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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Matrix metalloproteinase 7 (MMP-7) has been purified as an inactive zymogen of M(r) 28,000 (proMMP-7) from the culture medium of CaR-1 human rectal carcinoma cells. The NH2-terminal sequence of proMMP-7 is Lys-Pro-Lys-Pro-Gln-Glu, which is identical to that of matrilysin. The zymogen is activated by 4-aminophenylmercuric acetate (APMA), yielding an intermediate form of M(r) 21,000 and an active species of M(r) 19,000 which shows the new NH2-terminal sequence of Tyr78-Ser-Leu-Phe-Pro-Asn-Ser. Although trypsin fully activates the zymogen, the activation rate by plasmin or leukocyte elastase is confined to approximately 50%. ProMMP-7 can be activated by MMP-3 (stromelysin 1) to its full activity in a single-step mechanism and generates the same NH2 terminus obtained by APMA activation, whereas MMP-1 (tissue collagenase), MMP-2 (gelatinase A), and MMP-9 (gelatinase B) do not have such an effect. On the other hand, proMMP-1 is activated by MMP-7 to an activity similar to that obtained by APMA and the activation by MMP-7 is enhanced up to approximately 6.5 fold in the presence of APMA. This enhanced activity is donated by specific cleavage at the Gln80-Phe81 bond of proMMP-1. MMP-7 can also activate proMMP-9 up to approximately 50% of the full activity with a new NH2 terminus of Leu16-Arg-Thr-(Asn)-Leu. Incubation of proMMP-2 or proMMP-3 with MMP-7 results in no activation of these proMMPs. MMP-7 degrades type IV collagen, laminin-1, fibronectin, proteoglycan, type I gelatin, and insoluble elastin. These results suggest that in vivo MMP-7 may play a role in degradation of extracellular matrix macromolecules in concert with MMP-1, -3, and -9 under pathological conditions.
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PMID:Matrix metalloproteinase 7 (matrilysin) from human rectal carcinoma cells. Activation of the precursor, interaction with other matrix metalloproteinases and enzymic properties. 789 11

A series of dipeptides which contained phosphonate analogs of proline and piperidine-2-carboxylic acid (homoproline) have been synthesized and tested as inhibitors of DPP-IV. The rates of inhibition of DPP-IV by these compounds are moderate, but the inhibitors are quite specific. The best inhibitor in the series is Ala-PipP(OPh-4-Cl)2 (13), which has a k(inact) of 0.353 s-1 and KI of 236 microM. The DPP-IV inhibitors Ala-ProP(OPh)2 (6), Ala-ProP(OPh-4-Cl)2 (12), and Ala-PipP(OPh-4-Cl)2 (13) do not inhibit trypsin, human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), acetylcholinesterase, papain, and cathepsin B. However, compounds 12 and 13 inhibited chymotrypsin slowly. Most of these dipeptides containing a homoproline phosphonate residue (PipP) or a Pro phosphonate residue (ProP) at the P1 site are stable in a pH 7.8 buffer with half-lives of several hours to several days. DPP-IV inhibited by 6, 7 (Ala-PipP(OPh)2), 12, or 13 is quite stable, and no enzyme activity was recovered after removal of excess inhibitor and incubation in buffer for 1 day. Since the phosphonate inhibitors are specific toward DPP-IV and the inhibited enzymes are stable, they should be useful in establishing the biological functions of DPP-IV and may be useful therapeutically in the prevention of the rejection of transplanted tissue.
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PMID:Dipeptide phosphonates as inhibitors of dipeptidyl peptidase IV. 796 57

