Nathan L. Mata

Substrate specificities and 13-cis-retinoic acid inhibition of human, mouse and bovine cis-retinol dehydrogenases

Recent studies of the human, mouse and bovine genes for 11-cis-retinol dehydrogenase (11cRDH) and human and mouse 9-cis-retinol dehydrogenase (9cRDH) suggest that they are homologs of the same enzyme. This conclusion is inconsistent with earlier literature indicating that 11cRDH is expressed solely in the eye and does not utilize 9-cis-retinol as a substrate. We have compared directly the kinetic properties of recombinant human and mouse 9cRDH with those of bovine 11cRDH for 9-cis- and 11-cis-retinol and investigated the inhibitory properties of 13-cis-retinoic acid on each of these enzymes. Human and mouse 9cRDH and bovine 11cRDH have very similar kinetic properties towards 9-cis- and 11-cis-retinol oxidation and they respond identically to 13-cis-retinoic acid inhibition. Our biochemical data are consistent with the conclusion that 9cRDH and 11cRDH are the same enzyme.

Distribution of 11-cis LRAT, 11-cis RD and 11-cis REH in bovine retinal pigment epithelium membranes

Our recent finding of the co-localization of 11-cis retinyl esters and 11-cis retinyl ester hydrolase (11-cis REH) activity in bovine retinal pigment epithelium (RPE) plasma membrane (PM) has led us to explore the possibility that the PM may provide 11-cis retinal for rhodopsin regeneration. In the RPE, visual chromophore is synthesized via a membrane associated 11-cis retinol dehydrogenase (11-cis RD). Accordingly, bovine RPE membranes enriched with either endoplasmic reticulum (ER) or plasma membrane (PM) enzyme markers were prepared and assayed for visual cycle enzyme activities. Pronounced 11-cis RD activity was associated with both ER- and PM-enriched membrane fractions. In contrast, 11-cis REH activity was mostly recovered in PM-enriched fractions while LRAT activity was found only in ER-enriched membranes. The finding that both 11-cis retinol and 11-cis retinal can be produced at the PM of the bovine RPE strongly suggests that 11-cis retinyl esters at this subcellular locale serve as a precursor of visual chromophore for pigment regeneration.

Identification of 11-cis-retinal and demonstration of its light-induced isomerization in the chicken pineal gland

Direct evidence is not available that (1) rhodopsin-like photopigment exists in the chicken pineal and that (2) the visual pigment is responsible for the light sensitivity of the gland. Therefore, the objective of this study was to test for the existence of visual pigment in the chicken pineal by means of the identification of 11-cis-retinal in this organ. 11-cis- and all-trans retinoids were extracted from light- and dark-adapted chicken pineals and analyzed by high performance liquid chromatography (HPLC) using the formaldehyde method. 11-cis-Retinal was initially identified by coelution with an authentic standard. Further characterization was carried out by collecting the retinal from the HPLC eluant, subjecting it to reduction by sodium borohydride and then identifying the derived 11-cis-retinol using HPLC. Proportions of 11-cis-retinal to total pineal retinals were also studied from decapitated heads after light and dark adaptation. Analyses of dark-adapted, pooled chicken pineals revealed equal proportions of 11-cis and all-trans retinals at 2 h after dark and at night. Two hours of light adaptation resulted in the reduction of the 11-cis proportion (from 50%) to 26% of total retinals. These observations prove that 11-cis-retinal exists in the chicken pineal and that it undergoes light-induced cis to trans isomerization in a manner similar to the visual pigment chromophores in the vertebrate retina.

Substrate specificities of retinyl ester hydrolases in retinal pigment epithelium.

Publisher Summary Previous studies have shown that retinyl esters are located in the retinal pigment epithelium (RPE) of many vertebrate species from fishes, amphibians, reptilians, and birds to mammals including humans. To investigate the role of retinyl esters in the visual process, a simple hypothesis is porposed that in the RPE, there exists a substrate-specific retinyl ester hydrolase relevant to the visual cycle. This chapter presents biochemical methods employed in laboratory to investigate retinyl ester hydrolysis in the RPE, with an emphasis on substrate specificity. On the basis of results reported in the literature and on those from the investigation, the location and role of 11-cis-REH seem apparent. Starting from the branch point where 11-cis-retinyl esters are synthesized at the ER, they are somehow relocated to the plasma membrane, where they are stored. Hydrolysis of this pool of retinyl esters is carried out by a substrate-specific 11-cis-retinyl ester hydrolase and the product of the reaction, 11-cis- retinol, is then oxidized by the 11-cis-RD at the PM.