CHARACTERIZATION OF ACID PHOSPHATASE FROM PENICILLIUM FELLUTANUM

真菌糖肽加工是一系列复杂的事件， 导致细胞壁、细胞膜和细胞外的改变 结构。这个子项目的主要目标是探索 一种真菌酸性磷酸单酯酶家族的假说 Fellutanum青霉菌都参与了青霉菌的加工 来自细胞膜、细胞壁和细胞外的糖肽 分数。为了检验这一假设，我们建议(1) 用已建立的蛋白质分离法分离磷酸单酯酶 (2)鉴定蛋白质结构； (3)建立酶的动力学参数；(4)建立 该酶的天然底物通过测试其在 青霉菌细胞壁、质膜和胞外 糖肽。甘德和同事的早期报告确立了 至少存在三种不同的酸性磷酸酶蛋白和 某些青霉菌酸性磷酸酶活性在 磷酸盐限制的条件。来检验这一假设 有不同的诱导性和结构性酸性磷酸酶，我们 建议(1)分离和鉴定来自中国的 与真核酸同源的费氏青霉菌DNA (2)构建c DNA文库，并用 与酸性磷酸酶同源；(3)克隆了酸性磷酸酶基因(S) 并在合适的载体中表达它们；(4)比较 热休克、缺磷和pH值对红细胞膜生物量的影响 编码酸性磷酸酶基因的mRNA和DNA。我们也 建议用荧光显微镜对酸性磷酸酶进行定位 细胞、细胞膜和细胞壁。酸的作用的发现 真菌代谢和生理中的磷酸酶有望 提供可用于打击传播的信息 霉菌，如由马尔尼菲青霉菌引起的，在艾滋病毒- 感染和其他免疫功能受损的个人。

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.