Urokinase-plasminogen activator (uPA) associated with surface receptors of various types of tumor cells is generally considered to involve in cellular invasion by braking down proteins of extracellular matrix. Other proteases such as collagenases or gelatinases are also important in the destruction of the surrounding architecture. To answer the question whether uPA is able to activate the latent matrix metalloproteinases (MMP) associated with a metastatic carcinomatous cell line, Detroit 562, affinity purified MMPs on a column of gelatin-Sepharose from the conditioned medium or membrane fractions were used to test either by cellular phosphorylated uPA (p-uPA) or urinary uPA. In comparison with this, the latter was labeled with biotin and phosphorylated by the incubation with materials co-precipitated with antipp60c'src antibody or with p43v-aW. They were evaluated by blots of SDSPAGE gels by use of avidin-conjugated peroxidase or anti-mouse IgG conjugated with peroxidase following by enhanced chemiluminescence. MMPs were examined by gelatin-containing zymography and immunoblots using antibodies against MMPs. Both the plasminogen and uPA or p-uPA were immobilized on disks of nitrocellulose filters and incubated with the MMP. Activation of plasminogen was examined by use of a synthetic substrate, S-2251, and that of MMPs was by determinations at 420 nm of hydrolysates of a synthetic substrate (PZ-PLGPR). MMP-2 and MMP-9 were identified to be associated with membrane fractions and they were found to be activated solely by the p-uPA. Addition of plasminogen did not enhance the activation of MMPs. It was also able to activate plasminogen at a higher rate than that of unphosphorylated one. Inhibitors (PAI-1 and PAI-2) did not interfere the p-uPA activity. Thus, it suggests that inhibitor resistant p-uPA and MMPs may involve in a concomitant functional network of proteases on cell surfaces enabling an efficient hydrolysis of matrix proteins.
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