A comprehensive group of experiments has been designed to study the interactions with cellular components of a unique, intracellular class of lymphocyte stimulator, the C8-substituted guanine ribonucleosides. First, the existence of intracellular recognition structures or receptors specific for 8bromoguanosine (8BrGuo) will be evaluated. Using high specific activity (5-3H)8BrGuo, the saturability of binding to cellular components will be assessed. Average receptor number, apparent affinity, binding kinetics and receptor specificity will also be determined. These parameters will then be evaluated in B cells from nucleoside nonresponder mice and in a variety of B cell lines. Second, cellular receptors for 8BrGuo will be isolated and characterized. Cells will be biosynthetically labeled, disrupted by nitrogen cavitation, and passed over affinity columns. The eluate will be analyzed on HPLC gel filtration and reverse phase columns. Receptors in nucleoside nonresponder mice will be studied similarly. Alternatively, cellular constituents will be separated by electrophoresis, blotted onto nitrocellulose, overlaid with (U-14C)8BrGuo, and assessed by autoradiography. Receptors will be characterized by relative molecular mass, isoelectric point, pH sensitivity, apparent affinity, and rate of turnover. Third, direct interactions between 8BrGuo and cellular nucleic acid will be investigated. Binding studies with purified DNA and RNA will be performed, using nucleic acid from nucleoside nonresponder strains as controls. If binding is observed, it will be characterized in terms of the parameters listed below. Fourth, the interactions of C8-substituted guanine ribonucleosides with enzymes of purine metabolism will be studied. The role of phosphorylation of 8BrGuo, both by salvage pathway enzymes and by direct phosphorylation will be determined. The biologic and metabolic activity of a substituted synthetic nucleoside which cannot be directly phosphorylated will be evaluated. The effects of 8BrGuo on purine nucleotide generation and degradation, on the activity of salvage pathway enzymes, and on de novo purine biosynthesis will be evaluated. Finally, the biological interactions of C8-substituted guanine ribonucleosides with cytokines will be investigated both in terms of their ability to synergize with T cell-derived lymphokines and to induce secretion of monokines and lymphokines. Initial physical characterization of these activities will be undertaken.
