EARLY BIOCHEMICAL EVENTS DURING LYMPHOCYTE ACTIVATION

我们的目的是研究人类外周血中的早期生化改变 淋巴细胞，这可能是重要的传播和传播 淋巴细胞活化的信号从外到内 淋巴细胞 重点将放在环核苷酸代谢， 谷胱甘肽代谢和这两个系统之间的相互作用。 活化淋巴细胞中特异性蛋白质的磷酸化已被证实是一种重要的生物学机制。 存在于可溶性和颗粒部分中。 可溶性 来自与（32 p）孵育的淋巴细胞的65，000道尔顿磷蛋白（pp 65） 正磷酸盐并用促有丝分裂凝集素处理的细胞将用 硫酸铵沉淀，离子交换色谱和制备 SDS-聚丙烯酰胺凝胶电泳。 针对pp 65的抗体 将被制备并用于在添加 促有丝分裂剂和随后的活化。 颗粒磷蛋白 也将被纯化，针对每个物种的抗体将被 用于确定这些物种位置的变化。 使用 降低淋巴细胞还原型谷胱甘肽（GSH）水平特异性方法， 将检查谷胱甘肽含量降低对环磷酸腺苷的影响， cGMP积累，蛋白激酶活性，蛋白磷酸化，和 静息淋巴细胞、孵育淋巴细胞中磷蛋白的迁移 与促有丝分裂凝集素、非促有丝分裂凝集素和增加 细胞内cAMP，但不促有丝分裂。 使用内源性标记（35 S） 谷胱甘肽，我们将研究蛋白质谷胱甘肽的形成和溶解 混合二硫化物 与GSH相互作用并被标记的蛋白质 在淋巴细胞活化过程中将被识别并与蛋白质进行比较 新磷酸化的蛋白质。 此外，我们将确定这些 蛋白-GSH混合二硫化物是参与环核苷酸合成的酶。 产生或环核苷酸依赖性或非依赖性磷酸化。 我们期望这些研究将提供更多的见解， 进入活化过程，提供新的研究方法， 免疫缺陷状态，并最终导致新的变化， 操纵免疫反应的方法。

We purpose to study early biochemical alterations in human peripheral blood lymphocytes which may be important for the transmission and propagation of the signal for lymphocyte activation from the exterior to the interior of the lymphocyte. Major emphasis will be on cyclic nucleotide metabolism, glutathione metabolism and the interaction between these two systems. Phosphorylation of specific proteins in activated lymphocytes have been occurring in both the soluble and particulate fractions. The soluble 65,000 dalton phosphoprotein (pp65) from lymphocytes incubated with (32p) orthophospate and treated with mitogenic lectins will be purified using ammonium sulfate precipitation, ion exchange chromatography and preparative SDS-polyacrylamide gel electrophoresis. Antibodies directed against pp65 will be prepared and used to localize this protein prior to addition of mitogenic agents, and following activation. Particulate phosphoproteins will also be purified and antibodies directed against each species will be utilized to determine changes in the location of these species. Using a specific method for lowering lymphocyte reduced glutathione (GSH) levels we will examine the effect of depressed glutathione content on cyclic AMP and cGMP accumulation, protein kinase activity, protein phosphorylation, and migration of phosphoproteins in resting lymphocytes, lymphocytes incubated with mitogenic lectins, nonmitogenic lectins, and agents which increase intracellular cAMP but are not mitogenic. Using endogenously labeled (35S) glutathione we will examine the formation and dissolution of protein-GSH mixed disulfides. Proteins which interact with GSH and become labeled during lymphocyte activation will be identified and compared with proteins which are newly phosphorylated. In addition we will ascertain if these protein-GSH mixed disulfides are enzymes involved in cyclic nucleotide generation or cyclic nucleotide dependent or independent phosphorylation. It is our expectation that these studies will provide additional insight into the activation process, provide new approaches to the study of immunologic deficiency states in man and eventually to new changes and approaches to the manipulation of the immune response.