BIOCHEMISTRY OF PROPHENOL OXIDASE ACTIVATING PROTEINASE

酚氧化酶激活蛋白酶的生物化学

• 批准号: 6164832
• 负责人: HAOBO JIANG
• 金额: --
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6164832
• 关键词: Manduca; affinity chromatography; cofactor; complementary DNA; electrofocusing; enzyme activity; enzyme induction /repression; gel filtration chromatography; genetic library; hemolymph; high performance liquid chromatography; ion exchange chromatography; matrix assisted laser desorption ionization; molecular cloning; monophenol monooxygenase; protein sequence; recombinant proteins; serine proteinases; western blottings

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.

酚氧化酶原的激活是一个重要的生理过程。 参与昆虫防御机制的过程。这些包括： 角质层硬化、伤口愈合、包封和黑化 异物的产生，以及细胞毒性分子的产生。 知识 模型昆虫（如 Manduca sexta）的激活过程将 对于研究吸血昆虫的类似系统很有用 传播人类、疾病。 这项研究的目标是表征 酚氧化酶原激活蛋白酶及其机制研究 调节 M. sexta 中的酶。 项目的具体目标 是： 1. 酚氧化酶原的纯化和表征 激活蛋白酶。活化蛋白酶将从中纯化 血淋巴和角质层。 氨基酸序列及其他生化 将研究酶的特性。 酶学性质 有或没有辅因子（称为因子 A）的蛋白酶将是 研究了解前苯酚的活化机制 氧化酶。 2. 酚氧化酶原的分子克隆和表达 激活蛋白酶。 具有氨基酸序列信息和 可用的激活蛋白酶的抗血清，其 cDNA 将是 通过筛选M. sexta血细胞、脂肪体或表皮cDNA克隆 图书馆。 蛋白酶将被表达为重组蛋白 以其酶原形式用于上游激活剂和最终的研究 重建前酚氧化酶激活系统。 地点、时间、 水平，以及 M. sexta 中酶表达的诱导性将是 调查了。 3. 酚氧化酶原激活的调节 丝氨酸蛋白酶抑制剂抑制蛋白酶。 抑制反应 M. sexta 丝氨酸蛋白酶抑制剂和 Kunitz 型胰蛋白酶抑制剂对蛋白酶的影响 将进行研究以了解其生理调节 酶。 4. 因子 A 的纯化和表征。 因子 A 改变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。 它是 与激活蛋白酶和前酚氧化酶的相互作用将 被表征。 这项研究将有助于我们对生物化学的理解 前酚氧化酶激活的过程，并会产生有用的 用于研究人类昆虫媒介中类似系统的分子探针 疾病。