use as a substrate during the course of the reaction. Inhibition by cleavage products was not observed. Among several tissues which were examined, the RPE was the most active in rhodopsin-cleaving activity. Although stable as a crude extract, the activity of the rhodopsin-cleaving enzyme, as well as the bovine serum albumin (BSA)-cleaving enzyme (cathepsin-D) were rapidly lost upon purification by DEAE-Sephacel and pepstatin-Sepharose. Differences in relative specific activity among tissues as well as differential patterns of purification suggest that the rhodopsin-cleaving enzyme and the BSA-cleaving enzyme of the RPE may not be the same. Using N-retinylopsin as a substrate for the rhodopsin-cleaving enzyme, it was shown that the regions of the cleavage products containing vitamin A were not associated with the glycopeptide. Some of the properties of the product were examined. Its response to ion exchange resins, susceptibility to further cleavage by pronase, amino acid analysis, solubility properties, isoelectric point, and binding by concanavalin A (conA)-Sepharose, were consistent with the product being described as an acidic glycopeptide. The glycopeptide was reactive with the antibody prepared against bovine rhodopsin, and retained the full antigenicity of the intact molecule. This enzyme system may be one of the means whereby the RPE participates in maintaining the physiological concentration of rhodopsin which is synthesized in the retina and catabolized in the RPE.