Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The data presented in this paper show that when rabbit plasminogen is activated to plasmin by urokinase at least two peptide bonds are cleaved in the process. Urokinase first cleaves an internal peptide bond in plasminogen, leading to two-chain disulfide-linked plasmin molecule. The plasmin heavy chain of molecular weight 66,000 to 69,000 possesses an NH2-terminal amino acid sequence identical with the original plasminogen (molecular weight 88,000 to 92,000). The plasmin light chain of molecular weight 24,000 to 26,000 is known to be derived from the COOH-terminal portion of plasminogen. The plasmin generated during the activation of plasminogen is capable, by a feedback process, of cleaving a peptide of molecular weight 6,000 to 8,000 from the NH2 terminus of the heavy chain, producing a proteolytically modified heavy chain of molecular weight 58,000 to 62,000. Plasmin also can cleave this same peptide from the original plasminogen, yielding an altered plasminogen of molecular weight 82,000 to 86,000. This plasmin-altered plasminogen and the plasmin heavy chain derived from it by urokinase activation process NH2-terminal amino acid sequences which are identical with each other and with the plasminolytic product of the original plasmin heavy chain. These studies support a mechanism of activation of plasminogen by urokinase which involves loss of a peptide located on the NH2 terminus of plasminogen. However, these same results show that this NH2-terminal peptide need not be released from rabbit plasminogen prior to the cleavage of the internal peptide bond which leads to the two-chain plasmin molecule. Furthermore, these studies show that urokinase cannot remove this peptide from either the original rabbit plasminogen molecule or from the heavy chain of the initial plasmin formed.
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PMID:The mechanism of activation of rabbit plasminogen by urokinase. 12 29

A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase-activated plasminogen with 2-mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6-aminohexanoic acid (yield 60-65%). Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate-polyacrylamide electrophoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH2-terminal amino acid was predominantly threonine. 6-Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor-induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6-aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6-aminohexanoic acid.
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PMID:A new method of isolation and some properties of the heavy chain of human plasmin. 12 54

When human plasminogen (Glu-Pga) is activated by urokinase in the presence of pancreatic trypsin inhibitor, the plasmin produced (Glu-Pma) exclusively contains a heavy chain (Glu-Ha) derived intact from the original NH2 terminus of Glu-Pga. Similar activations, utilizing a low molecular weight synthetic plasmin acylating agent, p-nitrophenyl-p-(pyridiniummethyl) benzoate, still result in a plasmin molecule with approximately 50% of the plasmin heavy chain containing the intact NH2 terminus of the original Glu-Pga. Activations performed at high levels of urokinase in the absence of any inhibitors initially produce Glu-Pma. However, the final stable plasmin, Lys-Pmb, which is obtained contains a heavy chain (Lys-Hb) which arises by plasminolysis of a small peptide from the NH2 terminus of Glu-Ha. Alternatively, Lys-Pmb can be formed in a separate series of reactions initially involving plasminolysis of Glu-Pga to yield Lys-Pgb. The peptide removed in this step is identical to the peptide removed in the Glu-Ha to Lys-Hb reaction. Next, urokinase catalyzes the conversion of Lys-Pgb to Lys-Pmb without further loss of peptide material. This latter pathway involving Lys-Pgb is probably the major pathway for human Lys-Pmb generation. These studies support a mechanism of activation of human plasminogen which involves at least two bond cleavages in Glu-Pga. However, these same studies strongly indicate that the Nh2-terminal peptide need not be released from Glu-Pga prior to plasmin formation. Further, we feel that plasmin and not urokinase catalyzes cleavage of the NH2-terminal peptide bond from Glu-Pga and the Glu-Ha heavy chain of Glu-Pma.
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PMID:Mechanism of the urokinase-catalyzed activation of human plasminogen. 13 42

