REGULATION OF BIOSYNTHESIS OF DOPAMINE-BETA-HYDROXYLASE

阐明参与生物合成调控的机制 多巴胺β-羟基酶(DBH)是理解因子的核心 调控去甲肾上腺素能神经元的发育和表达 系统。拟议的研究将： 1.分离大鼠胸径基因克隆并测序，首次推测 时间，该酶的氨基酸序列。Lambdagt11表达式 从PC12嗜铬细胞瘤细胞mRNA中制备的cDNA文库将 用抗胸径抗体筛选大鼠胸径基因。 2.表征多个亚基之间的生化差异 最近由我们和其他人展示的胸径形式 PC12嗜铬细胞瘤细胞、牛肾上腺髓质和大鼠 大脑。我们将研究这种改变是否由于C-C- 或将77K形式锚定在膜上的N-末端多肽，以及 亚基形式是否被差异糖基化。第一 有可能利用基于氨基酸的特定肽图 序列。糖基化将通过内切糖苷酶消化来研究。 放射性标记胸径和特定的糖标记实验。可能的 还将检查O-连接低聚糖的存在。 3.确定翻译后加工的亚细胞位置 并研究其与神经肽加工的关系。 相对特定的抑制剂，如莫能菌素、氯喹和 将使用衣霉素。 4.林木胸径多亚基生物合成规律的研究 蓝斑，大脑中去甲肾上腺素能神经元的主要中枢。 改变大脑中去甲肾上腺素水平的因素的影响将是 检查过了。 5.研究在一定条件下胸径特异性基因表达水平的调控 这可能导致对胸径活性的长期调节。 肾上腺髓质中糖皮质激素的调节；b.个体发育 肾上腺髓质和蓝斑的胸径发育；c. 利血平对蓝斑和肾上腺髓质的影响 精神分裂症患者的胸径。 6.胸径基因的基因组结构测定。这应该是 提供有关可能存在的共同外显子的信息 儿茶酚胺生物合成酶。

Elucidating the mechanisms involved in the regulation of biosynthesis of dopamine beta-hydroxylase (DBH) is central in understanding factors controlling the development and expression of the noradrenergic neuronal system. The proposed research will: 1. Isolate and sequence a cDNA clone for rat DBH, to deduce, for the first time, the amino acid sequence of this enzyme. A lambdagt11 expression library of cDNA prepared from PC12 pheochromocytoma cell mRNA will be screened with antibodies to DBH to isolate the cDNA for rat DBH. 2. Characterize the biochemical differences between the multiple subunit forms of DBH which have recently been shown by us and others to be present in PC12 pheochromocytoma cells, and in bovine adrenal medulla and rat brain. We will examine whether the alteration is due to cleavage of a C- or N-terminal polypeptide anchoring the 77K form in the membrane, and whether the subunit forms are differentially glycosylated. The first possibility will utilize specific peptide mapping, based on the amino acid sequence. The glycosylation will be studied by endoglycosidase digestions of radiolabeled DBH, and specific sugar labeling experiments. The possible presence of O-linked oligosaccharide also will be examined. 3. Determine the subcellular site of the post-translational processing of DBH and examine its relationship to the processing of neuropeptides. Relatively specific inhibitors such as monensin, chloroquine, and tunicamycin will be used. 4. Study the regulation of biosynthesis of multiple subunit forms of DBH in the locus coeruleus, a major center of noradrenergic neurons in the brain. The effect of factors which alter noradrenaline levels in the brain will be examined. 5. Study the regulation of specific mRNA levels for DBH under conditions which have been suggested to lead to long-term regulation of DBH activity. a. Regulation by glucocorticoids in the adrenal medulla; b. Ontogenetic development of DBH in the adrenal medulla and locus coeruleus; c. Effect of reserpine on the locus coeruleus and the adrenal medulla; d. Alteration of DBH in schizophrenia. 6. Determination of the genomic organization of DBH genes. This should provide information regarding the possible existence of common exons for catcholamine biosynthetic enzymes.