The Escherichia coli HU-1 was cloned by use of mixed synthetic oligonucleotides (17-mer) predicted from a portion of its amino acid sequence. The amino acid sequence of the HU-1 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream from the translational initiation codon (GUG) of the HU-1 gene.

Two forms of urokinase [EC 3.4.99.26] with molecular weights of 51,600 and 34,500 were purified from human urine. The specific activities of the high molecular weight urokinase (HMW-UK) and low molecular weight urokinase (LMW-UK) were 157,400 and 246,700 International Units (IU/mg), respectively. Purified HMW-UK was 97% active and LMW-UK was 88% active, as judged by using p-nitrophenyl-p'-guanidinobenzoate. LMW-UK had five multiple isoelectric subforms, compared with HMW-UK which had only one. Not only HMW-UK but also LMW-UK was composed of two polypeptide chains linked by disulfide bond(s). The molecular weight of the heavy chain of both forms was the same (34,000 daltons), while the molecular weight of the light chain of HMW-UK was 17,600 and that of LMW-UK was approximately 1,200-3,400. Enzyme kinetic studies revealed that the kinetic constants, Km and Kcat, of both forms toward the synthetic substrates, acetyl-Gly-Lys-methylester (AGLMe) and glutaryl-Gly-Arg-4-methylcoumarin-7-amide (GGA-MCA), were almost the same, but the dissociation constant of HMW-UK toward Glu-plasminogen was 2.4-2.6 times less than that of LMW-UK. HMW-UK incubated at 37 degrees C was converted into LMW-UK in an autocatalytic digestion manner leading to no loss of the total activity. These results show that HMW-UK with a higher affinity toward Glu-plasminogen is converted into LMW-UK with a lower affinity, a greater portion of the light chain of HMW-UK splitting off.

Coronary thrombolysis was induced by infusion of highly purified human pro-urokinase isolated from a transformed kidney cell line (ACHN)

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