Effect of dithiothreitol on activity and protein structure of human urine urokinase

Human urine urokinase [EC 3.4.21.31] was found to be inactivated by dithiothreitol (DTT) much more severely than by 2-mercaptoethanol at the same concentration on the basis of -SH groups. Removal of DTT by dialysis restored the activities of esterase toward acetyl-glycyl-L-lysine methyl ester, plasminogen activation, and amidase toward 7-(glutaryl-glycyl-L-arginine-amido)-4-methyl coumarin. But the restoration of amidase activity was much less than that of esterase activity. The addition of DTT mediated the conversion of high molecular weight urokinase to low molecular weight urokinase, releasing several peptides. This suggests that the urokinase consists of several polypeptides linked by disulfide bonds. The molecular weight of urokinase produced with DTT was smaller than that of low molecular weight urokinase obtained by autodigestion of high molecular weight urokinase. The autodigestion was also accompanied by liberation of some peptides. But, those peptides released on autodigestion of high molecular weight urokinase were different from those appearing in the presence of DTT.     

Conformation of one- and two-chain high molecular weight urokinase analyzed by small-angle neutron scattering and vacuum ultraviolet circular dichroism

The structures of one- and two-chain high molecular weight human urokinase were analyzed by small-angle neutron scattering and vacuum ultraviolet circular dichroism. Both one- and two-chain high molecular weight urokinases exhibited a radius of gyration of 31 A and a maximum dimension of 90 A. Neither parameter was affected by the presence of lysine sufficient to saturate all the lysine-binding sites in human plasminogen. These physical parameters are consistent with the sedimentation coefficient of high molecular weight urokinase and indicate that both proteins are highly asymmetric. Neither protein contained much alpha-helix or parallel beta-sheet. Most of the secondary structure was in the form of antiparallel beta-sheet and beta-turns, very similar to the secondary structure of plasminogen. The macroscopic kinetic constants, Km and kcat, for the hydrolysis of (pyroGlu-Gly-Arg-NH)2-rhodamine by two-chain high molecular weight urokinase and low molecular weight urokinase which lacks the epidermal growth factor and kringle domains were similar. These structural and kinetic data are consistent with the domains in both forms of urokinase being independent structural and functional units.     

Random peptide bacteriophage display as a probe for urokinase receptor ligands

The urokinase receptor is a multi-functional protein that plays a central role in cell surface plasminogen activation, cell migration, and cell adhesion. We previously demonstrated that high affinity peptide ligands for the urokinase receptor, which are urokinase competitors, can be obtained from a 15mer peptide library (Goodson et al., 1994). In order to probe for additional urokinase receptor binding sites we affinity selected the same bacteriophage library on complexes of soluble urokinase receptor (suPAR) and the receptor binding domain of urokinase, residues 1-48 (uPA1-48). Bacteriophage were isolated which bound to suPAR and suPAR:uPA1-48 complexes with high yield. The peptide sequences encoded by these bacteriophage were distinct from those obtained previously on urokinase receptor expressing cells, and comprise two groups based upon effects on su-PAR:1-anilino-8-napthalene sulfonate (ANS) fluorescence, and vitronectin binding competition. Alanine scanning mutagensis of the soluble peptides was used to define minimal regions and key residues for suPAR binding by competition with the parent bacteriophage. A comparison of these results with sequences of domains of both vitronectin and integrin alpha-chains, which have been reported to be important for urokinase receptor binding, suggests that the homology with the peptide sequences selected is functionally significant.     

Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study

The kinetics of the activation of Glu- and Lys-plasminogen by single-chain urokinase (sc urokinase) derived from the transformed human kidney cell line TCL-598 have been studied and compared with two-chain urokinase (tc urokinase). Plasminogen activation was determined by the increase in fluorescence polarization of fluorescein-labeled aprotinin, a high affinity inhibitor of plasmin. This methodology allows plasmin generation by sc urokinase to be measured in functional isolation, with no interfering generation of tc urokinase, sc urokinase was found to activate plasminogen to plasmin with apparent Michaelis-Menten-type kinetics. The Km for Glu-plasminogen activation was 47.7 microM, with a catalytic constant of 2.91 min-1. Lys-plasminogen activation by sc urokinase was characterized by a Km of 11.7 microM and a kcat of 5.60 min-1. The Km values for the activation of Glu- and Lys-plasminogen by tc urokinase were found to be similar to those for activation by sc urokinase (36.8 and 9.0 microM, respectively), but the catalytic constants were higher at 36.0 and 118 min-1, respectively. Therefore, on the basis of the catalytic efficiency kcat/Km, sc urokinase seems to have 16-27-fold lower activity than tc urokinase. This activity of sc urokinase is in contrast to its lack of activity against a low molecular weight peptide substrate (less than 0.2% of the activity of sc urokinase). The activation of sc urokinase to tc urokinase by plasmin was also characterized (Km = 3.0 microM, kcat = 105 min-1). Using these data, it was possible to calculate the theoretical rate of plasminogen activation by sc urokinase in the absence of aprotinin, when tc urokinase is generated by the action of plasmin. The calculated rate was in good agreement with that determined experimentally using the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide. These data demonstrate that sc urokinase has properties which distinguish it from conventional serine protease zymogens. The lack of activity against low molecular weight peptide substrates demonstrates the inaccessibility of the substrate-binding pocket. However, there is a moderate activity against plasminogen, suggesting that plasminogen may be acting as both an effector and a substrate for sc urokinase.     

The matrix metalloproteinase pump-1 catalyzes formation of low molecular weight (pro)urokinase in cultures of normal human kidney cells.

The enzyme responsible for the metalloproteinase activity which cleaves the Glu143-Leu144 bond of (pro)urokinase has been isolated from the conditioned medium of cultured normal human kidney cells. Using S-Sepharose and Cibacron Blue-agarose chromatography, then C-4 reversed phase high pressure liquid chromatography, a protein of about 20,000 Da was isolated. Through an identical amino-terminal sequence, the protein was shown to be the matrix metalloproteinase previously referred to in the literature as "pump-1" (putative metalloproteinase). When aprotinin was added during the course of the purification, the major species isolated was the zymogen form (28,000 Da) of pump-1. Pump-1 has been shown to efficiently cleave the susceptible bond of both pro-urokinase (single-chain) and active (two-chain) urokinase and thereby produce the corresponding low molecular weight forms. The amino-terminal sequences of the A and B chains of low molecular weight urokinase prepared by action of pump-1 on recombinant high molecular weight urokinase are identical to those of the low molecular weight urokinase isolated from human kidney cell culture. Since the reaction of urokinase with this metalloproteinase results in separation of its serine proteinase region from the domain which mediates binding to the urokinase receptor, it may be of importance in the regulation of the functional activity of the plasminogen activator in cellular processes.     

Thrombolytic properties of an inactive proenzyme form of human urokinase secreted from human kidney cells

The relative fibrin-binding, fibrinolytic and fibrinogenolytic properties of single-chain pro-urokinase, an inactive proenzyme form of human urokinase purified from cultured human kidney cells, and urokinase were compared. The affinity of single-chain pro-urokinase for fibrin was much higher than that of urokinase. In Vitro thrombolytic studies showed that single-chain pro-urokinase is approximately three times more potent in fibrinolysis than urokinase and that it does not degrade fibrinogen in the plasma at a concentration, at which complete plasma clot lysis takes place; whereas, urokinase extensively degrades the fibrinogen in the plasma. These specific, potent thrombolytic properties of single-chain pro-urokinase seem to be due to its high affinity for fibrin and to its conversion from the inactive single-chain form to the active two-chain form on the thrombus by the catalytic amount of plasmin generated during coagulation. This single-chain pro-urokinase obtained from human kidney cells by tissue culture should prove advantageous than urokinase in thrombolytic therapy.     

Heparin binding to the urokinase kringle domain.

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.     

Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.

Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.     

