Immunochemical Studies of High and Low Molecular Forms of Urokinase

Sumi, Hiroyuki; Toki, Naotika; Maehara, Susumu; Kosugi, Tadayoshi; Akazawa, Kenji; Matsuo, Osamu; Mihara, Hisashi

doi:10.1159/000207073

Cited by 10 publications

(5 citation statements)

References 7 publications

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“…As reported previously [37], it has been found that UK antibody reacts with and can inhibit the fibri nolytic activity of all active molecular forms of UK. As illustrated in figure 5, by the dou ble immunodiffusion technique both anti no binding affinity for fibrin.…”

Section: Discussionsupporting

confidence: 74%

Fibrin-Binding Urokinase (or Precursor Form of Urokinase) with a Molecular Weight of About 100,000 in Fresh Human Urine

Sumi¹,

Tsushima²,

Maruyama³

et al. 1985

Enzyme

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Fresh human urine was found to contain at least three different molecular forms of fibrin-binding urokinase (UK) or its precursor, all of which were adsorbed on a fibrin/Celite column at neutral pH, and could be eluted with 0.3-1.0 mol/1 NaCl in phosphate buffer, followed by 0.2 mol/1, Arg, 2 mol/1 KSCN, and 2 mol/1 urea, respectively. The main molecular form isolated revealed a molecular weight (MW) of approximately 100,000 (UK-100), and the minor ones were estimated to have MW of 150,000-200,000 and 45,000. In contrast, commercially obtained UK preparations contained mostly active enzymes with MW of 53,000 and 32,000, respectively, and the remaining high molecular forms represented less than 2.0% of the total amount. Rabbit monospecific antibody (IgG) against UK subcomponent (active heavy chain; H-chain UK) reacted and inhibited the fibrinolytic activity of all the active UK molecules. The UK-100 isolated was relatively stable in solution at neutral pH and resistant to mild reduction, without molecular change. Although the preparation had a very low specific activity (ca. 300 IU/mg protein), both the pyro-Glu-Gly-Arg-pNA amidolytic and plasminogen activating activities could be partially enhanced by the addition of trace amounts of plasmin. In this process, the appearance of two additional active enzymes of MW 53,000 and 32,000 was also confirmed by zymography.

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Section: Discussionsupporting

confidence: 74%

Fibrin-Binding Urokinase (or Precursor Form of Urokinase) with a Molecular Weight of About 100,000 in Fresh Human Urine

Sumi¹,

Tsushima²,

Maruyama³

et al. 1985

Enzyme

Self Cite

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“…10) In addition, the light and heavy chains separated by reduction, and also the peptide fragment with a molecular weight of 21,000, which is released from the enzyme after the self-digestion, are catalytically inactive or only slightly active.…”

Section: Urokinase (Uk Ec 342131)mentioning

confidence: 99%

Chemical modification of urokinase with bis-imidoesters and properties of the intramolecularly cross-linked enzyme.

Yokoigawa

Tanizawa

Soda

1989

Agricultural and Biological Chemistry

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To prepare a chemically modified urokinase that does not dissociate into two peptide fragments upon reduction of its disulfide bridge, we cross-linked the enzyme intramolecular!} with various bifunctional imidoesters. The enzyme underwent the intramolecular cross-linking most moderately by the reaction at 4°C for 5 hr with 3mMdimethyl suberimidate in 0.1 M potassium phosphate buffer (pH 9.0). The cross-linked urokinase isolated by gel filtration with a yield of 25 % showed a specific activity of 76,000 International Units/mg protein, which corresponds to 53 %of that of the native enzyme. Although the modified enzymewas similar to the native urokinase in some properties such as the autocatalytic self-digestion and the low affinity to fibrin, it showed higher in vivo and in vitro stabilities than the native one.

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“…It has been reported that the single-chain form had a higher specific thromobolytic activity and better selectivity for fibrin than the two chain form when tested in vitro (Sumi et al , 1983). The single-chain form was purified from urine (Husain et al , 19831, plasma (Wun et al , 1982), kidney cells (Sumi et al , 1982) and certain tumor cells . The recombinant pro-UK was produced in E. coli (Homes et al , 1985)) Chinese hamster ovary cells (Nells et al , 1987) , COS-1 cells (Chcng et al , 1988) and in insect cells (K ing et al ,199l).…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of recombinant human pro-urokinase produced in silkworm using a baculovirus vector

Gao

1994

Biotechnol Tech

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A recombinant baculovirus was constructed by inserting the human pro-urokinase (proUK) cDNA into the genome of baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV > adjacent to the strong polyhedrin promoter. The recombinant virus replicated in silkworm larvae, which synthesized 3Opg pro-UK/ml in the haemolymph within 4 days post-infection.Purification to near homogeneity was accomplished by fractional precipitation with ammonium sulphate and immunoaffinity chromatography with an overall yield of 23% and a specific activity of 10O,OOOIU/mg in fibrin plate assay. This purified product was comprised of a single chain protein with approximately Mr. 50kDa as determined by SDS-PAGE gel. The N-Terminal amino acids sequence revealed that the secretion signal of pro-UK was correctly processed.

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Immunochemical Studies of High and Low Molecular Forms of Urokinase

Cited by 10 publications

References 7 publications

Fibrin-Binding Urokinase (or Precursor Form of Urokinase) with a Molecular Weight of About 100,000 in Fresh Human Urine

Fibrin-Binding Urokinase (or Precursor Form of Urokinase) with a Molecular Weight of About 100,000 in Fresh Human Urine

Chemical modification of urokinase with bis-imidoesters and properties of the intramolecularly cross-linked enzyme.

Purification and characterization of recombinant human pro-urokinase produced in silkworm using a baculovirus vector

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