SYNTHESIS OF DBH, CHROMOGRANINS AND CHROMAFFIN VESICLES

这个项目的长期目标是获得一个 对多巴胺合成调控因素的认识-- β-羟基酶和嗜铬粒蛋白A及其活性 结合到嗜铬小泡中。的原生文化 牛肾上腺髓质细胞将用于下列用途 具体目标：1)确定合成条件 膜结合型胸径、可溶性胸径和CGA及其相互作用 掺入嗜铬小泡；2)检测其影响 胆碱能刺激对DBH和CGA合成的影响及其机制 掺入嗜铬小泡；3)确定 铬囊泡膜的再利用；4)测定 无论是生长因子、皮质细胞还是蛋白质水平的变化 激酶活性影响DBH和CGA的合成和/或其 掺入嗜铬小泡；5)研究 DBH和CGA合成中的糖基化及其相互作用 结合到嗜铬小泡中。细胞培养将是 与(35S)蛋氨酸孵育及其在胸径中的整合 CgA将通过免疫沉淀和 单价多克隆抗血清。放射性纯度与定量 其中的免疫沉淀物将通过凝胶电泳法进行验证， 放射自显影和密度扫描。成立为法团 新合成的DBH和CGA将成为嗜铬囊泡 通过亚细胞分馏程序和 离心法纯化嗜铬小泡的研究 连续密度梯度。因为肾上腺髓质细胞 已经被证明是很好的生化和功能模型 外周和中枢儿茶酚胺能神经元，信息到 从这些研究中获得的将与这些组织相关，因为 井。CGA最近被证明广泛分布在 非儿茶酚胺类神经内分泌组织及其知识 合成并包装成嗜铬小泡也可以 与其他神经内分泌组织相关。

The long range objectives of this project are to obtain an understanding of the factors regulating the synthesis of dopamine- beta-hydroxylase (DBH) and chromogranin A (CgA) and their incorporation into chromaffin vesicles. Primary cultures of bovine adrenal medullary cells will be used to pursue the following specific objectives: 1) to define the conditions for the synthesis of membrane-bound DBH, soluble DBH and CgA and their incorporation into chromaffin vesicles; 2) to examine the effects of cholinergic stimulation on DBH and CgA synthesis and their incorporation into chromaffin vesicles; 3) to determine whether chromaffin vesicle membranes are reutilized; 4) to determine whether growth factors, cortical cells or altered levels of protein kinase activities affect DBH and CgA synthesis and/or their incorporation into chromaffin vesicles; 5) to examine the role of glycosylation in the synthesis of DBH and CgA and their incorporation into chromaffin vesicles. Cell cultures will be incubated with (35S)methionine and its incorporation into DBH and CgA will be quantified by immunoprecipitation with monovalent polyclonal anti-sera. The radiopurity and quantitation of the immunoprecipitates will be verified by gel electrophoresis, autoradiography and densitometric scanning. Incorporation of newly synthesized DBH and CgA into chromaffin vesicles will be determined by subcellular fractionation procedures and purification of chromaffin vesicles by centrifugation through continuous density gradients. Since adrenal medullary cells have been shown to be good biochemical and functional models of peripheral and CNS catecholaminergic neurons, the information to be gained from these studies will be relevant to these tissues as well. CgA has recently been shown to be widely distributed in non-catecholamine neuroendocrine tissues and knowledge of its synthesis and packaging into chromaffin vesicles may also be relevant to other neuroendocrine tissues.