The principal objective of this proposed research is to develop a new approach using immobilized fluorogenic substrates to study specific proteases and their relationships to some pathological conditions.
Specific aims of this project include: (1) To design and synthesize oligopeptide amides of 6-aminoquinolines as fluorogenic substrates that undergo selective hydrolysis by specific proteases such as plasminogen activators and elastases; (2) To test the specificity, sensitivity, and efficiency of the fluorogenic substrates by measurement of the rates of proteolysis in solution with purified enzymes; (3) To attach covalently the fluorogenic moiety of the substrates to solid supports such as polyacrylamide beads, collagen-hydroxyethylmethacrylate (HEMA) gels, and glass coverslips, thereby preventing diffusion into solution after enzymatic cleavage; (4) To evaluate effectiveness of immobilized fluorogenic substrates for proteolysis by quantitative fluorescence microscopy; (5) To develop new methods using immobilized fluorogenic substrates for biochemical characterization and single cell bioassay of proteases, and for identification of proteolytic activity in cells and tissues; and (6) To apply these methods to investigate the roles of plasminogen activators and elastases in the pathogenesis of neoplasia, inflammation, and pulmonary emphysema. The development of new methodology that can detect the release of proteases from individual cells, allow their cloning, and permit the investigation of factors that modulate protease secretion during various pathophysicalogical processes will be an important tool for cell biology as well as diagnostic and therapeutic research.
| Andrade-Gordon, P; Cox, M J; Brynes, P J et al. (1989) Immobilized fluorogenic substrates for proteinases: a method for visualization and quantitation of elastase release from human monocytes. Cell Mol Biol 35:431-40 |
