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.

长期目标是建立蛋白质否定酶，蛋白质硝酸酶（或相关酶）的保护/调节作用，以逆转某些NO/过氧性反应（例如硝酸盐反应）（例如硝基酪氨酸形成），并确定这些疾病的缺乏症与埃尔兹（Enzysis）的缺乏症之间的关系，使其具有疾病的缺乏症。和前列腺癌。具体目的是：（i）测试阿尔茨海默氏症和帕金森氏病的发病机理以及前列腺癌。具体目的是：（i）测试底物（PN处理的蛋白质：组蛋白H1，BSA等）针对α，PI，PI，PSI同种型S-转移酶（I型I型，冗余依赖性蛋白质氮）的新型蛋白质脱硝化活性的特异性。可以通过硝酸盐的产生和抗硝基酪氨酸的免疫反应性降低来监测分解活性。 （ii）比较牛脑皮质，中脑和小脑以及前列腺的蛋白质硝酸酶（II型，多余的独立）的亚细胞（颗粒和胞质）分布。 DEAE-纤维素色谱，硫酸铵分馏，分子排除色谱和亲和色谱法将用于从粗提取物中净化酶。表征将包括共同因素，pH，蛋白水解（通过M-钙蛋白酶）的影响，磷酸化（通过蛋白激酶：PKA，PKG和PKC），硝化和自身抑制作用以及分子量，亚基，底物特异性和同酶的测定。 （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/酶）的影响，以及在结合和DNAS电泳动机后的活性的可能改变/恢复。从这项为期四年的研究中获得的洞察力可能有助于在将来对上述疾病的有效预防/治疗。