SYNTHESIS OF DBH, CHROMOGRANINS AND CHROMAFFIN VESICLES

该项目的长期目标是获得 了解调节多巴胺合成的因素- β-羟化酶（DBH）和嗜铬粒蛋白A（CgA）及其 并入嗜铬小泡。 的原代培养物 牛肾上腺髓质细胞将用于以下目的 具体目标：1）确定合成条件 膜结合胸径、可溶性胸径和CgA及其 掺入嗜铬小泡; 2）检查影响 胆碱能刺激对DBH和CgA合成的影响 掺入嗜铬小泡; 3）以确定是否 嗜铬小泡膜被重新利用; 4）以确定 无论是生长因子、皮层细胞还是蛋白质水平的改变 激酶活性影响DBH和CgA合成和/或其 掺入嗜铬小泡; 5）检查的作用 DBH和CgA合成中的糖基化及其 并入嗜铬小泡。 细胞培养物进行 用（35 S）蛋氨酸孵育并掺入DBH 和CgA将通过免疫沉淀进行定量， 单价多克隆抗血清。 放射性纯度和定量 将通过凝胶电泳验证免疫沉淀物的量， 放射自显影和密度扫描。 掺入 新合成的DBH和CgA进入嗜铬小泡将是 通过亚细胞分级分离程序测定， 通过离心纯化嗜铬小泡， 连续密度梯度 因为肾上腺髓质细胞 已被证明是良好的生物化学和功能模型， 外周和中枢神经系统的儿茶酚胺能神经元，信息， 从这些研究中获得的信息将与这些组织相关， 好. CgA最近被证明广泛分布于 非儿茶酚胺神经内分泌组织及其知识 合成和包装到嗜铬囊泡中也可以 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.