CELLULAR REGUATION OF TYROSINE HYDRPOXYLASE

酪氨酸羟化酶的细胞调节

• 批准号: 3477005
• 负责人: JOHN W HAYCOCK
• 金额: $ 7.38万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3477005
• 关键词: acetylcholine; adrenal glands; catecholamines; chromaffin cells; chromatography; cow; enzyme inhibitors; enzyme mechanism; enzyme substrate; gel electrophoresis; high performance liquid chromatography; immunochemistry; microinjections; molecular site; neurotransmitter biosynthesis; nicotinic receptors; phosphorylation; protein kinase; radiotracer; secretion; tissue /cell culture; tyrosine 3 monooxygenase

The long-term objectives of the research project are to describe the mechanisms by which catecholamine-containing neurons compensate for the diminution of endogenous catecholamine stores consequent to activity-related secretion of catecholamines. The compensatory replenishment of catecholamine stores results from an increase in catecholamine biosynthesis, the underlying basis of which is an increase in the catalytic activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Stimulation-dependent phosphorylation of tyrosine hydroxylase is one mechanism that catecholaminergic cells employ to activate tyrosine hydroxylase. The specific objective of the proposed research is to determine the phosphorylation sites and the protein kinase system(s) involved in the phosphorylation of tyrosine hydroxylase in intact bovine adrenal chromaffin cells as stimulated by acetylcholine (the splanchnic nerve neurotransmitter). Limit tryptic digestion will be used to produce tyrosine hydroxylase phosphopeptides. After separation of the peptides, site-specific phosphorylation of tyrosine hydroxylase produced by acetylcholine in situ will be compared with site-specific phosphorylation of tyrosine hydroxylase produced in situ by selective activators of specific protein kinases and in vitro by various purified protein kinases. In addition, the effects of injecting purified protein kinases and protein kinase inhibitors directly into chromaffin cells will be evaluated for effects upon tyrosine hydroxylase phosphorylation and catecholamine biosynthesis. These data should allow identification of the protein kinase system(s) involved in regulating tyrosine hydroxylase phosphorylation in situ. Such studies should provide important insight into the regulation of catecholamine biosynthesis as well as a firm biochemical basis for the further study of the relationship between the phosphorylation and catalytic activity of tyrosine hydroxylase with reference specifically to its regulation in situ.

该研究项目的长期目标是描述 含有儿茶酚胺的神经元 补偿内源性儿茶酚胺的减少 由于活动相关的儿茶酚胺分泌而储存。 补充的儿茶酚胺商店的结果 由于儿茶酚胺生物合成的增加， 其基础是酪氨酸的催化活性增加 羟化酶，儿茶酚胺的限速酶 生物合成 刺激依赖性酪氨酸磷酸化 羟化酶是儿茶酚胺能细胞 用于激活酪氨酸羟化酶。 具体目标 这项研究的目的是确定磷酸化位点 和参与磷酸化的蛋白激酶系统， 完整牛肾上腺嗜铬细胞酪氨酸羟化酶 由乙酰胆碱（内脏神经）刺激 神经递质）。 限制胰蛋白酶消化将用于产生酪氨酸 羟化酶磷酸肽。 在肽分离后， 酪氨酸羟化酶的位点特异性磷酸化 乙酰胆碱原位将与位点特异性 酪氨酸羟化酶的磷酸化 特异性蛋白激酶的选择性激活剂， 各种纯化的蛋白激酶。 此外， 注射纯化的蛋白激酶和蛋白激酶抑制剂 将评估直接进入嗜铬细胞对 酪氨酸羟化酶磷酸化和儿茶酚胺 生物合成 这些数据应允许鉴定蛋白激酶 参与调节酪氨酸羟化酶的系统 原位磷酸化。 这些研究将提供重要的 对儿茶酚胺生物合成的调节也有了新的认识 作为进一步研究的坚实的生物化学基础， 磷酸化和催化活性之间的关系 酪氨酸羟化酶及其调节 在原地。