COLLAGEN FOLDING IN PRESENCE OF CIS-TRANS ISOMERASE

顺反异构酶存在下胶原蛋白折叠

• 批准号: 3158046
• 负责人: HANS PETER BACHINGER
• 金额: $ 6.58万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3158046
• 关键词: acidity /alkalinity; affinity chromatography; cis trans isomerization; collagen; enzyme mechanism; enzyme structure; fluidity; immunoelectron microscopy; immunofluorescence technique; isomerase; laboratory rabbit; molecular shape; molecular weight; procollagen; proline; protein biosynthesis; protein sequence; tissue /cell culture

Folding is an essential step of biosynthesis of proteins because biological function can only be obtained by specific structures. This proposal focuses on the question how a recently discovered enzyme activity termed peptidyl-prolyl cis-transisomerase influences the triple helix formation of collagens in vitro and in vivo. The activity is to be purified to homogeneity from pig kidneys and characterized in regard to molecular weight, hydrodynamic parameters, aminoacid composition, aminoterminal aminoacid sequence, spectroscopic properties and enzyme kinetics. Enzymatic activity is determined against the cis to trans isomerization of succinyl-Ala-Ala-Pro-Phe-4- methlycumaryl-7-amide by means of a two-step process using chymotrypsin as the trans substrate cleaving activity. This enzyme probably helps folding of protein which has been shown to be rate limited by isomerization of proline containing peptide bonds. To test this hypothesis the influence of peptidyl-prolyl cis- trans isomerase on in vitro folding of type III collagen is investigated. Refolding of type III collagen was shown to be rate limited by cis to trans isomerization of peptide bonds. The influence of ionic strength and pH on the kinetics of peptidyl- prolyl cis-trans isomerase is investigated as well as the intercellular localization by immunoelectronmicroscopy. To see whether the enzyme is actively involved in in vivo folding during biosynthesis, the enzyme level is investigated for correlation with 4-prolyl hydroxylase in a developing system with varying degrees of collagen synthesis. What happens if folding is impaired has been shown recently in cases of Osteogenesis Imperfecta where a single aminoacid substitution for a glycine residue in the pro alpha 1 chain of type I procollagen leads to procollagen molecules with decreased melting temperature, increased posttranslational, modification altered rate of secretion, increased intracellular degradation, and decreased collagen production.

折叠是蛋白质生物合成的重要步骤，因为 生物学功能只能通过特定的结构来获得。 该提案关注的问题是最近发现的 称为肽基脯氨酰顺转异构酶的酶活性 影响体外和体内胶原蛋白三螺旋的形成 体内。 活性需从猪中纯化至同质 肾脏并以分子量为特征， 流体动力学参数、氨基酸组成、氨基末端 氨基酸序列、光谱特性和酶 动力学。 酶活性根据顺式测定 琥珀酰-Ala-Ala-Pro-Phe-4-的反式异构化 甲基枯芳基-7-酰胺通过两步法使用 糜蛋白酶作为反式底物裂解活性。 这 酶可能有助于蛋白质的折叠，这已被证明 速率受到含脯氨酸肽异构化的限制 债券。 为了检验这一假设，肽基-脯氨酰顺式的影响 反式异构酶对 III 型胶原体外折叠的作用是 调查了。 III 型胶原蛋白的重折叠率 受肽键顺式异构化和反式异构化的限制。 这 离子强度和pH值对肽基动力学的影响 研究了脯氨酰顺反异构酶 通过免疫电子显微镜进行细胞间定位。 查看 该酶是否积极参与体内折叠过程 生物合成，研究酶水平与 4-脯氨酰羟化酶在不同程度的发育系统中 胶原蛋白的合成。 如果折叠受损会发生什么 最近在成骨不全的病例中显示， pro α 中甘氨酸残基的单个氨基酸取代 1 条 I 型原胶原链导致原胶原分子 熔化温度降低，翻译后增加， 修饰改变了分泌率，增加了细胞内 降解，并减少胶原蛋白的产生。