The long-range goal of this research is to regulate the loss of nitrogen, wasting complications and tissue destruction associated with the juvenile-onset insulin-dependent from of diabetes mellitus and other diseases. Proteinases are intrinsically involved in normal and pathological processes of extensive protein degradation, as well as in limited degradation that occurs in protein maturation, enzyme and polypeptide activation or inactivation, and processes that occur at the cell surface. My approach is to identify and characterize cellular proteinases and mechanisms used by living systems to hydrolyze polypeptides, and to determine factors that control the rate of degradation of specific enzymes according to the metabolic needs of the organism. Aspects of this goal include determination of the initial or rate-limiting steps in the process of enzyme and polypeptide degradation, the influence of hormones and the diabetic state on protein turnover, types of proteinases associated with degradation in cells, and the identification of physiological substrates and specific functions of proteinases. In this project, we propose to microinject radiolabelled muscle aldolase into mammalian cells in culture and use this protein as a model cytosolic protein to determine how hormones and the diabetic state affect degradation. The technique allows us to study intermediate forms. Native and oxidized (denatured) forms of aldolase will be microinjected into cells from normal and diabetic mice to determine if the denatured forms are recognized differentially in the two types of cells. The effects of various ratios of insulin:glucagon, amino acid concentrations in the media, and proteinase inhibitors on degradation of aldolase will be analyzed. In addition, the role of a plasma membrane proteinase, meprin, in polypeptide hormone degradation and action will be investigated. Meprin is one of the few plasma membrane proteinases that has been purified and characterized. Most recently we discovered that active and latent forms of the enzyme exist is plasma membranes. We propose to study the interaction of these forms with insulin and other polypeptide hormomes, to compare the proteinase with plasma membrane polypeptide receptors, and to determine the relationship of meprin to a kinase-splitting membranous proteinase. These studies will provide fundamental knowledge on the role of proteinases in the mediation o cellular events, in hormone action, and in control of protein turnover in mammalian cells that related to our long-term goal of regulating the degradative process.
