CHARACTERIZATION OF RECOMBINANT HUMAN SINGLE-CHAIN LOW MOLECULAR WEIGHT UROKINASE (RE-SC-LUK)
RE-SC-LUK obtained from recomoinant b. con Bacteria showed a molecular mass similar to that of recombinant two-chain LUK (RE-TC-LUK) as judged from SDS-PAGE. By “Western“ blot analysis immunoreactivity of RE-SC-LUK was observed with monoclonal antibodies directed against the B chain but not with those against the A1 chain of urokinase. N-terminal sequence analysis c RE-SC-LUK showed identity to the A, chain of RE-TC_LUK and provided evidence for its single-chain nature, i.e. integrity of the Lys-Ile bond which is split in TC-UK. In all other respects structural identity of RE-SC-LUK and RE-TC-LUK was demonstrated by fingerprinting of fragments. Similar to recombinant pro-urokinase (RE-SCU-PA), RE-SC-LUK exhibits only marginal amidolytic activity, which is greatly enhanced by treatment with plasmin, but considerable fibrinolytic activity in a fibrin agar plate test.Thus, RE-SC-LUK is characterized as a fragment (residues 136 -411) of RE-SCU-PA, which lacks the “growth factor” and “kringle” domains. Moreover further evidence is provided that a free N-terminus of the B chain is essential for amidolytic but not for fibrinolytic activity of urokinase in more complex systems.