No abstract

Immunocytochemistry, using rabbit antibodies to a urokinase-type 48-Kdalton Mr mouse plasminogen activator, showed that enzyme immunoreactivity is widely distributed in the normal mouse. Strong staining was obtained in widely disseminated connective tissue cells with a fibroblast-like morphology . Such cells occurred in high numbers in the lamina propria mucosae of the gastrointestinal tract, and in moderate numbers in the connective tissue septa of the pancreas . A few such cells were detected around the larynx, trachea, and bronchi . Immunoreactivity also occurred in epithelial cells of the proximal and distal kidney tubules, the ductus deferens, and in pulmonary pneumocytes . In addition, presumably extracellular staining was seen irregularly along the basement membrane and fibrillar structures in the lamina propria of the small and large intestines . Moreover, decidual cells of the mouse placenta stained strongly, and a moderate staining was observed in epithelial cells of involuting mammary glands, but not in those of noninvoluting glands . No immunoreactivity was observed in endothelial cells.Control experiments included absorption of the antibodies against highly-purified mouse plasminogen activator and the corresponding proenzyme, and the finding of a good correspondence between the number of immunoreactive cells and measurable enzymatic activity determined in adjacent tissue sections . Separation by SDS PAGE followed by immunoblotting revealed only one immunochemically stainable protein band with Mr^-48 Kdaltons in extracts from tissues showing immunoreactivity .

No abstract

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.