Regulatory mechanisms of protein synthesis and secretion are investigated by combining biochemical and molecular biology techniques with morphological studies. New in vitro translation products whose synthesis is direct by mRNA after stimulation by agents whose action is mediated via the cyclase system, are identified using characterized monoclonal antibodies to acinar cell secretory proteins. Molecular hybridization studies are carried out using a cDNA probe prepared by the reverse transcription of poly A+ RNA. Intracellular staging of stimulus-responsive secretory protein synthesis and processing is followed electron microscopically using immunological probes labeled with colloidal gold. Cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, E.C. 2.7.1. 37) activity, localization and endogenous substrate responses are being measured in various tissues, under different stimulation conditions ranging from in viro effects of pharmacologic agents to changes resulting from altered gravity conditions of animals subjected to space flight. Comparisons of posttranslational modifying enzyme effects are made afer measuring activities of transglutaminase. Exocytosis can be regulated by agents which either undergo receptor interactions or involve cell membrane adenylate cyclase directly. Mechanisms of action of these stimuli appear to be cyclic AMP-dependent reactions involving gene regulation by cyclic AMP-mediated phosphorylation of cellular proteins.
