STRUCTURE OF VITREOUS HUMOR AND ZONULAR APPARATUS

玻璃体和小带装置的结构

• 批准号: 3267234
• 负责人: RICHARD MAYNE
• 金额: $ 16.3万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3267234
• 关键词: affinity chromatography; binding proteins; cartilage; chick embryo; chickens; collagen; cow; crosslink; embryogenesis; extracellular matrix proteins; hyaluronate; immunoelectron microscopy; monoclonal antibody; protein structure; proteoglycan; vitreous body

Biochemical and morphological analyses will be performed of the proteins and proteoglycans that form the structure of the vitreous humor and zonular apparatus. Collagen fibrils will be isolated from bovine and chicken vitreous and the organization of the three collagen types (type II, IX and V/XI collagen) present in these fibrils will be investigated. Analyses will be performed of the relative amounts of type V collagen [alpha1(V) and alpha2(V) chains] and type XI collagen [alpha1(XI) and alpha2(XI) and alpha3(XI) chains] that are present in the chicken and bovine vitreous. Using specific monoclonal antibodies, we will investigate if these type V/XI molecules form a core "microfibril" around which the fibril of type II collagen is assembled. Crosslinked peptides (containing both hydroxypyridinium and reducible crosslinks) of the collagen fibrils of bovine and chicken vitreous will be isolated and the location of crosslinks will be determined by N-terminal amino acid sequencing. The location of potential crosslinks between type II and type V/XI collagen or type IX collagen will be determined. Proteins or proteoglycans that are associated with collagen fibrils and are released after extensive digestion with bacterial collagenase will be investigated. Such proteins will be identified by N-terminal amino acid sequencing after SDS-PAGE. Specific analyses will be made for cartilage matrix protein (CMP) in the vitreous and its association with collagen fibrils. Other experiments with bovine vitreous will investigate the binding of novel proteins to hyaluronan and to investigate such proteins biochemically. Collagen fibrils of bovine vitreous will be digested with collagenase, stromelysin, gelatinase, plasmin, etc. using recombinant forms of the enzymes and the mixture of enzymes required for the complete dissolution of a collagen fibril will be determined. Studies will also investigate the release of type IX collagen from the surface of the collagen fibril by limited pepsin digestion and to examine such preparations by rotary shadowing for crosslinking to type II collagen. Type IX collagen will be isolated from chicken vitreous and the N-terminal amino acid sequence of the alpha1(IX) chain will be obtained in order to demonstrate that the downstream promotor is used during the formation of vitreous type IX collagen. The formation of the vitreous humor and its associated collagen fibrils will be investigated during eye development in chicken embryos. Zonular fibrils will be dissected from adult bovine eyes and experiments performed to determine the structure of these fibrils. Zonular fibrils will be digested with a variety of proteases and unique fragments identified after SDS PAGE and N-terminal amino acid sequencing. By use of monoclonal antibodies previously obtained to fibrillin, or monoclonal antibodies to other and potentially novel proteins of the zonule, our present model for the structure of these fibrils will be further investigated.

对蛋白质进行生化和形态学分析 以及形成玻璃体和小带结构的蛋白多糖 设备。 将从牛和鸡中分离出胶原纤维 玻璃体和三种胶原蛋白类型（II 型、IX 型和 将研究这些原纤维中存在的 V/XI 胶原蛋白。 分析 将进行 V 型胶原蛋白 [α1(V) 和 alpha2(V) 链] 和 XI 型胶原蛋白 [alpha1(XI) 和 alpha2(XI) 和 α3(XI)链]存在于鸡和牛玻璃体中。 使用特定的单克隆抗体，我们将研究这些类型是否 V/XI 分子形成核心“微原纤维”，II 型原纤维围绕其周围 胶原蛋白被组装。 交联肽（含有 胶原原纤维的羟基吡啶鎓和可还原交联） 牛和鸡的玻璃体将被分离并确定交联的位置 将通过N端氨基酸测序来确定。 的位置 II 型和 V/XI 型胶原蛋白或 IX 型之间的潜在交联 胶原蛋白将被测定。 相关的蛋白质或蛋白聚糖 与胶原纤维，并在广泛消化后释放 将研究细菌胶原酶。 这样的蛋白质将是 SDS-PAGE 后通过 N 端氨基酸测序进行鉴定。 具体的 对玻璃体中的软骨基质蛋白（CMP）进行分析 及其与胶原纤维的关联。 其他牛实验 玻璃体将研究新型蛋白质与透明质酸的结合， 对此类蛋白质进行生化研究。 牛胶原原纤维 玻璃体将被胶原酶、溶基质素、明胶酶消化， 使用重组形式的酶和混合物的纤溶酶等 完全溶解胶原纤维所需的酶是 决定。 研究还将调查 IX 型胶原蛋白的释放 通过有限的胃蛋白酶消化从胶原纤维表面分离出来 通过旋转阴影检查此类制剂是否与 II 型交联 胶原。 IX 型胶原蛋白将从鸡玻璃体中分离出来 α1(IX)链的N端氨基酸序列将在 为了证明下游启动子在 玻璃体 IX 型胶原蛋白的形成。 玻璃体的形成 眼科期间将研究体液及其相关的胶原纤维 鸡胚胎的发育。 小带原纤维将从 成年牛眼睛和为确定其结构而进行的实验 这些原纤维。 小带原纤维会被多种物质消化 SDS PAGE 和 N 末端后鉴定的蛋白酶和独特片段 氨基酸测序。 通过使用先前获得的单克隆抗体 原纤维蛋白或其他潜在新颖的单克隆抗体 小带的蛋白质，我们目前的这些结构模型 原纤维将被进一步研究。