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.

长期的目标是确定蛋白脱硝酶、蛋白硝酸酶(或相关酶)在逆转某些NO/过氧硝酸盐(PN)介导的反应(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氏症、帕金森氏病以及前列腺癌的发病机制之间的关系。具体目标是：(I)测试阿尔茨海默氏症和帕金森氏症以及前列腺癌的发病机制。具体目的是：(I)测试底物(经PN处理的蛋白质：组蛋白H1、BSA等)。谷胱甘肽S转移酶(I型，冗余依赖蛋白硝酸酶)的α、pi、psi亚型新的蛋白脱氮活性的特异性。反硝化活性可以通过硝酸盐的产生和Western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、小脑和狗前列腺中蛋白硝酸酶(II型，冗余独立)的亚细胞(颗粒和胞浆)分布。采用DEAE-纤维素层析法、硫酸铵分级层析法、分子排斥层析法和亲和层析法从粗提物中分离纯化该酶。表征将包括辅助因素、pH、蛋白分解(由m-calain)、磷酸化(由蛋白激酶：PKA、PKG和PKC)、硝化和自动反硝化的影响，以及分子质量、亚基、底物专一性和同工酶的测定。(Iv)研究过氧亚硝酸盐处理对cAMP依赖的蛋白激酶(PKA)、钙调素和拓扑异构酶-1(或其他蛋白质/酶)活性的影响，以及它们结合后活性和DNA凝胶迁移率的可能变化/恢复。通过这项为期4年的研究所获得的洞察力，可能有助于今后开展上述疾病的有效预防/治疗。