The long-term objective of this project is to learn how hormones stimulate steroidogenesis. During this project, we have been studying the ability of ACTH and cyclic AMP to stimulate steroidogenesis in gamma-1 adrenal tumor cells. In addition, to assess the generality of the findings, we have been comparing the results obtained with those obtained after gonadotropin (hCG) induction of steroidogenesis in Leydig cells. These cell types were chosen because they provide model systems for studying cyclic AMP-dependent and -independent steroidogenesis. The protocols used were designed to measure protein phosphorylation in response to various stimuli, using a newly devised two-dimensional electrophoresis double-labeling procedure. This procedure results in the radiolabeling of 200 to 300 phosphoproteins which can be separated and quantified. Results obtained indicate that the amounts of at least six phosphoproteins are reproducibly increased in response to ACTH, hCG or cyclic AMP. Alterations in radiolabeling have thus far been detected by visual inspection of autoradiographs of the two-dimensional gels. Given the complex interaction between cyclic AMP-dependent and -independent protein kinases, we anticipate that the phosphorylation state of larger numbers of proteins is altered after stimulation. To detect this and to make full use of the advantages of the double-label procedure, we are developing a computer-assisted optical image scanning system which has the ability to accurately quantify the density of all phosphoprotein spots seen in the autoradiographs and then to calculate the amount of each radiolabel in each spot. Studies with standards indicate that the double-label procedure will enable us to detect changes of 10% or less. A second aspect of these studies was to learn if the hormone-induced changes in phosphoproteins were reversible when the stimulus was removed. To accomplish this goal, we began to develop monoclonal antibody methods which could be used to rapidly strip hormones from the cell surface. During these studies, we observed that mixtures of monoclonal antibodies form a unique high-affinity complex with antigens. Aside from the experimental use we have for this observation, it provides the potential for a very sensitive assay of hCG which may have a practical medical use. Studies with pure hCG standards indicate that this assay may be useful for diagnosing and following patients with hCG-secreting tumors.