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J. Biol. Chem., Vol. 266, Issue 13, 8476-8482, 05, 1991
DS Adams, LA Griffin, WR Nachajko, VB Reddy and CM Wei
Using DNA synthesis technology we constructed two synthetic DNAs,
designated syn-uPA-DNA and mut-uPA-DNA. Syn-uPA-DNA contains the complete
coding sequence of human presecretion form of single-chain u- PA.
Mut-uPA-DNA was derived from syn-uPA-DNA by deleting 18 base pairs coding
for amino acids Arg179-Ser184. Each synthetic DNA was inserted into a
bovine papilloma viral genome-based expression vector to obtain expression
in mouse cells. The results indicate that both syn-uPA and mut-uPA proteins
are secreted predominantly in single-chain form. The single-chain form of
both enzymes can be completely converted to two- chain form by treatment
with plasmin. Both enzymes are as active as natural urokinase (std-uPA)
isolated from urine. Analysis of enzymatic activity indicates that under
conditions where syn-uPA and std-uPA are completely inhibited by
endothelial-type plasminogen activator inhibitor (PAI-1), mut-uPA retains
90% activity. In identical experiments with placental-type PAI (PAI-2),
mut-uPA retains 80% activity. Syn-uPA is capable of forming a approximately
100-kDa complex with PAI, whereas mut-uPA can not. PAI-treated mut-uPA has
kinetic properties similar to untreated syn-uPA or std-uPA. Overall, the
data indicate that amino acids Arg179-Ser184 function at least in part as a
binding site for PAI. Resistance to PAI inhibition may increase the potency
of mut-uPA as a thrombolytic agent.
A synthetic DNA encoding a modified human urokinase resistant to inhibition by serum plasminogen activator inhibitor
Transgenic Sciences, Inc., Worcester, Massachusetts 01608.
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