Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [125I]EDP I, [125I]Glu-plasminogen, and [125I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [125I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and 1730 microM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region (EDP I, Glu-plasminogen, Lys-plasminogen, and the plasmin heavy chain) and did not react with those lacking an EDP I region [miniplasminogen, the plasmin light chain or EDP II (kringle 4)] or with tissue plasminogen activator or prothrombin, which also contain kringles. By immunoblotting analyses, a chymotryptic degradation product of Mr 20,000 was derived from EDP I that retained reactivity with the antibody. The high-affinity lysine binding site was equally available to the antibody probe in Glu- and Lys-plasminogen and also appeared to be unoccupied in the plasmin-alpha 2-antiplasmin complex. alpha 2-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe. 309 30

The plasminogen activator urokinase was linked covalently to a monoclonal antibody specific for the amino terminus of the beta chain of human fibrin by means of the unidirectional cross-linking reagent N-succinimidyl-3-(2-pyridyldithio)propionate. N-Succinimidyl-3-(2-pyridyldithio)propionate allowed the amino groups on urokinase to be coupled to the sulfhydryl groups on iminothiolane (which had been introduced into the antibody before the coupling reaction). The inter-heavy chain sulfhydryl of the Fab' of this antibody was also linked to N-succinimidyl-3-(2-pyridyldithio)propionate-substituted urokinase. The antibody- or Fab'-urokinase complexes were purified by two affinity chromatography steps. In the first, benzamidine was used as ligand for urokinase, in the second, a heptapeptide consisting of the 7 amino-terminal residues of the beta chain of fibrin (beta peptide) was used as ligand for the antibody. The activity of the purified conjugates was compared with that of urokinase alone in an assay measuring lysis of 125I-fibrin monomer covalently linked to Sepharose CL-4B. For any concentration of either urokinase alone or urokinase-antifibrin antibody conjugate, an equivalent amount of lysis (release of labeled peptide from fibrin monomer-Sepharose) was obtained with 1/250 the concentration (with respect to urokinase content) of antifibrin antibody-urokinase conjugate. The antifibrin Fab'-urokinase conjugate exhibited a similar enhancement of activity in comparison with urokinase. Enhanced fibrinolysis was fully inhibited by beta peptide. These results suggest that antibody targeting enhances the concentration of urokinase in the vicinity of immobilized fibrin monomer, thereby also increasing the local conversion of plasminogen to plasmin, which in turn degrades its substrate, fibrin. Univalent antigen-antibody binding is sufficient for optimal efficiency.
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PMID:Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin. 361 Oct 93

When Glu-plasminogen (Glu-plg) was incubated with plasmin for various time intervals, and the mixture was activated by urokinase (UK), the activation rate increased gradually as incubation time increased. The presence of fibrin not only enhanced the activation rate of Glu-plg but also that of proteolytically modified form to some extent. The results of SDS-PAGE indicated that the release of N-terminal peptides from Glu-plg or Glu-plasmin takes place gradually when the concentration of plg was about 1 microM, and that Glu-plasmin I of larger molecular weight is more slowly converted to Lys-plasmin than Glu-plasmin II of smaller molecular weight. The amounts of carbohydrate moieties on the heavy chain of plasmin may influence the release of N-terminal peptide from Glu-plasmin. Kinetic studies indicate that Lys-plasmin has smaller km than Glu-plasmin, thus the former being better enzymatically than the latter.
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PMID:Activation pathway of glu-plasminogen to Lys-plasmin by urokinase. 612 17

Urokinase-activated human plasma was analysed by acetic acid/urea/polyacrylamide-gel electrophoresis. The bands representing plasminogen, the plasmin-alpha 2-plasmin inhibitor and plasmin-alpha 2-macroglobulin complexes were identified by immunoprecipitation with specific antibodies and by comparison with purified components. Plasminogen and the plasmin-inhibitor complexes were isolated from plasma or thrombin-clotted plasma containing 125I-labelled Glu-plasminogen (residues 1-790) and urokinase. The plasma was kept at 37 degrees C for 0.5 and 10 times the lysis time of the clotted plasma, the clotted plasma until lysis. The plasmin heavy chain from the plasmin-inhibitor complexes was subsequently prepared. Only in one case could a low-grade proteolytic conversion of Glu- forms into Lys/Met/Val-forms (residues 77-790, 68-790 and 78-790 respectively) during the preparations be detected. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and N-terminal sequence analysis of the purified plasminogen and plasmin heavy chain showed the following. The plasminogen in plasma was on the Glu- form. Glu-plasmin constituted 0.74 and 0.58 of the plasmin bound to the alpha 2-plasmin inhibitor in plasma after brief and prolonged activation respectively. The rest was Lys/Met/Val-plasmin. The clotted plasma contained about equal amounts of Glu-plasminogen and Lys/Met/Val-plasminogen, and predominantly Lys/Met/Val-plasmin complexed to alpha 2-plasmin inhibitor and alpha 2-macroglobulin. The results of the analysis of the purified material substantiated the identity of radioactive protein bands in the gel after acetic acid/urea/polyacrylamide-gel electrophoresis.
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PMID:Identification of molecular forms of plasminogen and plasmin-inhibitor complexes in urokinase-activated human plasma. 620 93