A complex between plasmin and an inhibitor was isolated by affinity chromatography from urokinase-activated human plasma. The complex did not react with antibodies against any of the known proteinase inhibitors in plasma. A rabbit antiserum against the complex was produced. It contained antibodies agianst plasminogen+plasmin and an alpha2 protein. By crossed immunoelectrophoresis the alpha2 protein was shown to form a complex with plasmin, when generated by urokinase in plasma, and with purified plasmin. The alpha2 protein was eluted by Sephadex G-200 gel filtration with KD approx. 0.35, different from the other inhibitors of plasmin in plasma, and corresponding to an apparent relative molecular mass (Mr) of about 75000. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Mr of the complex was found to be approx. 130000. After reduction of the complex two main bands of protein were observed, with Mr, about 72000 and 66000, probably representing an acyl-enzyme complex of plasmin-light chain and inhibitor-heavy chain, and a plasmin-heavy chain. A weak band with Mr 9000 was possibly an inhibitor-light chain. The inhibitor was partially purified and used to titrate purified plasmin of known active-site concentration. The inhibitor bound plasmin rapidly and strongly. Assuming an equimolar combining ratio, the concentration of active inhibitor in normal human plasma was estimated to be 1.1 mumol/1. A fraction about 0.3 of the antigenic inhibitor protein appeared to be functionally inactive. In plasma, plasmin is primarily bound to the inhibitor. Only after its saturation does lysis of fibrinogen and fibrin occur and a complex between plasmin and alpha2 macroglobulin appear.
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PMID:The primary inhibitor of plasmin in human plasma. 13 18

The heavy polypeptide chains of human Glu-plasmin and human Lys-plasmin have been isolated in native solvents, after partial reduction and carboxymethylation of the corresponding plasmins. Two major forms of each heavy chain can be eluted, after adsorption to Sepharose/lysine, utilizing a gradient of epsilon-aminocaproic acid as the eluant. The elution profile of these heavy chains is practically identical to the elution behavior previously observed for human Glu- and Lys-plasminogen, and human Glu- and Lys-plasmin adsorbed to these columns. Sedimentation velocity analysis of the heavy chain of human Glu-plasmin, in the presence of epsilon-aminocaproic acid, demonstrated that a gross conformational alteration occurs in this peptide accompanying binding of this amino acid. A much smaller conformational alteration occurs under similar circumstances with the human Lys-plasmin heavy chain. We find that the NH2-terminal peptide released in the Glu-plasminogen to Lys-plasminogen and Glu-plasmin to Lys-plasmin conversions is also released in the Glu-plasmin heavy chain to Lys-plasmin heavy chain conversion. This reaction is catalyzed at a significant rate only by plasmin and not by urokinase. Finally, no strong interaction between streptokinase and the isolated plasmin heavy chains is observed.
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PMID:Purification and some properties of the Glu- and Lys-human plasmin heavy chains. 13 7

Affinity chromatography on agmatine-Sepharose was used for the separation of two active forms of urokinase (EC 3.4.99.26) from partially purified human urinary urokinase. The approximate molecular weight of the heavier form was 47 000 and of the lighter 33 400. Both forms were homogeneous by sodium dodecyl sulfate gel electrophoresis and by 3H-labeled diisopropylphosphorofluoridate and 14C-labeled p-nitrophenyl-p'-guanidinobenzoate incorporation studies. The 33 400 mol. wt. form had a single chain, and the 47 000 mol. wt. form had two chains (33 100 and 18 600 mol. wt.) linked by disulfide bonds. The specific activity of the heavier form was 104 000 CTA units/mg protein, compared with 226 000 units/mg for the lighter form but the activities per mmol of active site (molar activities) of the two forms were almost identical (9.6-10(9) and 10.2-10(9) CTA units/mmol). Isoelectric focusing on gels showed that the 47 000 material contained one major subform with a pI of 8.60 and a minor subform with a pI of 8.90, while the 33 400 material had three major subforms with pI values of 8.35, 8.60 and 8.70, respectively, and a minor subform with a pI of 8.05. 3H-labeled diisopropylphosphorofluoridate incorporation studies revealed an active-site serine residue in the heavy chain.
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PMID:Purification and characterization of two forms of urokinase. 97 2

A 38-residue fragment is isolated from carboxymethylated plasminogen. Residues 29-38 have the same sequence as the amino-terminal end of the light chain of plasmin. The sequence 1-28 is therefore the sequence of the carboxyl-terminal end of the heavy chain and contains the specific sequence at which urokinase (EC 3.4.99.26) and other plasminogen-activating serine proteases split. Two of the five carboxymethyl-cysteine residues in the isolated fragment are situated close to the cleavage site and the fragment is not itself a substrate for plasminogen-activators. Residues 1-11 show extensive sequence homology with residues 137-147 and 242-252 in prothrombin, which are located in corresponding regions of the two internally homologous 83-residue structures in the non-thrombin part of the molecule, indicating that such structures may be a common feature of the non-protease part of the larger serine protease zymogens.
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PMID:Amino-acid sequence of activation cleavage site in plasminogen: homology with "pro" part of prothrombin. 105 75

