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.
