BIOCHEMISTRY OF PROPHENOL OXIDASE ACTIVATING PROTEINASE

前酚氧化酶的激活是一种重要的生理机制， 参与昆虫防御机制的过程。其中包括： 角质层硬化、伤口愈合、包囊化和黑化 异物和细胞毒性分子的产生。 知识 从模式昆虫，如天蛾的激活过程， 对于研究吸血昆虫的类似系统是有用的， 传播人类疾病。 本研究的目的是描述 酚氧化酶原激活蛋白酶及其机制研究 调节M.六。 项目的具体目标 是：1. 酚氧化酶原的纯化及性质研究 激活蛋白酶。活化蛋白酶将从 血淋巴和角质层。 氨基酸序列和其他生物化学 将研究酶的特性。 酶学性质 具有和不具有称为因子A的辅因子的蛋白酶将被 研究了苯酚的活化机理 氧化酶。 2. 酚氧化酶原基因的克隆与表达 激活蛋白酶。 与氨基酸序列信息和 激活蛋白酶的抗血清可用，其cDNA将是 通过筛选M.六血细胞、脂肪体或表皮cDNA 图书馆. 蛋白酶将表达为重组蛋白 以其酶原形式用于上游激活剂的研究， 前酚氧化酶激活系统的重建。 地点，时间， 水平，并诱导M. Sexta将是 研究了 3. 前酚氧化酶激活的调节 丝氨酸蛋白酶抑制剂。 抑制反应 蛋白酶的M. sexta丝氨酸蛋白酶抑制剂和Kunitz型胰蛋白酶抑制剂 将进行调查，以了解生理调节的 酵素 4. 因子A的纯化和鉴定。因子A 这改变了酚氧化酶原的活性和特异性 活化蛋白酶将从角质层和血淋巴中纯化。 其 与活化蛋白酶和酚氧化酶原的相互作用将 被定性。 这项研究将有助于我们了解生物化学 前酚氧化酶活化的过程，并将产生有用的 用于研究人类昆虫载体中类似系统的分子探针 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.