Two forms of urokinase (EC 3.4.99.26) with apparent molecular weights of 33 400 and 47 000 purified by affinity chromatography have been modified specifically with newly synthesized peptide chloroketones by affinity labeline. Rapid inactivation of the enzyme preparations was observed with Ac-Gly-Lys-CH2 Cl and Nle-Gly-Lys-CH2 Cl which might be associated with a change in which a histidine residue is lost. After performic acid oxidation, an equivalent amount of 3-carboxymethyl histidine could be recovered, indicating alkylation at the N-3 of a histidine residue. In the case of the norleucine derivative, norleucine was concomitantly incorporated into the protein. It is thus likely that urokinase belongs in the class of enzymes utilizing the Asp..His..Ser triad for their catalytic action. The two active site residues so far identified, serine and histidine, were located in the heavy chain (33 100 mol. wt) of the 47 000 molecular weight form and in the 33 400 molecular weight form, the molecular weight of which remained constant.
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PMID:Identification of an active site histidine in urokinase. 126 31

Hybrid molecules containing the catalytic domain of either tissue plasminogen activator (tPA) or single chain urokinase-type plasminogen activator (scuPA), and the fibrin binding domain of a murine antifibrin monoclonal antibody were constructed using either cDNA or genomic DNA encoding the plasminogen activator and genomic DNA encoding antifibrin monoclonal antibody 59D8. In order to optimize expression of these fusion proteins in hybridoma cells, we compared plasminogen activator 3' UT domains (which decrease mRNA stability) with immunoglobulin and beta globin 3' UT domains (which increase mRNA stability). The presence of the plasminogen activator 3' UT domain resulted in approximately tenfold lower steady-state mRNA levels, and 300 to 500-fold lower levels of expressed functional protein. The initial goal of these studies was to increase the fibrinolytic potency and selectivity of tPA or scuPA. Fusion proteins comprising an antifibrin antibody domain and the catalytic domain of either tPA or scuPA were expressed and shown to have very different properties. The fusion protein that comprised the Fab portion of an antifibrin antibody and the catalytic domain of tPA, while displaying antigen binding properties indistinguishable from those of the parent antibody and amidolytic activity similar to that of tPA, was not more efficient than tPA in an in vitro clot lysis assay. In contrast, it had been shown that tPA chemically coupled to the same antibody was four- to sixfold more efficient in fibrinolysis both in vitro and in vivo. A recombinant scuPA-antifibrin antibody hybrid, however, was sixfold more potent than scuPA in vitro and 20-fold more potent in a rabbit thrombolysis model. An explanation for this apparent discrepancy may relate to the requirement for stimulation by fibrin in order for tPA to achieve its maximal catalytic activity, a property that was demonstrated to have been lost in the antifibrin-tPA fusion protein. In contrast, the activity of urokinase is independent of the presence of fibrin. This may explain the greater success achieved in enhancing catalytic activity in the urokinase-antifibrin fusion protein. It is of additional interest that fibrin or soluble fibrin fragments stimulate the catalytic activity of both tPA and the isolated tPA B chain, demonstrating that at least part of the enhanced catalytic activity of tPA observed in the presence of fibrin is independent of fibrin binding either by the tPA kringles or finger domain (or any heavy chain domain). These data indicate that it is possible to construct recombinant hybrid molecules in which both plasminogen activator catalytic function and antibody binding are preserved.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Hybrid molecules: insights into plasminogen activator function. 180 66

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 absorbed on a fibrin/Celite column at neutral pH, and could be eluted with 0.3-1.0 mol/l NaCl in phosphate buffer, followed by 0.2 mol/l, Arg, 2 mol/l KSCN, and 2 mol/l 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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PMID:Fibrin-binding urokinase (or precursor form of urokinase) with a molecular weight of about 100,000 in fresh human urine. 294 Dec 73


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