双专－性抗体介导的纤溶作用

用双功能团试剂将抗尿激酶单克隆抗体Ｎ３４的ＩｇＧ和抗人活化血小板α－颗粒膜蛋白ＧＭＰ－１４０单克隆抗体ＳＺ－５１的Ｆａｂ片段通过二硫键共价偶联,偶联的抗体保留了对各自抗原的亲和性。这种对尿激酶和血栓同时具有亲和活性的双专一性抗体（Ｎ３４－ＳＺ－５１）提高低分子量尿激酶的溶栓效率３８倍,且对血浆中纤维蛋白原的含量基本上不影响。     

Urokinase-related proteins in human urine. Isolation and characterization of single-chain urokinase (pro-urokinase) and urokinase-inhibitor complex

Urokinase-related proteins were purified from 60-liter batches of human urine collected into the protease inhibitor aprotinin to prevent proteolytic degradation. Three homogeneous species were obtained by chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, Sephadex G-100, benzamidine-Sepharose, and immunoadsorption on a murine anti-human urokinase monoclonal antibody. One urokinase-related protein with Mr 95,000 representing a complex of two-chain urokinase with an inhibitor accounts for about 70% of the total urokinase-related antigen in urine. Nucleophilic agents dissociate the complex into active two-chain urokinase and a protein with Mr 45,000-50,000 which is immunologically distinct from urokinase. Approximately 25% of the urinary urokinase-related antigen represents a single-chain molecule with Mr 54,000. This highly purified single-chain molecule was obtained with a yield of 5 micrograms/liter of urine. Only trace amounts (less than 5%) of the urokinase-related antigen were recovered as free two-chain urokinase. The urinary single-chain urokinase-related protein has no specific affinity for fibrin. It has a very low activity on Pyroglu-Gly-Arg-p-nitroanilide, a urokinase-specific synthetic substrate, but directly activates plasminogen following Michaelis-Menten kinetics with Km = 0.7 microM and kcat = 0.0011 S-1. The single-chain molecule is rapidly converted to active two-chain urokinase by plasmin. Active two-chain urinary urokinase has a very high amidolytic activity and activates plasminogen with Km = 60 microM and kcat = 1.4 S-1. It is concluded that the urokinase-related proteins in human urine consist of about 25% of single-chain urokinase (10-20 micrograms/liter) and of about 75% two-chain urokinase (40-50 micrograms/liter), the bulk of which is complexed to an inhibitor. Because even in freshly voided urine most of the urokinase-related antigen is already converted to two-chain urokinase, urine does not seem to be a suitable source for the large-scale purification of single-chain urokinase. In view of the very significant intrinsic plasminogen-activating properties of single-chain urokinase, it should not be considered to be a proenzyme form of urokinase. The dramatic differences of its kinetic constants from those of urokinase render the designation single-chain urokinase equally inadequate. Consequently, the designation "single-chain urokinase-type plasminogen activator" was recently adopted by the International Committee on Thrombosis and Haemostasis (Annual Meeting, San Diego, CA, July 13-14, 1985).     

Urokinase receptors in human monocytes

Receptors for the 54 kDa plasminogen activator urokinase were characterized in freshly isolated and 5-14 day cultured human monocytes. The half saturation constant was about 55 pM in freshly isolated monocytes at 4 degrees C and 140 pM at 37 degrees C. Diisopropylfluorophosphate-inactivated urokinase was bound with the same affinity as catalytically active urokinase. Binding per cell of 2-5 pM urokinase increased progressively during cell culture with a concomitant decrease in the apparent affinity. By 14 days, binding had increased 5-7-fold and the half-saturation constant had increased to 500 pM at 4 degrees C, indicating a large increase in the binding capacity. Affinity cross-linking of labelled urokinase to receptors showed a 110 kDa complex in both freshly isolated and cultured monocytes. When cells with labelled urokinase (prebound at 4 degrees C) were incubated at 37 degrees C, about 80% of the urokinase dissociated as the intact molecule, whereas about 20% was degraded to iodide and iodotyrosine. Electron microscopic autoradiography of cultured monocytes incubated at 4 degrees C showed a marked heterogeneity between cells with regard to bound urokinase. Autoradiographic grains were mainly seen over the plasma membrane in areas rich in microvilli and invaginations. Transfer of the cells to 37 degrees C caused no major alteration in the distribution of grains. Thus, freshly prepared monocytes have urokinase receptors (approx. 55 kDa) of high affinity. Development to macrophage-like cells in culture causes a decrease in affinity and a large increase in capacity. The receptors are confined mainly to certain areas of the plasma membrane. Internalization and degradation of the ligand occurs only to a minor extent.     

