PROTEIN NITRATASE DENITRATION ENZYME FROM BRAIN/PROSTATE

来自脑/前列腺的蛋白质硝酸酶脱硝酶

• 批准号: 6325858
• 负责人: WU-NAN KUO
• 金额: $ 4.85万
• 依托单位: BETHUNE-COOKMAN COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325858
• 关键词: Alzheimer's disease; SDS polyacrylamide gel electrophoresis; affinity chromatography; animal tissue; autoradiography; biological signal transduction; brain; enzyme activity; enzyme substrate; gel mobility shift assay; immunoprecipitation; ion exchange chromatography; nitrification; pathologic process; phosphorylation; prostate; prostate neoplasms; protease inhibitor; protein kinase; protein purification; transcription factor; western blottings

The long-term objective is to establish the protective/regulatory role of protein denitration enzymes, protein nitratases (or related enzymes), in reversing certain NO/peroxynitrate (PN)-mediated reactions (eg nitrotyrosine formation) and to elucidate a relationship between the deficiency of these enzymes and the pathogenesis of Alzheimer's and Parkinson's diseases, and prostate cancer. The specific aims are: (I) To test pathogenesis of Alzheimer's and Parkinson's diseases, and prostate cancer. The specific aims are: (I) To test substrate (PN-treated proteins: Histone H1, BSA, etc.) specificity for the novel protein-denitration activity of alpha, pi, psi isoforms of glutathione S-transferase (type I, redundant-dependent protein nitrases). The denitration activity can be monitored by nitrate production, and by the decreased anti-nitrotyrosine immunoreactivity in Western blot. (II) To compare subcellular (particulate and cytosolic) distribution of protein nitratases (type II, redundant independent) among bovine cerebral cortex, midbrain and cerebellum, and dog prostate. DEAE-cellulose chromatography, ammonium sulfate fractionation, molecular exclusion chromatography and affinity chromatography will be employed to purify the enzyme from crude extract. Characterization will include the effects of co-factors, pH, proteolysis (by m-calpain), phosphorylation (by protein kinases: PKA, PKG and PKC), nitration and auto-denitration, and the determination of molecular mass, subunits, substrate specificity, and isozymes. (IV) To study the effect of peroxynitrite-treatment on the activities of cAMP- dependent protein kinase (PKA), calmodulin and topoisomerase-1 (or other proteins/enzymes), and possible alteration/restoration of their activities after binding and DNAS electrophoretic mobility, respectively. The insight gained from this 4-year study might facilitate developing effective prevention/treatment of the above mentioned diseases in the future.

长期目标是确定蛋白质脱硝酶、蛋白质硝酸酶（或相关酶）在逆转某些一氧化氮/过氧硝酸盐（PN）介导的反应（例如硝基酪氨酸形成）中的保护/调节作用，并阐明这些酶的缺乏与阿尔茨海默病和帕金森病发病机制之间的关系， 和前列腺癌。具体目标是：（一）检测阿尔茨海默病、帕金森病以及前列腺癌的发病机制。具体目标是： (I) 测试底物（PN 处理的蛋白质：组蛋白 H1、BSA 等）对谷胱甘肽 S-转移酶（I 型，冗余依赖性蛋白质硝酸酶）的 α、pi、psi 亚型的新型蛋白质脱硝活性的特异性。脱硝活性可以通过硝酸盐的产生以及蛋白质印迹中抗硝基酪氨酸免疫反应性的降低来监测。 (II) 比较牛大脑皮层、中脑和小脑以及狗前列腺中蛋白硝酸酶（II 型，冗余独立）的亚细胞（颗粒和胞质）分布。 DEAE-纤维素层析、硫酸铵分级分离、分子排阻层析和亲和层析将用于从粗提取物中纯化酶。表征将包括辅因子、pH、蛋白水解（通过 m-钙蛋白酶）、磷酸化（通过蛋白激酶：PKA、PKG 和 PKC）、硝化和自动脱硝的影响，以及分子量、亚基、底物特异性和同工酶的测定。 (IV) 研究过氧亚硝酸盐处理对 cAMP 依赖性蛋白激酶 (PKA)、钙调蛋白和拓扑异构酶 1（或其他蛋白质/酶）活性的影响，以及它们在结合和 DNAS 电泳迁移率后活性的可能改变/恢复。从这项为期四年的研究中获得的见解可能有助于将来开发出有效预防/治疗上述疾病的方法。