A proposal is made for an in depth study of family of high molecular weight plasminogen activators present in a variety of culture fluids from human cancer cell lines, and in a number of extracts of human solid tumors. The activators of interest range in size from 400,000 to several million daltons. The proposal covers 4 closely related topics: 1. Investigation of the structural organizaation. Preliminary analysis by SDS PAGE and column chromatography suggests that these enzymes represent a disulfide-linked family of multimers of an approx.2000,000 dalton peptide chain. Reduction with increasing concentrations of dithiothreitol in the presence and absence of SDS will be used to verify this interpretation. Since the enzymes are partially inhibitable by anti-urokinase antibody, emphasis will be laid on answering the question whether the activity is due to an Mr 55,000 urokinase (or urokinase-like chain) which is disulfide-linked to a carrier, or whether the activator sequence is an integral part of the 212,000 chain. The approach will be a combination of SDS-PAGE of both reduced and unreduced enzyme in conjunction with zymographic visualization of enzyme activity, and autoradiographic localization of 3H-DFP label in the native and reduced chains on the same material. DFP-labeling will also be used to determine the number of active sites/mg protein. Furthermore, we will investigate whether the remarkable change in substrate specificity in some of the members of the group upon exposure to SDS, or limited tryptic digestion, are accompanied by changes in active site reactivity as measured by 3H-DFP uptake. 2. Monoclonal antibodies against the large anzymes. These will be raised to be used in the development of an ELISA for screening pathological material for the presence of these enzymes, and to be used a immunosorbents for affinity purification. 3. cDNA cloning. In order to determine the amino acid sequence of the basic chain constituent of these enzymes, and compare it with that of urokinase, cloning of cDNA will be undertaken, starting with the isolation of poly A+ mRNA from a cell line which produces large amounts of these activators, using antibodies against the latter. The authentic plasminogen activator gene inserts will be selected from the resulting cDNA-containing clones by a) hybridization of cDNA-containing plasmid DNA-s to authentic mRNA and identifying in vitro synthesized protein by specific binding to antibody, or b) binding antibody to proteins made by bacterial colonies which contain cDNA that had been inserted into an expression vector.
