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J. Biol. Chem., Vol. 262, Issue 12, 5682-5689, 04, 1987
L Nelles, HR Lijnen, D Collen and WE Holmes
The cDNA encoding full-length single chain urokinase-type plasminogen
activator (scu-PA) was cloned and sequenced, and the recombinant scu-PA
(rscu-PA) was expressed in Chinese hamster ovary cells. Two mutants,
constructed by in vitro site-specific mutagenesis of Lys158 in rscu-PA to
Gly158 (rscu-PA-Gly158) or to Glu158 (rscu-PA-Glu158), were also expressed
in Chinese hamster ovary cells. Wild type and mutant rscu-PAs were purified
to homogeneity by immunoadsorption on an insolubilized monoclonal antibody
raised against natural scu-PA (nscu-PA), followed by gel filtration. The
specific activity of the mutant scu-PAs on fibrin plates is very low (less
than 1,000 IU/mg) compared to that of the wild type rscu-PA (44,000 IU/mg).
The mutants, in contrast to the wild type rscu-PA, are not converted to
amidolytically active two chain u-PA (tcu-PA) by plasmin and do not cause
lysis of a 125I-fibrin- labeled plasma clot immersed in citrated plasma.
However, in a purified system, both rscu-PA-Gly158 and rscu-PA-Glu158
activate plasminogen following Michaelis-Menten kinetics, with a much lower
affinity (Km = 60-80 microM) but with a higher turnover rate constant (k2 =
0.01 s-1) as compared to the wild type rscu-PA (Km = 1.0 microM, k2 = 0.002
s-1). We conclude that conversion of scu-PA to tcu-PA is not a prerequisite
for the activation of plasminogen. Substitution of Lys158 by Gly158 or
Glu158 does, however, markedly decrease the stability of the Michaelis
complex.
Characterization of recombinant human single chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of lysine 158
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