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)

Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins.
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PMID:Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells. 312 94

Human plasminogen is a beta-globulin (2% carbohydrate, molecular weight 90 KD), which in its native form has NH2-terminal glutamic acid (Glu-plasminogen) whose primary structure is known (31, 37, 38). From human plasma plasminogen can easily be isolated by affinity chromatography techniques (10, 25, and Table 1). Plasminogen is synthesized in many organs. The production site of the zymogen may be the liver (21), the eosinophiles (3) or the kidney (15). The plasma-plasminogen level is low in newborns (22) and even lower in the premature infant (2). In healthy adults it is found in plasma or serum in a concentration of 200 mg/l (= 2 microM, 22, 39). The half-life of the native (Glu-) plasminogen is 2.24 +/- 0.29 days (6). Two types of Glu-plasminogen occur in human plasma, which differ in their carbohydrate composition as well as in their content of sialic acid. Genetic variants (see Mayr, 3.1.); of plasminogen have been reported (16) after isoelectric focusing of human plasma in polyacrylamide gels. Three patterns were found, two completely different and the third most likely a mixture of the other two. Characteristical functional properties of plasminogen are related to its molecular structure, e.g. its in vivo specificity for fibrin in contrast to the fairly unspecific in vitro activity of plasmin. Glu-plasminogen is easily converted by limited plasmic digestion to modified forms with NH2-terminal lysine, valine or methionine, which are commonly designated "Lys-plasminogen" displaying a plasma half-life time of 0.8 days.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Plasminogen: a brief introduction into its biochemistry and function. 328 Apr 26

The specificity and reactivity of human alpha 1-proteinase inhibitor has been investigated by in vitro mutagenesis of the reactive site P1 methionine 358 residue to alanine 358 and cysteine 358. A comparison of the second-order association rates of both uncharged mutants with 9 serine proteinases indicated that each reacted similarly to either the normal plasma inhibitor or to a mutant containing valine in this position (Travis, J., Owen, M., George, P., Carrell, R., Rosenberg, S., Hallewell, R. A., and Barr, P. J. (1985) J. Biol. Chem. 260, 4384-4389) when tested against either neutrophil or pancreatic elastase. However, oxidation, carboxymethylation, or aminoethylation of the cysteine mutant to yield a charged P1 residue resulted in a significant decrease in association rates with both elastolytic enzymes, and aminoethylation created an excellent trypsin and plasmin inhibitor. These results indicate that the specificity of alpha 1-proteinase inhibitor is determined in a general manner by the class of amino acid residue in the P1 position. Substitution within the same category, such as from valine to alanine or cysteine among the aliphatic hydrophobic residues, has little effect on association rates with the elastolytic enzymes tested. However, alteration from an uncharged to a charged residue may cause considerable changes in both inhibitor specificity and reactivity as noted here with the cysteine derivatives and also previously with a natural variant in which methionine 358 to arginine 358 conversion resulted in the production of a potent thrombin inhibitor (Owen, M. C., Brennan, S. O., Lewis, J. H., and Carrell, R. W. (1983) N. Engl. J. Med. 309, 694-698).
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PMID:Recombinant DNA-derived forms of human alpha 1-proteinase inhibitor. Studies on the alanine 358 and cysteine 358 substituted mutants. 352 44

The immunochemical specificity of rabbit antisera to human fibrinopeptide-B (FPB) has been studied by comparing the relative abilities of FPB and of various proteins and peptides containing the NH2-terminal segment of the B beta-chain of human fibrinogen to inhibit the binding of a radioiodinated FPB derivative by each of seven anti-FPB sera. Anti-FBP sera varied in the extent to which they cross-reacted with fibrinogen, the NH2-terminal disulfide knot of fibrinogen (N-DSK), B beta 1(Pyr)-118(Met), B beta 1(Pyr)-42(Arg), and desarginyl-FPB. Anti-FPB sera have been identified that discriminate effectively between FPB and larger FBP-containing peptides; such antisera can be used to measure FPB in the absence of the larger peptides or to demonstrate the presence of larger peptides such as B beta 1(Pyr)-42(Arg) in extracts of clinical plasma samples by means of an increase in FPB immunoreactivity following thrombin treatment. One anti-FPB serum has been identified that is capable of detecting desarginyl-FPB, and this antiserum has been used in the development of a radioimmunoassay for desarginyl-FPB. Thus, by precisely defining the specificity of anti-FPB sera, it has been possible to identify antisera that are useful, not only in the measurement of FPB, but also in the detection of other important related molecules, such as B beta 1(Pyr)-42(Arg) and desarginyl-FPB. The immunochemical detection of these FPB-related peptides should provide useful information concerning the action of proteolytic enzymes, such as plasmin on the NH2-terminal segment of the B beta-chain of fibrinogen, and of carboxypeptidase-B on free FPB, in human plasma.
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PMID:Immunochemical studies of antisera to human fibrinopeptide-B. 617 83

