Howell D N, Valnickova Z, Oury T D, Miller S E, Sanfilippo F P, Enghild J J
Department of Pathology, Veterans Affairs Medical Center, Durham, NC 27705, USA.
Mol Vis. 1998 Sep 8;4:15.
To examine the molecular structure and ultrastructural distribution of a novel amine oxidase in human ciliary body.
Human ciliary bodies were solubilized with a nonionic detergent. The solubilized material was subjected to affinity chromatography with 2B4.14.1, a monoclonal antibody which recognizes a family of ciliary body glycoproteins. Proteins eluted from the affinity column were further separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Peptides produced from a 2B4.14. 1-reactive protein with an approximate molecular weight of 100 kDa were analyzed by Edman degradation. The protein thus identified was further examined by Western blotting and immunoelectron microscopy with anti-peptide antisera.
Peptide sequences from the 100 kDa ciliary body protein were identical to the predicted protein sequence of an amine oxidase identified recently in a human placental cDNA library. The identity of the ciliary body protein was confirmed by Western blotting with rabbit antiserum generated against the predicted carboxy-terminal peptide of human placenta amine oxidase. Western blotting under nonreducing conditions and following glycosidase digestion indicated that the native enzyme is a disulfide-linked homodimer with multiple N-linked oligosaccharide side chains. By immunoelectron microscopy, the ciliary body amine oxidase was localized to the plasma membranes of inner epithelial cells.
Human placenta amine oxidase is present on the plasma membranes of ciliary body inner epithelial cells. This finding provides a potential explanation for amine oxidase enzyme activity detected in previous studies of anterior segment tissues. Though the functional role of human placenta amine oxidase in the eye is unclear, it may contribute to the production of H2O2 in aqueous humor.
研究人睫状体中一种新型胺氧化酶的分子结构和超微结构分布。
用人睫状体用非离子去污剂溶解。溶解后的物质用2B4.14.1进行亲和层析，2B4.14.1是一种识别睫状体糖蛋白家族的单克隆抗体。从亲和柱上洗脱的蛋白质通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳进一步分离。对一种分子量约为100 kDa的2B4.14.1反应性蛋白产生的肽进行埃德曼降解分析。用抗肽抗血清通过蛋白质印迹法和免疫电子显微镜对鉴定出的蛋白质进行进一步检测。
100 kDa睫状体蛋白的肽序列与最近在人胎盘cDNA文库中鉴定出的一种胺氧化酶的预测蛋白序列相同。用人胎盘胺氧化酶预测的羧基末端肽产生的兔抗血清进行蛋白质印迹法，证实了睫状体蛋白的同一性。在非还原条件下和糖苷酶消化后的蛋白质印迹法表明，天然酶是一种具有多个N-连接寡糖侧链的二硫键连接的同型二聚体。通过免疫电子显微镜观察，睫状体胺氧化酶定位于内上皮细胞的质膜上。
人胎盘胺氧化酶存在于睫状体内上皮细胞质膜上。这一发现为先前在前节组织研究中检测到的胺氧化酶活性提供了一种潜在的解释。虽然人胎盘胺氧化酶在眼中的功能作用尚不清楚，但它可能有助于房水中过氧化氢的产生。
