CELLULAR REGULATION OF TYROSINE HYDROXYLASE

研究项目的长期目标是描述 儿茶酚胺神经元的作用机制 弥补内源性儿茶酚胺的减少 由于与活动相关的儿茶酚胺的分泌而产生的存储。 补充儿茶酚胺的效果 从儿茶酚胺生物合成的增加，潜在的 其基础是酪氨酸的催化活性增加 儿茶酚胺中的限速酶--羟基酶 生物合成。刺激依赖的酪氨酸磷酸化 羟基酶是儿茶酚胺能细胞的机制之一 用来激活酪氨酸羟基酶。具体目标 建议的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(S)。 正常牛肾上腺嗜铬细胞酪氨酸羟化酶的研究 乙酰胆碱(内脏神经)刺激 神经递质)。 有限的胰酶消化将用于生产酪氨酸 羟基酶磷酸肽。在分离多肽后， 产酪氨酸羟化酶的位点特异性磷酸化 将原位乙酰胆碱与特定部位的乙酰胆碱进行比较 原位合成酪氨酸羟基酶的磷酸化 特异性蛋白激酶的选择性激活剂和体外 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 直接进入嗜铬细胞的效果将被评估 酪氨酸羟基酶磷酸化与儿茶酚胺 生物合成。 这些数据应该可以用来鉴定蛋白激酶 参与酪氨酸羟化酶调节的系统(S) 原位磷酸化。这样的研究应该提供重要的 对儿茶酚胺生物合成调控的认识 作为进一步研究的坚实的生化基础。 磷酸化与催化活性的关系 酪氨酸羟基酶及其调控 在原地。

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.