Driessen C A, Janssen B P, Winkens H J, van Vugt A H, de Leeuw T L, Janssen J J
Institute of Ophthalmology, University of Nijmegen, The Netherlands.
Invest Ophthalmol Vis Sci. 1995 Sep;36(10):1988-96.
Identification of a 32-kd protein in the bovine retinal pigment epithelium.
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.
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.
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.
鉴定牛视网膜色素上皮细胞中的一种32 kd蛋白。
构建噬菌体λZAP Express牛视网膜色素上皮细胞cDNA文库。使用一种名为21-C3/AV的单克隆抗体分离编码21-C3/AV抗原的cDNA。对一个名为21-C3RDH/CD的阳性全长克隆进行测序。采用Northern印迹分析确定mRNA的长度和组织表达模式。利用克隆21-C3RDH/CD的完整开放阅读框分离重组杆状病毒克隆，并在草地贪夜蛾昆虫细胞中表达。研究其对11-顺式视黄醛的酶活性。
获得了21-C3RDH/CD的完整核苷酸序列。推导的氨基酸序列显示与短链醇脱氢酶具有同源性。Northern印迹分析检测到一个1.2 kb的转录本。尽管用于分离21-C3RDH/CD的单克隆抗体也与其他眼组织和非眼组织发生反应，但作者未能证明其与从不同（非）眼组织分离的RNA样本有任何反应性。重组杆状病毒感染的昆虫细胞合成了21-C3/AV抗原。该蛋白显示出11-顺式视黄醇脱氢酶活性。
与人类D-β-羟丁酸脱氢酶前体和其他醇脱氢酶的同源性表明，21-C3RDH/CD编码一种短链醇脱氢酶。此外，抗原的组织特异性和分子量表明，21-C3RDH/CD编码牛视网膜色素上皮11-顺式视黄醇脱氢酶。直接证据来自我们使用基于杆状病毒的表达系统体外合成21-C3RDH/CD编码的蛋白的实验。发现从重组杆状病毒感染的昆虫细胞中获得的蛋白提取物能够还原11-顺式视黄醛。
