蛇毒丝氨酸蛋白酶多样性──底物专一性免疫化学及序列比较研究

本文报道烙铁头（Ｔｒｉｍｅｒｅｓｕｒｕｓｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒纤维蛋白原溶酶（ＴＭＶＦｇ），眼镜王蛇（Ｏｐｈｉｏｐｈａｇｕｓｈａｎｎａｈ）蛇毒纤维蛋白原溶酶（ｏｈＳ１），竹叶青（Ｔｒｉｍｅｒｅｓｕｒｕｓｓｔｅｊｎｅｇｅｒｉ）蛇毒专一纤溶酶原激活剂（ｓｖ－ｐＡ）对５种小分子多肽底物的底物专一性，及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用，并和其它蛇毒丝氨酸蛋白酶如矛头蝮（Ｂｏｔｈｒｏｐｓａｔｒｏｘ）蛇毒凝血酶样酶（Ｂａｔｒｏｘｏｂｉｎ）、铜头蝮（Ａｇｋｉｓｔｒｏｄｏｎｃｏｎｔｏｒｔｒｉｘｃｏｎｔｏｒｔｒｉｘ）蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇（Ｖｉｐｅｒａｒｕｓｓｅｌｌｉ）毒第Ⅴ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫交叉反应，并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。     

The expression of tissue and urokinase-type plasminogen activators in neural development suggests different modes of proteolytic involvement in neuronal growth.

Tissue and urokinase-type plasminogen activators are serine proteases with highly restricted specificity, their best characterised role being to release the broad specificity protease plasmin from inactive plasminogen. It has frequently been suggested that these, and similar proteases, are involved in axonal growth and tissue remodelling associated with neural development. To help define what this role might be, we have studied the expression of the plasminogen activators in developing rat nervous tissue. Urokinase-type plasminogen activator mRNA is strongly expressed by many classes of neurons in peripheral and central nervous system. We have analysed its appearance in spinal cord and sensory ganglia, and found the mRNA is detectable by in situ hybridisation very early in neuronal development (by embryonic day 12.5), at a stage compatible with it playing a role in axonal or dendritic growth. Tissue plasminogen activator mRNA, on the other hand, is expressed only by cells of the floor plate in the developing nervous system, from embryonic day 10.5 and thereafter. Immunohistochemical and enzymatic analysis showed that active tissue plasminogen activator is produced by, and retained within, the floor plate. A mechanism is suggested by which high levels of tissue plasminogen activator produced by the stationary cells of the floor plate could influence the direction of growth of commissural axons as they pass through this midline structure.     

Cellular mechanisms regulating non-haemostatic plasmin generation

A variety of proteases have the potential to degrade the extracellular matrix (ECM), thereby influencing the behaviour of cells by removing physical barriers to cell migration, altering cell-ECM interactions or releasing ECM-associated growth factors. The plasminogen activation system of serine proteases is particularly implicated in this pericellular proteolysis and is involved in pathologies ranging from cancer invasion and metastasis to fibroproliferative vascular disorders and neurodegeneration. A central mechanism for regulating plasmin generation is through the binding of the two plasminogen activators to specific cellular receptors: urokinase-type plasminogen activator to the glycolipid-anchored membrane protein uPAR, and tissue plasminogen activator to a type-II transmembrane protein recently identified on vascular smooth muscle cells. These binary complexes interact with membrane-associated plasminogen to form higher order activation complexes that greatly reduce the K(m) for plasminogen activation and, in some cases, protect the proteases from their cognate serpin inhibitors. Various other proteins that are involved in cell adhesion and migration also interact with these complexes, modulating the activity of this efficient and spatially restricted proteolytic system. Recent observations demonstrate that certain forms of the prion protein can stimulate tissue plasminogen activator-catalysed plasminogen activation, which raises the possibility that these proteases may also have a role in the pathogenesis of the transmissible spongiform encephalopathies.     

Activity-based protein profiling reveals active serine proteases that drive malignancy of human ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.     

Characterization of a plasma membrane-associated plasminogen activator on thymocytes

Plasma membranes isolated from normal thymocytes of hamster and rats were found to exhibit neutral protease activity toward 125I-labeled casein. The plasma membrane-associated proteases were completely inhibited by the serine protease inhibitors, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and p-nitrophenyl-p-guanidinobenzoate, partially inhibited by soybean trypsin inhibitor and antipain, but were only weakly inhibited by L-1-tosylamino-2-phenylethyl chloromethyl ketone. The plasma membrane-associated proteases were also completely inhibited by ZnCl2 (75--100 mu M), but they were not affected by several other divalent cations. The plasma membrane fraction contained a plasminogen activator activity which was specifically localized in this fraction. The plasma membrane-associated plasminogen activator activity was inhibited by all of the inhibitors which inhibited plasma membrane-associated proteases except L-1-tosylamido-2-phenylethyl chloromethyl ketone. Labeling of plasma membrane-associated serine esterases with [3H] diisopropyl fluorophosphate followed by separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that this fraction contained a single major 3H-labeled protein of Mr 105 000. Both the plasminogen activator and the Mr 105 000 esterase were shown to be glycoproteins by affinity chromatography on lentil lectin-Sepharose. These results indicate that the plasminogen activator of thymocytes is a glycosylated serine protease with an active site-containing subunit of Mr 105 000 which is specifically localized in the plasma membrane.     

