AMELOGENIN PROCESSING AND APATITE CRYSTAL GROWTH

釉蛋白加工和磷灰石晶体生长

• 批准号: 6379818
• 负责人: Janet M. Oldak
• 金额: $ 11.45万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379818
• 关键词: acidity /alkalinity; active sites; amelogenin; calcium; carbonates; chemical association; chemical binding; chemical cleavage; chymotrypsin; conformation; endopeptidases; enzyme activity; enzyme substrate; eukaryote; fluorine; high performance liquid chromatography; hydroxyapatites; phosphates; protein degradation; protein sequence; proteolysis; recombinant DNA; recombinant proteins; temperature; tooth enamel

This R29 application is designed to investigate the correlation between proteolytic processing of the enamel extracellular matrix protein amelogenin and growth of apatite crystals, using an in vitro system. The applicant identified a serine protease, designated ameloprotease-I, as a cleavage product of one of the larger non-amelogenin proteins. This protease was shown by the applicant to degrade amelogenin proteins in vitro. The hypotheses to be examined are: that the activity of ameloprotease-I correlates with formation and maturation of enamel apatite crystals; that ameloprotease-I is activated in the extracellular matrix through proteolytic cleavage of its precursors; and that adsorption and dissociation of both enzyme and substrate onto apatite crystals are involved in proteolytic processing. Four specific aims are proposed: (I) To examine autocatalytic action of the precursor 89 kDa enamelin in release of the 32 kDA protease in the absence and presence of apatite crystals; (II) To investigate effects of calcium, phosphate, carbonate and fluoride on activity of ameloprotease-I; (III) To determine cleavage sites within amelogenin after proteolysis +/-apatite crystals, and to monitor adsorption of enzyme and substrate to the apatite; and (IV) To investigate cleavage of amelogenin by other serine proteases +/- apatite crystals, to determine site of enzyme accessibility on the protein surface.

该R29应用程序旨在研究 搪瓷细胞外基质蛋白的蛋白水解加工 使用体外系统的氨基蛋白酶和磷灰石晶体的生长。 申请人确定了丝氨酸蛋白酶，指定为ameleprotease-i， 作为较大的非淀粉蛋白蛋白之一的裂解产物。 申请人显示了这种蛋白酶以降解蛋白质蛋白 体外。 要研究的假设是： Ameleprotease-I与搪瓷的形成和成熟相关 磷灰石晶体； ameleprotease-i在细胞外激活 通过其前体的蛋白水解裂解的基质；那 酶和底物在磷灰石上的吸附和解离 晶体参与蛋白水解加工。 四个具体目标是 提议：（i）检查前体89 kDa的自催化作用 在不存在和存在的情况下释放32 kDa蛋白酶的搪瓷蛋白 磷灰石晶体； （ii）研究钙，磷酸盐的影响 碳酸盐和氟化物对绿蛋白酶I的活性； （iii）至 确定蛋白水解后氨基蛋白酶内的切割位点+/-磷灰石 晶体，并监测酶和底物的吸附到 磷灰石; （iv）研究其他丝氨酸蛋白蛋白的裂解 蛋白酶+/-磷灰石晶体，以确定酶位点 蛋白质表面的可及性。