A Comparative Study of High Molecular Weight Urokinase and Low Molecular Weight Urokinase

Abstract

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 sub-forms, 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 dal-tons), 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-methylcou-marin-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(o)C was converted into LMW-UK in an auto-catalytic 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.