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Investigative Ophthalmology & Visual Science, Vol 36, 1988-1996, Copyright © 1995 by Association for Research in Vision and Ophthalmology
ARTICLES AND REPORTS |
CA Driessen, BP Janssen, HJ Winkens, AH van Vugt, TL de Leeuw and JJ Janssen
Institute of Ophthalmology, University of Nijmegen, The Netherlands.
PURPOSE. Identification of a 32-kd protein in the bovine retinal pigment epithelium. METHODS. A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. RESULTS. The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21- C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus- infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity. CONCLUSIONS. Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.
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