Fibrinolysis by urokinase endowed with magnetic property

The activated magnetic modifier was synthesized from magnetite, alpha, omega-dicarboxymethylpoly(oxyethylene) and N-hydroxysuccinimide (Biochem. Biophys. Res. Commun., 145, 908-914, 1987). Urokinase was directly coupled with the activated magnetic modifier to obtain magnetic urokinase. The magnetic urokinase dispersed in saline and exerted high fibrinolytic activity (13.8 X 10(4) IU/mg protein), and was readily recovered from saline by magnetic force of 250 Oe. By applying magnetic force, the urokinase was attracted at our will and local fibrinolysis was achieved on fibrin gel in a petri dish.     

Urokinase: a chemotactic factor for polymorphonuclear leukocytes in vivo

The effects of injecting urokinase into subdermal air sacs on the back of mice was studied. Urokinase was leukotactic in the concentration range of 2 X 10(-13) to 2 X 10(-15) M. This response was absolutely dependent on the enzyme activity of the serine esterase, but was found to be independent of generation of the chemotactic complement split product C5a. At high doses of urokinase (greater than 2 X 10(-12) M), no cellular infiltration was observed. Injection of 2 X 10(-10) M urokinase i.p. led to the systemic desensitization of mice when challenged in the skin with a lower dose (2 X 10(-14) M) of urokinase. Urokinase desensitization did not alter the ability of mice to respond to the chemical chemotactic factor f-met-leu-phe or to respond to C5a-dependent chemotactic stimuli. Urokinase desensitized mice failed to demonstrate a chemotactic response to nerve growth factor, thrombin, plasmin, or factor X activating enzyme, all of which were chemotactic in non-urokinase pre-treated animals. The results of these studies indicate the presence of three physiologically independent inflammatory pathways in mice: independent of C5 and not influenced by pretreatment with urokinase, independent of C5 and inhibited by pretreatment with urokinase, and dependent on C5 and not influenced by pretreatment with urokinase.     

A bispecific antibody enhances the fibrinolytic potency of single-chain urokinase.

A monoclonal antibody specific for an epitope at the amino terminus of the beta chain of fibrin and a monoclonal antibody that binds both one- and two-chain high molecular weight urokinase were chemically cross-linked [using N-succinimidyl 3-(2-pyridyldithio)propionate and 2-iminothiolane]. The chemically modified material was heterogeneous, ranging in molecular size from tetramers to monomers and containing the two antibodies in various ratios. Nevertheless, fractions of a molecular size larger than a monomer were capable of binding fibrin and urokinase simultaneously in a radioimmunoassay. These fractions also enhanced fibrinolysis by high molecular weight single-chain urokinase (scuPA) by 50-fold and plasma clot lysis by 5-fold. Whereas scuPA significantly decreased the concentration of fibrinogen in plasma clot assay supernatants, scuPA in association with the bispecific antibody did not.     

抗尿激酶单克隆抗体识别相应抗原决定簇的研究

 尿激酶是一种纤溶酶原激活剂,临床上用于治疗血栓。为了有效地用单克隆抗体亲和柱纯化尿激酶,我们对一组抗尿激酶单克隆抗体识别相应抗原决定簇的特性进行了研究。Western Blotting试验表明:S_(13)、S_(26)、N_(14)、N_(30)、N_(17-2)、N_(34)、N_(36)七个单克隆抗体主要抗54000道尔顿的高分子量尿激酶(HUK)。除N_(30)外,其余抗体还同时不同程度地抗33000道尔顿的低分子量尿激酶(LUK)。N_(30)除识别HUK外,还识别分子量为18000道尔顿的多肽链。竞争性结合试验证明:七个单克隆抗体分别抗五个不同的抗原决定簇,但它们都不抗尿激酶的活力中心。     

Effect of butyrate on the expression of the human preprourokinase gene introduced into Chinese hamster ovary cells

A plasmid containing the human preprourokinase gene cDNA under the control of the simian virus 40 early region promoter was introduced into CHO-K1 cells and recombinant cell lines secreting a relatively high level of urokinase were obtained. In the course of studying the effects of various agents on the recombinant cell lines, we found that exposure of recombinant cells to 5 mM butyrate for 24 hours resulted in a 2-3 fold increase in urokinase production. The induction by butyrate was dose-dependent. The half maximal dose was approximately 2 mM; maximal stimulation occurred at 5-10 mM. Cell growth, on the other hand, was inhibited by butyrate concentrations greater than 2.5 mM. The response of cells to butyrate was rapid: a significant increase in urokinase production was observed 6 hours after exposure to 5 mM butyrate. Butyrate treatment increased not only the extracellular level but also the intracellular level of urokinase.     

