CHARACTERIZATION OF ACID PHOSPHATASE FROM PENICILLIUM FELLUTANUM

费鲁坦青霉酸性磷酸酶的表征

• 批准号: 6442288
• 负责人: Sandra Bonetti
• 金额: $ 28.07万
• 依托单位: COLORADO STATE UNIVERSITY-PUEBLO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6442288
• 关键词: Moniliales; SDS polyacrylamide gel electrophoresis; affinity chromatography; cell membrane; cell wall; enzyme activity; enzyme inhibitors; extracellular matrix; extracellular matrix proteins; fluorescence microscopy; gas chromatography mass spectrometry; gene expression; genetic library; glycopeptides; high performance liquid chromatography; molecular cloning; nuclear magnetic resonance spectroscopy; paper chromatography; phosphomonoesterases; polymerase chain reaction; posttranslational modifications; protein biosynthesis; protein sequence; protein structure

Fungal glycopeptide processing is a series of complex events that result in the modification of cell wall, membrane, and extracellular structures. The major goal of this subproject is to explore the hypothesis that a family of fungal acid phosphomonoesterase derived from Penicillium fellutanum are involved in the processing of glycopeptides from the membrane, cell wall, and extracellular fractions. In order to test this hypothesis, we propose to (1) isolate the phosphomonoesterase using established protein isolation (chromatographic) methods; (2) characterize the protein structure; (3) establish kinetic parameters for the enzyme; and (4) establish natural substrates for the enzyme by testing its activity on Penicillium cell wall, plasma membrane, and extracellular glycopeptides. Early reports by Gander and coworkers established the existence of at least three distinct acid phosphatase proteins and that some Penicillium acid phosphatase activities are inducible under conditions of phosphate limitation. To test the hypothesis that there are distinct inducible and constitutive acid phosphatases, we propose to (1) isolate and characterize gene sequences from Penicillium fellutanum DNA with homology to eukaryotic acid phosphatases; (2) create a cDNA library and identify cDNAs with homology to acid phosphatases; (3) clone the gene(s) for the acid phosphatases and express them in a suitable vector; (4) compare the effects of heat shock, phosphate deprivation, and pH on the amount of mRNA and DNA encoding for the acid phosphatase genes. We also propose to localize the acid phosphatases by fluorescence microscopy of cells, membranes, and cell walls. Discovery of the role of acid phosphatases in fungal metabolism and physiology is expected to provide information that can be applied to combat disseminated mycoses, such as those caused by Penicillium marneffei, in HIV- infected and other immunocompromised individuals.

真菌糖肽加工是一系列复杂的过程， 导致细胞壁、膜和细胞外的修饰 结构. 这个子项目的主要目标是探索 假设真菌酸性磷酸单酯酶家族衍生 来自Penicillium fellutanum的细菌参与了 来自膜、细胞壁和细胞外的糖肽 分数 为了验证这一假设，我们建议（1） 使用已建立的蛋白质分离法分离磷酸单酯酶 （色谱）方法;（2）表征蛋白质结构; (3)建立酶的动力学参数;和（4）建立 天然底物的酶通过测试其活性对 青霉菌细胞壁、质膜和细胞外 糖肽 甘德和同事的早期报告确定了 存在至少三种不同的酸性磷酸酶蛋白， 一些青霉酸性磷酸酶活性是可诱导的 磷酸盐限制条件。 为了验证这个假设， 有不同的诱导型和组成型酸性磷酸酶，我们 建议（1）分离和表征来自 一种与真核酸同源的青霉DNA （2）建立cDNA文库并鉴定cDNA， 与酸性磷酸酶同源;（3）克隆酸性磷酸酶的基因 磷酸酶并在合适的载体中表达它们;（4）比较 热休克，磷酸盐剥夺和pH值对 编码酸性磷酸酶基因的mRNA和DNA。 我们也 建议用荧光显微镜定位酸性磷酸酶 细胞、细胞膜和细胞壁。 发现酸的作用 磷酸酶在真菌代谢和生理学中的作用， 提供可用于打击传播的 真菌病，如由马尔尼菲青霉菌引起的真菌病， 感染者和其他免疫功能低下的个体。