AMELOGENIN PROCESSING BY SERINE PROTEINASE(S) IN ENAMEL

牙釉质中丝氨酸蛋白酶对釉原蛋白的加工

• 批准号: 2131504
• 负责人: Pamela K Den Besten
• 金额: $ 20.34万
• 依托单位: EASTMAN DENTAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2131504
• 关键词: SDS polyacrylamide gel electrophoresis; acid base balance; active sites; ameloblasts; animal genetic material tag; animal tissue; complementary DNA; enamel organ; enzyme activity; enzyme substrate; extracellular matrix proteins; gel filtration chromatography; genetic library; high performance liquid chromatography; hydrolysis; in situ hybridization; messenger RNA; nucleic acid sequence; polymerase chain reaction; protein purification; protein sequence; proteolysis; serine proteinases; thermostability; tooth enamel; SDS polyacrylamide gel electrophoresis; acid base balance; active sites; ameloblasts; animal genetic material tag; animal tissue; complementary DNA; enamel organ; enzyme activity; enzyme substrate; extracellular matrix proteins; gel filtration chromatography; genetic library; high performance liquid chromatography; hydrolysis; in situ hybridization; messenger RNA; nucleic acid sequence; polymerase chain reaction; protein purification; protein sequence; proteolysis; serine proteinases; thermostability; tooth enamel

Tooth enamel is secreted as a protein matrix which is removed as the enamel matures. The largest fraction of the protein matrix consists of amelogenins, which are selectively removed from the enamel as maturation progresses. This processing of amelogenins is important for regulating crystal growth in the mineralizing enamel matrix. The overall objective in this proposal is to better understand amelogenin processing by identifying and characterizing amelogeninases in the developing enamel matrix. The specific hypothesis to be tested is that a serine proteinase, expressed by ameloblasts, is responsible for the hydrolysis of amelogenin protein in the developing enamel matrix. The specific aims are: 1) to isolate and purify the serine proteinase in enamel which actively hydrolyzes amelogenin; 2) to characterize the amelogeninase for substrate specificity, pH optima and thermal stabilities; 3) to determine the specific cleavage sites of the amelogeninase by using purified 25,000 MW amelogenin as a susbstrate; 4) to amplify, clone and sequence the cDNA(s) from bovine enamel organ mRNA which contains the conserved active site for a serine proteinase; 5) to clone a full-length cDNA for the serine proteinase/amelogeninase from a bovine enamel organ lambda phage cDNA library and to begin in situ hybridization studies. Purification and characterization of the serine proteinase/amelogeninase will be completed by isoelectric focusing, gel electrophoresis and column chromatography. Specificity of the amelogeninase will be determined by hydrolysis of a purified amelogenin substrate. The cDNA for the serine proteinase will be cloned to allow comparisons between the initially expressed mRNA transcript and the active form of the proteinase present in the enamel matrix. These studies to identify and characterize the serine proteinase/amelogeninase in developing enamel will lead to a better understanding of the mechanisms by which amelogenesis occurs. They will also further our understanding of defects which occur in enamel formation, such as in enamel fluorosis.

牙齿搪瓷被分泌为蛋白质基质，被去除为 搪瓷成熟。蛋白质基质的最大分数包括 氨基蛋白蛋白，随着成熟的牙釉质选择性去除 进展。 氨基蛋白的这种处理对于调节很重要 矿物化搪瓷基质中的晶体生长。 总体目标 在此提案中是通过 识别和表征发育中的牙釉质中的氨基蛋白酶 矩阵。要测试的特定假设是丝氨酸蛋白酶， 由成成布表示，负责氨基蛋白的水解 发育中的搪瓷基质中的蛋白质。 具体目的是：1）分离和纯化丝氨酸蛋白酶 积极水解蛋白质蛋白的搪瓷； 2）表征 用于底物特异性，pH Optia和热的氨基蛋白酶酶 稳定； 3）确定特定的切割位点 通过使用纯化的25,000 MW氨基蛋白蛋白作为susbrate，氨基蛋白酶酶； 4） 从牛搪瓷器官mRNA放大，克隆并序列cDNA（S） 其中包含丝氨酸蛋白酶的保守活性位点； 5）到 克隆从A 牛搪瓷器官lambda噬菌体cDNA库，然后开始原位 杂交研究。 丝氨酸蛋白酶/阿梅原酶的纯化和表征 将通过等电聚焦，凝胶电泳和色谱柱完成 色谱法。阿梅原酶的特异性将由 纯化的氨基蛋白蛋白底物的水解。丝氨酸的cDNA 蛋白酶将被克隆以允许最初的比较 表达的mRNA转录和存在于 搪瓷矩阵。 这些研究以识别和表征丝氨酸 在发育搪瓷中的蛋白酶/氨基蛋白酶酶将导致更好 了解发生无ele灭病的机制。 他们会的 还进一步了解了我们对搪瓷形成中发生的缺陷的理解， 例如在搪瓷氟中毒中。