Selective proteolysis has been used to delineate the hemoglobin-binding site on haptoglobin heavy chain. Haptoglobin was cleaved specifically by plasmin, trypsin, chymotrypsin, staphylococcal protease, and thermolysin. Haptoglobin-hemoglobin complex was treated with these enzymes to determine which sites were protected from cleavage by the hemoglobin. The modified haptoglobins were tested for changes in their hemoglobin and hemoglobin alpha chain-binding properties. The sites of proteolytic cleavage were identified from the newly generated NH2 termini by automated Edman degradation amino acid-sequencing techniques. The results suggest that residues 128 through 131, 136 and 137, as well as 9 and 10 of the heavy chain may be involved in the binding of hemoglobin. On the other hand, residues 159 and 160, which lie in the 17-residue additional loop that is unique to haptoglobin among its homologous serine protease family, and residues 73 and 74, which lie close to the carbohydrate-binding residues, appear to be remote from the hemoglobin-binding site.
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PMID:Hemoglobin-binding site on haptoglobin probed by selective proteolysis. 621 62

Two plasmin inactivators, plasminase A and B, and their inhibitor embrinogen were isolated from embryonal carcinoma F9 cells by preparative two-dimensional electrophoresis. Plasminases A and B have molecular weights of 160,000 and 82,000, respectively. Both are serine proteinases which digest the light chain of plasmin in a time dependent inactivation process. The heavy chain of plasmin is not affected by this action. Plasminases A and B show similar specificity towards synthetic and natural polypeptide inhibitors. The interaction of the two enzymes leads to their inhibition. Embrinogen (m.w. 84,000) inhibits both plasminases A and B as well as urokinase and plasmin. Its activation by trypsin creates embrin, a proteinase directed against plasmin heavy chain.
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PMID:Plasmin regulating system from embryonal carcinoma F9 cells: plasminases A, B and embrinogen. 623 28

It was demonstrated that plasminogen and the plasmin heavy chain form a complex with an immobilized fibrinogen fragment E. The E-fragment interacts, in its turn, with the immobilized heavy chain; this interaction is provided for by the lysin binding sites of the plasminogen molecule. The plasmin light chain having no lysin binding sites is specifically absorbed on the immobilized fragment D, whereas the D-fragment--on the immobilized light chain. The elution is caused by arginine or benzamidine; 6-aminohexanoic acid does not affect this interaction. It is assumed that the interaction of plasminogen and plasmin with fibrin is provided for not only by the lysine binding but also by the benzamidine binding sites of the plasminogen molecule.
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PMID:[Interaction of heavy and light chains of plasmin with fibrinogen E and D fragments]. 624 Sep 93

Human factor XII was activated by limited proteolysis with trypsin, and the resulting beta-factor XIIa (Mr = 30,000) was isolated by DEAE-Sephacel column chromatography. The complete amino acid sequence of beta-factor XIIa was then determined on peptides produced by enzymatic digestion with either trypsin, chymotrypsin, or Staphylococcus aureus V8 protease and by chemical cleavage at methionyl and tryptophyl bonds. beta-Factor XIIa is a glycoprotein composed of a heavy chain (243 amino acid residues) and a light chain (9 amino acid residues), and these two chains are held together by a disulfide bond. The carbohydrate is attached to asparagine residue 61 in the heavy chain. The amino acid sequence of the heavy chain shows a high degree of homology to the corresponding regions of other plasma serine proteases, such as plasmin, thrombin, factor IXa and factor Xa, as well as the pancreatic digestive enzymes. These results demonstrate that factor XII is the precursor of a typical serine protease that participates in the coagulation cascade.
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PMID:Amino acid sequence of human beta-factor XIIa. 660 55

Low-molecular-weight urokinase (molecular weight 33100) was separated by analytical and preparative isoelectric focusing into five major subforms with isoelectric points between 8.7 and 9.6. These subforms are very similar in molecular weight, specific activity, amino acid composition and content of amino sugar and their N-terminal sequence constellation is identical. Low-molecular-weight urokinase consists of two polypeptide chains connected by a single disulfide bridge. The N-terminal region of the heavy chain (calculated Mr 30700) exhibits homology within the first 46 residues analyzed, with the known primary structure of other serine proteases. The mini chain (Mr 2426), whose complete sequence was determined, consists of 21 residues which show homology with the primary structure of the C-terminal region of the plasmin heavy chain. Based on sequence data and homology criteria with serine proteases a single-chain urokinase precursor is postulated having a peptide bond constellation between heavy and light chain region compatible with the requirements for serine protease activation.
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PMID:Human low-molecular-weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains. 674 91

The single polypeptide chain of native plasminogen (molecular weight approx. 90000) after CNBr-cleavage and gel filtration (Sephadex G-75) yielded a high molecular weight core fraction of fragments linked by disulfide bridges and three fragments of lower molecular weight (N-terminal and C-terminal CNBr-fragments and dodecapeptide). From the reduced and S-carboxamidomethylated core fraction an additional seven fragments with molecular weights between 2000 and 38000 were obtained. The CNBr-fragments were aligned in the porcine plasminogen polypeptide chain according to sequence homologies with the known primary structure of human plasminogen. Due to the lack of two methionine residues in kringle 1 and in the N-terminal part of the light chain region and to an additional methionine residue in kringle 2 the CNBr-fragment pattern differs from that of human plasminogen. Affinity chromatography of elastase-digested, native plasminogen yielded three fragments with intact lysine binding sites, originating from the heavy chain region and a non-adsorbable fragment, corresponding to human 'mini'-plasminogen. This fragment was converted to urokinase into a proteolytically active protein which served for the isolation of the porcine plasmin light chain. With the aid of the fragments produced by the CNBr and elastase cleavage approx. 350 residues were sequenced, of which about 80% showed identity with the sequence of human plasminogen. This percentage varied depending on the region of the molecule, with the highest extent of identity (80--90%) found in the analyzed kringles 2 and 4.
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PMID:Primary structure of porcine plasminogen. Isolation and characterization of CNBr-fragments and their alignment within the polypeptide chain. 734 Dec 39


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