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

Porcine tissue-type plasminogen activator (t-PA) increases the binding of 125I-glu-plasminogen to clots made from human plasma or purified fibrinogen in a time and t-PA concentration dependent fashion. The accumulation of plasminogen was faster and greater on noncrosslinked plasma clots than on clots which had been crosslinked by Factor XIIIa. Furthermore, the uptake of plasminogen to crosslinked fibrin clots occurred at a slower rate in the presence of alpha 2-plasmin inhibitor (alpha 2 PI) than in its absence. The kinetics of the uptake of 125I-plasminogen were analyzed using SDS-polyacrylamide gel electrophoresis and radioautography of solubilized plasma clots formed in the presence of t-PA. During the initial phase there was a decrease of clot-bound glu-plasminogen; simultaneously, there was a slight increase in clot-bound glu-plasmin and in plasmin complexed to alpha 2 PI that was crosslinked to alpha-chain polymers of fibrin. This was followed by a marked increase in clot-bound plasminogen having glutamic acid as NH2-terminal (glu-plasminogen) and gluplasmin. t-PA-induced enhancement of glu-plasminogen uptake appears to be mediated by plasmin but does not require the conversion of glu-plasminogen to plasminogen having lysine or methionine as NH2-terminal. The described mechanism assures an adequate supply of clot-bound plasmin, which is the enzyme ultimately involved in the degradation of fibrin.
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PMID:Tissue-type plasminogen activator increases the binding of glu-plasminogen to clots. 621 Mar 7

It has been previously demonstrated that commercial bacterial fibrinolysin (EC 3.4.21.7) selectively cleaves the bond between Met-53 and Ala-54 in ovine prolactin (199 amino acids). A one-step purification procedure on DEAE-cellulose for Protease F, which is the active component of bacterial fibrinolysin, and properties of the purified enzyme are reported. The enzyme is homogeneous as judged by acrylamide gel electrophoresis. Its molecular weight, calculated from gel filtration experiments on Sephadex G-100, is around 13,800. Amino acid analyses do not reveal the presence of any half-cystines. The presence of one tryptophan residue per enzyme molecule was resolved from the fluorescence spectrum. Amino terminal analysis showed that leucine was at the amino terminal position. Protease F hydrolyzes casein and synthetic specific substrates for chymotrypsin and elastase esterases but not for trypsin esterases. It is fully inhibited by phenylmethylsulfonyl fluoride, by chicken ovoinhibitor, and by Chymotrypsin Inhibitor I from potatoes but not by the trypsin-chymotrypsin inhibitors from soybeans and chick peas or by tosyl-L-phenylalanine chloromethyl ketone. The enzyme is stable at room temperature and in the cold, it is not affected by dialysis or by freezing and thawing, but it is inactivated during freeze-drying. The circular dichroism spectra of Protease F indicate an approximate 20% alpha-helix content of the enzyme with a considerable similarity to those of subtilisin, elastase, and beta-trypsin. The relatively low molecular weight of Protease F, the absence of intrachain disulfide bridges, and the fact that it is inhibited by several, but not all, chymotrypsin inhibitors suggest that it may differ phylogenetically from the known serine proteases.
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PMID:Purification and properties of protease F, a bacterial enzyme with chymotrypsin and elastase specificities. 622 44

The site of synthesis of alpha 2-plasmin inhibitor (alpha 2-PI), a physiologic inhibitor of plasmin, is not known with certainty. We have studied the production and secretion of alpha 2-PI by three established human liver cell lines derived from hepatocellular carcinoma and hepatoblastoma (Hep G2, Hep 3B, and PLC/PRF/5). As measured by a specific radioimmunoassay, the titer of alpha 2-PI increased in the medium of Hep G2 and Hep 3B cells with time, but no significant amount of alpha 2-PI was found in the medium of PLC/PRF/5. There was no evidence for a significant intracellular pool of this protein. On immunodiffusion against anti-alpha 2-PI serum, alpha 2-PI secreted by Hep G2 (G2 alpha 2-PI) formed a simple precipitin line of complete identity with the alpha 2-PI present in plasma (plasma alpha 2-PI). G2 alpha 2-PI behaved similarly to plasma alpha 2-PI in Sephadex G-150 gel filtration, sucrose density-gradient centrifugation, and crossed immunoelectrophoresis. G2 alpha 2-PI inhibited plasmin activity instantaneously in a functional assay and formed a complex with plasmin demonstrable by crossed immunoelectrophoresis. De novo synthesis of alpha 2-PI was shown by the presence of specific immunoprecipitable radioactivity in the medium after 5 hr of labeling of the cells with [35S]methionine. Analysis of the immunoprecipitates by NaDodSO4/polyacrylamide gel electrophoresis showed a single peak of radioactivity corresponding to Mr 68,000. These results indicate that the liver is a site of alpha 2-PI production.
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PMID:Synthesis and secretion of alpha 2-plasmin inhibitor by established human liver cell lines. 629 Oct 58

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.
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PMID:The complete amino acid sequence of low molecular mass urokinase from human urine. 675 72

The association rate constants for the interaction of alpha-1-proteinase inhibitor, oxidized alpha-1-proteinase inhibitor, and alpha-1-antichymotrypsin with several mammalian serine proteinases have been determined. The results indicate that leukocyte elastase reacts more rapidly with alpha-1-proteinase inhibitor than any other proteinase tested, while leukocyte cathepsin G shows the strongest association with alpha-1-antichymotrypsin. Oxidation of the critical methionine residue of alpha-1-proteinase inhibitor reduces the association with leukocyte elastase by a factor of more than 2000 and also lowers the association with all of the other enzymes tested with the exception of chymotrypsin. Significantly, oxidation completely abolishes any interaction of alpha-1-proteinase inhibitor with porcine elastase, human plasmin or human thrombin. These data support previous results (Johnson, D., and Travis, J. (1979) J. Biol. Chem. 254, 4022-4026) which indicated that oxidation of human alpha-1-proteinase inhibitor in vivo could reduce the effectiveness of this inhibitor in controlling proteolysis. In the lung, in particular, oxidizing agents of both chemical and biological sources could, indirectly, augment elastolysis in this tissue, resulting in the development of pulmonary emphysema.
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PMID:Kinetics of association of serine proteinases with native and oxidized alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin. 698 30


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