REGULATION OF BIOSYNTHESIS OF DOPAMINE BETA-HYDROXYLASE

阐明了参与调节生物合成的机制， 多巴胺β-羟化酶（DBH）是理解 控制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.分离并测序了大鼠胸径的cDNA克隆， 时间，这种酶的氨基酸序列。 一个古怪的表情 从PC 12嗜铬细胞瘤细胞mRNA制备的cDNA文库将被 用抗DBH抗体筛选以分离大鼠DBH的cDNA。 2.描述多亚基之间的生化差异 我们和其他人最近已经证明存在的DBH形式 在PC 12嗜铬细胞瘤细胞、牛肾上腺髓质和大鼠中 个脑袋 我们将研究这种改变是否是由于C- 或将77 K形式锚定在膜中的N-末端多肽，和 无论亚基形式是否被差异糖基化。 第一 可能性将利用基于氨基酸的特异性肽图谱， 顺序 糖基化将通过内切糖苷酶二聚体进行研究 放射性标记的DBH，和特定的糖标记实验。 可能的 还将检查O-连接的寡糖的存在。 3.确定翻译后加工的亚细胞位点 DBH和检查其与神经肽加工的关系。 相对特异性的抑制剂，如莫能菌素、氯喹， 将使用衣霉素。 4.研究不同亚基形式胸径生物合成的调控 蓝斑，大脑中去甲肾上腺素能神经元的主要中心。 改变大脑中去甲肾上腺素水平的因素的影响将是 考察 5.研究不同条件下对胸径特异性mRNA水平的调控 这已经被认为导致DBH活性的长期调节。 a.肾上腺髓质中糖皮质激素的调节; B.个体发育 肾上腺髓质和蓝斑中DBH的发展; c.的影响 蓝斑和肾上腺髓质上的利血平; d.改变 精神分裂症的DBH 6. DBH基因的基因组结构的确定。 这应该 提供关于可能存在共同外显子的信息， catcholamine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.