BIOCHEMISTRY OF PROPHENOL OXIDASE ACTIVATING PROTEINASE

酚氧化酶原的激活是一种重要的生理过程 昆虫中参与防御机制的过程。这些措施包括： 角质层硬化、伤口愈合、包膜和黑化 外来物质和细胞毒性分子的产生。知识 模式昆虫的激活过程，如六叶蝉Will 对于研究吸血昆虫的类似系统是有用的 传播人类疾病。这项研究的目标是描述 酚氧化酶原激活蛋白水解酶及其机制的研究 来调节六分枝杆菌中的酶。该项目的具体目标 主要内容有：1.原酚氧化酶的纯化及性质 激活蛋白酶。激活的蛋白水解酶将从 包括血淋巴和角质层。氨基酸序列和其他生化 将研究该酶的特性。酶促性质的研究 含有和不含有辅因子的蛋白水解酶，称为因子A，将是 了解原苯酚活化机理的研究 氧化物酶。2.原酚氧化酶的分子克隆与表达 激活蛋白酶。具有氨基酸序列信息和 如果有激活蛋白水解酶的抗血清，它的cDNA会被 筛选六倍体血细胞、脂肪体或表皮基因克隆 图书馆。该蛋白酶将以重组蛋白的形式表达。 以其酶原形式用于上游激活剂和最终的 原酚氧化酶活化系统的重建。地点、时间、 该酶在六分枝杆菌中的表达水平和可诱导性将是 调查过了。3.原酚氧化酶活性的调节 丝氨酸蛋白酶抑制剂。抑制反应 蛇形分枝杆菌和库尼茨型胰酶抑制剂对蛋白酶的影响 将被调查以了解细胞的生理调节 酵素。4.A因子的分离纯化及性质研究 它改变了原酚氧化酶的活性和特异性 激活蛋白水解酶将从角质层和血淋巴中提纯。它的 与激活的蛋白水解酶和原酚氧化酶的相互作用 被刻画出来。 这项研究将有助于我们对生物化学的理解。 原酚氧化酶的活化过程，并会产生有用的 研究人类昆虫媒介相似系统的分子探针 疾病。

The activation of pro-phenol oxidase is an important physiological process involved in defense mechanisms in insects. These include: cuticle sclerotization, wound healing, encapsulation and melanization of foreign materials, and generation of cytotoxic molecules. Knowledge of the activation process from model insects such as Manduca sexta will be useful for studying similar systems in blood-sucking insects that transmit human, diseases. Goals of this research are to characterize the pro-phenol oxidase activating proteinase and to study mechanisms that regulate the enzyme in M. sexta. The specific aims of the project are: 1. Purification and characterization of the pro-phenol oxidase activating proteinase. The activating proteinase will be purified from both hemolymph and cuticle. Amino acid sequence and other biochemical characteristics of the enzyme will be studied. Enzymatic properties of the proteinase with and without a cofactor, named factor A, will be investigated to understand the activation mechanism of pro-phenol oxidase. 2. Molecular cloning and expression of the pro-phenol oxidase activating proteinase. With the amino acid sequence information and antiserum to the activating proteinase available, its cDNA will be cloned by screening M. sexta hemocyte, fat body or epidermis cDNA libraries. The proteinase will be expressed as a recombinant protein in its zymogen form for a study of upstream activators and eventual reconstitution of the pro-phenol oxidase activating system. Site, time, level, and inducibility of expression of the enzyme in M. sexta will be investigated. 3. Regulation of the pro-phenol oxidase activating proteinase by serine proteinase inhibitors. The inhibition reactions of the proteinase by M. sexta serpins and Kunitz-type trypsin inhibitors will be investigated to understand the physiological regulation of the enzyme. 4. Purification and characterization of factor A. Factor A which alters the activity and specificity of pro-phenol oxidase activating proteinase will be purified from cuticle and hemolymph. Its interactions with the activating proteinase and pro-phenol oxidase will be characterized. This research will contribute to our understanding of the biochemical process of pro-phenol oxidase activation, and will generate useful molecular probes for studying similar systems in insect vectors of human diseases.