Mutational and immunochemical analysis of plasminogen activator inhibitor 1

We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.     

Evolution of Thrombin and Other Hemostatic Proteases by Survey of Protochordate, Hemichordate, and Echinoderm Genomes

Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications.     

Neuronal extracellular proteases facilitate cell migration, axonal growth, and pathfinding

The release of extracellular proteases by the axonal growth cone has been proposed to facilitate its movement by digesting cell-cell and cell-matrix contacts in the path of the advancing growth cone. The serine protease plasminogen activator (PA) has been shown to be secreted and focally concentrated at axonal growth cones of cultured mammalian neurons. Thus, PAs are well-placed to play an active role in growth cone movement and axonal pathfinding in development and regeneration. We discuss recent findings that suggest that the biological action of these proteases is more complex than originally thought. Received: 15 May 1997 / Accepted: 4 June 1997     

Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.     

Serine proteases of the fibrinolysis pathway are not involved in lethal hepatitis and fibrinogen breakdown induced by tumor necrosis factor

Tumor necrosis factor (TNF) plays a key role in several types of fulminant and acute hepatitis, and induces massive apoptosis and necrosis of hepatocytes. Our previous studies described the central role played by several matrix metalloproteinases (MMPs) and one or more unknown serine proteases. The aim of this study was to investigate the involvement of serine proteases of the fibrinolytic pathway, known to be activators of several MMPs, in TNF-induced hepatitis and fibrinogen (FG) breakdown. Experiments were performed in a model of TNF-induced hepatitis, consisting of administration of TNF in combination with D-(+)-galactosamine (GalN) to mice deficient in urokinase-type plasminogen (PG) activator (u-PA), tissue-type PG activator (t-PA) or PG. Lethality, transaminase release, increased plasma clotting time and FG levels were measured. In PA- and PG-deficient mice, TNF/GalN still induced hepatitis, as well as increased clotting time and FG breakdown. MMP-9 activation still occurred in the liver despite the lack of plasmin. The data suggest that the serine proteases involved in TNF-induced lethal hepatitis are no constituents of the fibrinolytic cascade.     

Hepatocyte growth factor plasma levels after myocardial infarction are not affected by recombinant tissue-type plasminogen-activator therapy

Hepatocyte growth factor (HGF), a cytokine involved in tissue regeneration, angiogenesis and lateral vessel growth, is secreted as a biological-inactive, single-chain precursor named pro-HGF. In case, of tissue injury pro-HGF is proteolytically cleaved at the extracellular locus by serine proteases. Results obtained from in vitro experiments showed that urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) can cleave single-chain HGF. In this study we measured serum HGF levels in patients with acute myocardial infarction (MCI). Two groups of patients were compared. One group (n = 7) was treated with a conventional therapy and the other group (n = 7) was subjected to a thrombolytic therapy with recombinant tissue-type plasminogen activator (rtPA). Serum samples were collected at time of admission and subsequently 12-16 hours, 20-30 hours and 50-60 hours after onset of chest pain. At admission and before administration of rtPA, serum HGF levels peaked at 16.8 +/- 2.2 ng/ml in the lysed group and at 20.7 +/- 6.5 ng/ml in the non-lysed group. Levels then continuously declined, reaching lowest values 50-60 hours after onset of chest pain (3.2 +/- 1.3 ng/ml in the group treated with rtPA versus 4.4 +/- 0.9 ng/ml in the non-lysed group). No statistical significant difference could be detected between the two groups at any time. We suggest that serine proteases other than tPA are involved in HGF activation in vivo.     

Tissue-type plasminogen activator gene is on chromosome 8

Tissue plasminogen activator is one of the two plasminogen activators, both serine proteases, that catalyze the conversion of inactive plasminogen to plasmin, which then degrades the fibrin network of blood clots. By combining somatic cell genetics, in situ hybridization, and Southern blot hybridization, we localized the human tissue plasminogen activator gene to the pericentromeric region of chromosome 8.     

Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains

Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation.     

Role of P225 and the C136-C201 disulfide bond in tissue plasminogen activator

The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+ was discovered. The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM. In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases.     

Growth factor-dependent proliferation and invasion of muscle satellite cells require the cell-associated fibrinolytic system

