STRUCTURE OF VITREOUS HUMOR AND ZONULAR APPARATUS

玻璃体和小带装置的结构

• 批准号: 2019852
• 负责人: RICHARD MAYNE
• 金额: $ 19.34万
• 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 1998-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2019852
• 关键词: affinity chromatography; binding proteins; cartilage; chick embryo; chickens; collagen; cow; crosslink; embryogenesis; extracellular matrix proteins; hyaluronate; immunoelectron microscopy; monoclonal antibody; protein sequence; 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.

将对蛋白质进行生化和形态分析 和蛋白聚糖形成玻璃体幽默和Zonular的结构 设备。 胶原原纤维将从牛和鸡肉中隔离 玻璃体和三种胶原蛋白类型的组织（II型，IX和类型 将研究这些原纤维中存在的v/xi胶原蛋白）。 分析 将对V型胶原蛋白的相对量进行[alpha1（v）和 alpha2（v）链]和XI型胶原蛋白[Alpha1（XI）和Alpha2（XI）和 鸡肉和牛玻璃体中存在的α3（XI）链。 使用特定的单克隆抗体，我们将研究这些类型 v/xi分子形成核心“微纤维” 胶原蛋白是组装的。 交联的肽（两者都包含 的羟基吡啶和还原的交联） 牛和鸡肉玻璃将被隔离，交联的位置 将由N末端氨基酸测序确定。 位置 II型和类型V/XI胶原蛋白或类型IX之间的潜在交联 将确定胶原蛋白。 相关的蛋白质或蛋白聚糖 胶原蛋白原纤维，并在大量消化后释放 将研究细菌胶原酶。 这样的蛋白质将是 SDS-PAGE后通过N末端氨基酸测序鉴定。 具体的 将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。 牛的其他实验 玻璃体将研究新蛋白与透明质酸和 在生化上研究这种蛋白质。 牛的胶原纤维 玻璃体将用胶原酶，斯特罗米辛，明胶酶，明胶，明胶酶消化 纤溶酶等使用重组形式的酶和混合物的混合物 完全溶解胶原原纤维所需的酶是 决定。 研究还将研究IX型胶原蛋白的释放 通过有限的胃蛋白酶消化，从胶原原纤维的表面和 通过旋转阴影来检查此类制剂，以交联的II型 胶原。 IX型胶原蛋白将与鸡肉玻璃体隔离 α1（IX）链的N末端氨基酸序列将在 为了证明下游启动子在 玻璃体IX胶原蛋白的形成。 玻璃体的形成 幽默及其相关的胶原蛋白原纤维将在眼睛期间进行研究 鸡肉胚胎的发育。 将从 成人牛眼和实验以确定的结构 这些原纤维。 占地纤维将被各种消化 SD页面和N末端后确定的蛋白酶和独特的片段 氨基酸测序。 通过使用先前获得的单克隆抗体 到原纤维蛋白或其他新型和潜在新颖的单克隆抗体 分子的蛋白质，我们目前的这些结构模型 原纤维将进一步研究。

项目成果

Transglutaminase activity in the eye: cross-linking in epithelia and connective tissue structures.

眼睛中的转谷氨酰胺酶活性：上皮和结缔组织结构中的交联。

• 发表时间: 1999
• 期刊: Investigative ophthalmology & visual science.
• 作者: Raghunath,M;Cankay,R;Kubitscheck,U;Fauteck,JD;Mayne,R;Aeschlimann,D;Schlotzer-Schrehardt,U
• 通讯作者: Schlotzer-Schrehardt,U