BIOLOGICAL ROLE OF NEUROCATIN--NOVEL BRAIN PEPTIDE

这项研究计划的目的是描述一种神经调节 从哺乳动物脑中分离出来的一种因子（神经兴奋素）。 神经激动素调节 多巴胺和去甲肾上腺素的释放和代谢， 从大鼠脑中分离的突触体。 它似乎至少在一定程度上， 通过增加质膜的钙渗透性。 的 该项目的具体目标是： 1. 纯化的neurocatin的表征。 神经麻醉已经被净化了 纯度>90%，并测定了其氨基酸组成。 由于电流 如果有证据表明它是一种肽，所采用的程序将包括： a）。 氨基酸序列测定。 B）。 通过标准方法从氨基酸合成神经卡汀或 肽合成 （c）。 验证合成化合物的活性相同 与天然化合物相同。 在确定化学结构后，放射性标记的神经激动素 将通过将放射性氨基酸掺入 合成. 该放射性标记化合物将用于测量 高亲和力结合位点存在于突触体的膜， 表征受体结合并放射自显影绘制 受体（高亲和力结合位点）在大鼠中的分布 个脑袋 将开发神经卡素的抗体，这些抗体将用于筛选 脑组织中是否存在其他表达交叉的分子 与神经卡素抗体的反应性。 免疫组织化学定位 将被用来确定，在大鼠大脑中的物种分布 与神经卡素抗体反应 2. 纯化因子生物学作用的测定。 证据 已经发现神经阻滞素增加了儿茶酚胺的释放 并诱导其代谢的重大改变。 的 改变包括抑制多巴胺的氧化脱氨基作用， 减少3，4-二羟基苯乙酸（DOPAC）的形成，以及 增加去甲肾上腺素及其N-甲基衍生物的形成。 我们将确定代谢途径的步骤， 神经激动素的表达以及每种神经激动素的活性的机制 反应被调制。 我们的研究将包括表征 质膜和蛋白质的Ca 2+渗透性变化 在神经激动素的存在下磷酸化。 关于后者， 我们将：1。 定量蛋白质的相对标记， SDS-PAGE凝胶电泳后的密度测定， 放射自显影，2. 测量诱导的磷酸化的剂量反应 通过添加因子PW，3. 确定蛋白激酶的类型， 活化的（无论磷酸化发生在丝氨酸、酪氨酸或 苏氨酸残基）和4.确定途径中的酶， 儿茶酚胺合成和可能被磷酸化的儿茶酚。

The aim of this research program is to characterize a neuroregulatory factor (neurocatin) isolated from mammalian brain. Neurocatin modulates both the release and metabolism of dopamine and norepinephrine in synaptosomes isolated from rat brain. It appears to act, at least in part, by increasing the calcium permeability of the plasma membrane. The specific goals of this project are: 1. Characterization of purified neurocatin. Neurocating has been purified to >90% purity and its amino acid composition determined. Since current evidence indicates it is a peptide, the procedures employed will include: a). Determination of the amino acid sequerice. b). Synthesis of neurocatine from amino acids by standard methods or peptide synthesis. c). Verification that the activity of the synthetic compound is the same as that of the native compound. Following determination of the chemical structure, radiolabeled neurocatin will be prepared by incorporation of radioactive amino acid in the synthesis. This radiolabeled compound will be used to measure the number of high affinity binding sites present in the membranes of synaptosomes, to characterize the receptor binding and to autoradiographically map the distribution of the receptors (high affinity binding sites) in the rat brain. Antibodies will be developed to neurocatin and these will be used to screen brain tissue for the presence of other molecules expressing cross reactivity with the antibodies to neurocatin. Immunohistochemical mapping will be used to determine, in rat brain the distribution of species reacting with antibodies to neurocatin. 2. Determination of the biological role of purified factor. Evidence has already been obtained that neurocatin increase release of catecholamines from synaptosomes and induces major alterations in their metabolism. The alteration include inhibition of oxidative deamination of dopamine, decreased formation of 3,4-dihydroxyphenylacetic acid (DOPAC), and increased formation of norepinephrine and its N-methyl derivatives. We will determine the step of the metabolic pathways at which control by neurocatine is expressed and the mechanism(s) by which the activity of each reaction is modulated. Our studies will include characterization of the Ca2+ permeability changes in the plasma membrane and of the proteins phosphorylated in the presence of neurocatine. With respect to the latter, we will: 1. quantitate the relative labelling of the proteins by densitometry after SDS-PAGE gel electrophoresis followed by autoradiography, 2. measure the dose-response of phosphorylation induced by addition of factor PW, 3. determine the type of protein kinase which is activated (whether phosphorylation occurs at the serine, tyrosine or threonine residues) and 4. identify enzymes in the pathway for catecholamine synthesis and catabolism which may be phosphorylated.