多通道流动电泳技术及其在蛋白质、酶和抗体纯化中的应用研究

提出一种新型制备电泳方法即多通道流动电动电泳技术,建立了微机控制的电泳设备,考查了操作条件对设备分离效率的影响。应用该技术进行牛血清清蛋白(BSA)-牛血红蛋白(HBB)混合物的连续分离,每小时从含BSA、HBB各37.0mg的蛋白质混合物中分离出13.6mg的BSA和20.0mg的HBB。应用该技术进行尿激酶粗品的纯化,可将其比活力由4000IU/mg提高到4×10~4IU/mg以上,产量在10×10~4IU/h以上。将该技术与(NH_4)SO_4沉淀方法结合,从15ml免疫鼠血清中分离出12.5mg高纯度尿激酶抗体IgG,产量为2.1mg/h。上述结果证明多通道流动电泳技术具有分离速度快、精度高和可操作性好等优点,在生物产品分离领域中具有广阔的应用前景。     

The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type

The capacity of cells to interact with the plasminogen activator, urokinase, and the zymogen, plasminogen, was assessed using the promyeloid leukemic U937 cell line and the diploid fetal lung GM1380 fibroblast cell line. Urokinase bound to both cell lines in a time- dependent, specific, and saturable manner (Kd = 0.8-2.0 nM). An active catalytic site was not required for urokinase binding to the cells, and 55,000-mol-wt urokinase was selectively recognized. Plasminogen also bound to the two cell lines in a specific and saturable manner. This interaction occurred with a Kd of 0.8-0.9 microM and was of very high capacity (1.6-3.1 X 10(7) molecules bound/cell). The interaction of plasminogen with both cell types was partially sensitive to trypsinization of the cells and required an unoccupied high affinity lysine-binding site in the ligand. When plasminogen was added to the GM1380 cells, a line with high intrinsic plasminogen activator activity, the bound ligand was comprised of both plasminogen and plasmin. Urokinase, in catalytically active or inactive form, enhanced plasminogen binding to the two cell lines by 1.4-3.3-fold. Plasmin was the predominant form of the bound ligand when active urokinase was added, and preformed plasmin can also bind directly to the cells. Plasmin on the cell surface was also protected from its primary inhibitor, alpha 2-antiplasmin. These results indicate that the two cell lines possess specific binding sites for plasminogen and urokinase, and a family of widely distributed cellular receptors for these components may be considered. Endogenous or exogenous plasminogen activators can generate plasmin on cell surfaces, and such activation may provide a mechanism for arming cell surfaces with the broad proteolytic activity of this enzyme.     

Gangliosides interact directly with plasminogen and urokinase and may mediate binding of these fibrinolytic components to cells

Receptors for the fibrinolytic molecules plasminogen and urokinase are expressed at high capacity on a wide variety of peripheral blood cells and transformed cell lines. We have considered whether gangliosides, components of the outer leaflets of cell membranes, may modulate the interactions of these fibrinolytic ligands with cells. Radiolabeled plasminogen and urokinase bound directly to insolubilized gangliosides. The interactions were saturable and were 50% inhibited by 2.2 microM unlabeled plasminogen or 12 nM unlabeled urokinase, respectively. A panel of gangliosides inhibited binding of both ligands to U937 monocytoid cells, and the order of decreasing inhibitory effectiveness was GD1a greater than GM1 greater than GT1b greater than GM2, while GM3 was minimally effective. The individual components of gangliosides, hexoses, hexosamines, sialic acid, GM1 pentasaccharide, ceramides, and glucocerebrosides were ineffective in in inhibiting the binding of plasminogen and urokinase either to cells or to insolubilized gangliosides. Binding of both ligands to endothelial cells and granulocytes and binding of plasminogen to platelets were also inhibited by gangliosides. U937 cells were cultured with gangliosides to allow incorporation of these glycolipids into the cell membranes. After 3 days of culture, both urokinase binding and plasminogen binding to the cells became enhanced. These results suggest that gangliosides can directly bind to these fibrinolytic components and may mediate or modulate the interactions of plasminogen and urokinase with a variety of cell types.