METALLOPROTEIN BASED INTERFACIAL SYSTEMS

基于金属蛋白的界面系统

• 批准号: 6345237
• 负责人: PATRICIA A MABROUK
• 金额: $ 0.13万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6345237
• 关键词: bacteria; bioengineering /biomedical engineering; biological products; biomaterials; biomedical resource; biotechnology; carbohydrates; infection; proteins; structural biology; technology /technique

The long term objectives of the project are characterization of the enzymatic activities and gene products involved in synthesis of the peptidoglycan cell wall of bacterial endospores and examination of the role of each type of peptidoglycan structural modification in determining endospore resistance properties. The specific aims are 1) to determine the structure of the endospore peptidoglycan in the first stages of its synthesis and to track the structure to its final form in the dormant spore, 2) to identify changes in synthesis of this structure produced by mutation or loss of each penicillin-binding protein, autolysin, and sporulation-associated gene product, and 3) to purify and characterize in vitro the enzymatic activities involved in peptidoglycan side chain cleavage and muramic lactarn production. Knowledge of principles of endospore resistance properties, dormancy, and longevity may contribute to better decontamination methods and to methods for storage and transport of drugs and vaccines. Peptidoglycan synthesis in general is an attractive target for antibiotic action, further studies of this process will contribute to methods for identification of new antibiotics. Peptidoglycan is purified from sporulating cultures of Bacillus subtilis by chemical and enzymatic treatments, digested with muramidase, and analyzed using high-pressure liquid chromatography (HPLC) using methods previously developed for analysis of dormant spore peptidoglycan. Novel muropeptides are identified using amino acid analysis and mass spectrometry. Appearance and loss of peptide side chain alanine and glycine residues, muramic lactarn production, and changes in peptide cross-linking are quantified throughout the sporulation process. The analysis will be repeated using strains with altered or absent penicillin-binding proteins, autolysins, and sporulation-associated gene products. The cwlD and other gene products found to be associated with muramic lactam production will be assayed in vitro using extracts of sporulating cells or following over-expression in B. subtilis or Escherichia coli. Immature peptidoglycan samples from mutant strains and purified muropeptides will serve as substrates in these assays. Reaction progress will be monitored using the HPLC method and by development of a more rapid capillary electrophoresis method.

项目的长期目标是表征 涉及合成的酶活性和基因产物 细菌内孢子的肽聚糖细胞壁和检查 每种类型的肽聚糖结构修饰的作用 确定内孢子抗性特性。 具体目标是1） 确定第一个内孢子肽聚糖的结构 其合成的阶段并跟踪结构到最终形式 在休眠孢子中，2）确定合成的变化 通过突变或每种青霉素结合的损失产生的结构 蛋白质，自脂蛋白和孢子相关的基因产物，3）至 在体外纯化和表征涉及的酶活性 肽聚糖的侧链裂解和穆拉米克乳糖产生。 知识内孢子抗性特性，休眠， 寿命可能有助于更好的净化方法和 储存和运输药物和疫苗的方法。 通常，肽聚糖合成是一个有吸引力的目标 抗生素作用，对这一过程的进一步研究将有助于 鉴定新抗生素的方法。 肽聚糖是 从化学物质中纯化枯草芽孢杆菌培养物 和酶促处理，用muramidase消化，并使用 先前使用方法的高压液相色谱（HPLC） 开发用于分析休眠孢子肽聚糖。 小说 使用氨基酸分析和质量来鉴定杂肽 光谱法。 肽侧链丙氨酸的外观和损失 甘氨酸残基，穆拉米克乳酸产生和肽的变化 在整个孢子形过程中，对交联。 这 将使用变化或不存在的菌株重复分析 青霉素结合蛋白，自动蛋白和孢子蛋白相关 基因产品。 发现CWLD和其他基因产品是 将在体外测定与穆拉米克乳酸的产生 使用孢子细胞的提取物或遵循B中的过表达。 枯草或大肠杆菌。 未成熟的肽聚糖样品 突变菌株和纯化的杂肽将用作底物 这些测定。 反应进度将使用HPLC监测 方法和通过开发更快的毛细管电泳 方法。