The process of muscle regeneration in normal and dystrophic muscle depends on locally produced cytokines and growth factors and requires the activity of the urokinase plasminogen activator/urokinase plasminogen activator receptor/plasminogen activator inhibitor-1 system. In this study we tested the effect of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and transforming growth factor-beta (TGFbeta) on the fibrinolytic pattern of normal and dystrophic satellite cells, their mitogenic and motogenic activities and the dependence of such activities on the cell-associated fibrinolytic system. We have observed that the urokinase plasminogen activator (u-PA) receptor is weakly upregulated by bFGF in normal satellite cells, while it is strongly up-regulated by TGFbeta, mainly in dystrophic myoblasts. bFGF up-regulated u-PA in both normal and dystrophic myoblasts grown in primary culture, while a striking down-regulation was observed with TGFbeta. TGFbeta was the only growth factor able to exceptionally up-regulate plasminogen activator inhibitor-1 (PAI-1), mainly in dystrophic satellite cells. HGF did not show any activity on the fibrinolytic system. Proliferation and invasion into Matrigel matrices of normal and dystrophic cells occurred regardless of the growth factor-dependent regulation of the fibrinolytic system. Nevertheless, each growth factor required the efficiency of the constitutive cell-associated fibrinolytic system to operate, as shown by impairment of growth factor activity with antagonists of u-PA and of its receptor. Noteworthy, TGFbeta induced a dose-dependent increase of Matrigel invasion only in dystrophic myoblasts. Since TGFbeta-challenged dystrophic myoblasts undergo an exceptional up-regulation of the receptor and of PAI-1, we propose the possibility that the TGFbeta-induced fibrinolytic pattern (low urokinase plasminogen activator, high receptor and high PAI-1) may be exploited to promote survival and spreading of transplanted engineered myoblasts in Duchenne muscular dystrophy.     

Potent inhibition and global co-localization implicate the transmembrane Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-2 in the regulation of epithelial matriptase activity

Hepatocyte growth factor activator inhibitors (HAI)-1 and -2 are recently identified and closely related Kunitz-type transmembrane serine protease inhibitors. Whereas HAI-1 is well established as an inhibitor of the serine proteases matriptase and hepatocyte growth factor activator, the physiological targets of HAI-2 are unknown. Here we show that HAI-2 displays potent inhibitory activity toward matriptase, forms SDS-stable complexes with the serine protease, and blocks matriptase-dependent activation of its candidate physiological substrates proprostasin and cell surface-bound pro-urokinase plasminogen activator. To further explore the potential functional relationship between HAI-2 and matriptase, we generated a transgenic mouse strain with a promoterless beta-galactosidase marker gene inserted into the endogenous locus encoding HAI-2 protein and performed a global high resolution mapping of the expression of HAI-2, matriptase, and HAI-1 proteins in all adult tissues. This analysis showed striking co-localization of HAI-2 with matriptase and HAI-1 in epithelial cells of all major organ systems, thus strongly supporting a role of HAI-2 as a physiological regulator of matriptase activity, possibly acting in a redundant or partially redundant manner with HAI-1. Unlike HAI-1 and matriptase, however, HAI-2 expression was also detected in non-epithelial cells of brain and lymph nodes, suggesting that HAI-2 may also be involved in inhibition of serine proteases other than matriptase.     

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAl)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo. © 1995 Wiley-Liss Inc.     

Proteolytic cleavage of single-chain pro-urokinase induces conformational change which follows activation of the zymogen and reduction of its high affinity for fibrin

A plasminogen activator secreted from human kidney cells was highly purified by affinity chromatography on an anti-urokinase IgG-Sepharose column. The purified plasminogen activator was inactive and had a single-chain structure and a Mr of 50,000. It not only did not incorporate diisopropyl fluorophosphate, which reacts with active site serine residue in urokinase, but also did not bind to p-aminobenzamidine-immobilized CH-Sepharose, to which urokinase bind via its side-chain binding pocket present in active center. The plasminogen activator was converted to the active two-chain form with the same Mr by catalytic amounts of plasmin. Its potential enzymatic activity was quenched completely by anti-urokinase IgG, but not by anti-tissue plasminogen activator Ig. These results indicate that the plasminogen activator is an inactive proenzyme form of human urokinase. Therefore, the plasminogen activator was termed single-chain pro-urokinase. The cleavage of single-chain pro-urokinase by plasmin induced conformational change which followed the generation of reactive serine residue at active site, the increase enzyme activity and the reduction of its high affinity for fibrin. These findings suggest that conformational change occurs in both regions responsible for enzyme activity and affinity for fibrin upon activation of single-chain pro-urokinase.     

Isolation of rat hepatoma cell variants selectively resistant to dexamethasone inhibition of plasminogen activator.

Glucocorticoids induce several phenotypic changes in rat hepatoma cells in tissue culture, including the inhibition of plasminogen activator activity. Variant cell lines resistant to dexamethasone inhibition of plasminogen activator activity have been isolated using an agar-fibrin overlay technique to identify colonies with fibrinolytic (plasminogen activator) activity. The variants are resistant to concentrations of dexamethasone 1,000 times that necessary to completely inhibit plasminogen activator activity in wild-type cells. The variant phenotype has been inherited in a stable manner for more than 300 generations in continuous culture in the absence of dexamethasone. These variants are unique in that the resistance is not secondary to defective or absent glucocorticoid receptors but is due to a lesion specific for regulation of plasminogen activator. Fluctuation analyses support the hypothesis that resistance to dexamethasone arises randomly and is not induced by dexamethasone. Because HTC cells are heteroploid and karyotypically highly variable, variants are thought to arise primarily by chromosomal segregation events. These variants provide a valuable tool for studying the mechanism of hormonal regulation of plasminogen activator as well as the role of proteases in hormonal regulation of membrane functions.