The second domain (R-020) of human urinary trypsin inhibitor (UTI) exerts similar inhibitory activities on trypsin, alpha-chymotrypsin, leukocyte elastase, and plasmin to those of UTI itself, and additionally inhibits coagulation factor Xa (FXa) and plasma kallikrein, on both of which UTI has no inhibitory effect. In the present study, we attempted to increase this FXa-inhibitory activity by modeling the structure of R-020-FXa complex and substituting one or two amino acids in R-020 using recombinant DNA technology. Molecular modeling of R-020 and FXa was performed with reference to X-ray analysis of the complex of bovine pancreatic trypsin inhibitor (BPTI) and bovine trypsin to determine the site of amino acid modification. The expression plasmids into which R-020 genes with base substitution were inserted were prepared and introduced into Escherichia coli to express R-020 variants. The resulting variants were purified and their enzyme inhibitory activities were measured. The FXa-inhibitory activity was increased in four variants with single amino acid substitution. With another four variants having two amino acid substitutions involving combinations of the above single amino acid substitutions, the FXa-inhibitory activity was further increased. Because the electrostatic interaction within R-020-FXa complex seemed stronger in these R-020 variants, this increase in FXa-inhibitory effect was speculated to be a consequence of more potent binding between the enzyme and the inhibitor.
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PMID:Design of variants of the second domain of urinary trypsin inhibitor (R-020) with increased factor Xa inhibitory activity. 798 90

Serum amylase continues to be the most widely used test to diagnose acute pancreatitis; however, its popularity does not appear to be justified. The serum amylase test has a poor sensitivity and specificity. Furthermore, it has an extremely low sensitivity in detecting acute alcoholic pancreatitis, which is the most common cause of acute pancreatitis in city hospitals. Older assay techniques for serum lipase were cumbersome and time-consuming. The newer methods seem to have overcome the disadvantages of the previous techniques. They are quick, reliable, and inexpensive. Recent studies indicate that serum lipase may be a better test to diagnose acute pancreatitis. Therefore, serum lipase should be used more frequently in the diagnosis of acute pancreatitis. Serum trypsin, although sensitive, is difficult to estimate and is not routinely available. Serum elastase offers no additional benefit over the serum amylase or lipase tests. Markers such as alpha 2-macroglobulin, RNase, phospholipase, and polymorphonuclear elastase predict severity of disease, but assay techniques for these agents are still experimental and confined to specialized centers. C-reactive protein is a reasonably reliable indicator of severity and, because it is universally available, should be used more frequently. Of the imaging techniques, computerized tomography scanning is the best method to delineate the pancreas; however, ultrasound is more cost-effective in clinical practice.
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PMID:Diagnostic tests for acute pancreatitis. 805 37

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
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PMID:Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. 814 99

Multiple forms of Bowman-Birk soybean inhibitor have for the first time been isolated from commercial soya flour and purified to homogeneity. Amino acid compositions and isoelectric points of the inhibitors were determined. The isolated inhibitors are shown to be related to classic (M 8000 Da, 2-II) and high molecular mass glycine-rich (M 17 000 Da, 3-II, 5-II) Bowman-Birk inhibitors. The inhibitor (2-II) was found to have two reactive sites and bind trypsin at one centre and alpha-chymotrypsin, cathepsin G and human leukocyte elastase at the other. Rate constants of the complex formation (ka) and complex dissociation (kd) were determined by following the kinetics of approaching to the steady state in a system including the enzyme, the substrate and various concentrations of the inhibitor.
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PMID:[High molecular weight soy isoinhibitors of the Bowman-Birk type. Isolation, characteristics, and kinetics of interaction with proteinases]. 816 55

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.
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PMID:Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. 819 13

alpha 1-Proteinase inhibitor (alpha 1-PI) is the major endogenous inhibitor of human leukocyte elastase (HLE). We have employed two different methods to quantitate the binding of alpha 1-PI to extracellular matrix (ECM), composed of 51% glycoproteins and proteoglycans, 37% types I and III collagen, and 12% elastin, derived from rat heart smooth muscle cells. alpha 1-PI is tightly bound to ECM via a saturable adsorption process; the bound protein fails to dissociate from the matrix after repeated washing. Binding of alpha 1-PI is unaffected by the prior removal of ECM glycoproteins with trypsin. Binding to ECM is not decreased in the presence of high salt but is decreased at low pH. A 40-fold excess of unlabeled alpha 1-PI displaces only 50% of [125I]alpha 1-PI prebound to ECM. A 30% decrease in the levels of alpha 1-PI bound to ECM is observed after DTT washes of ECM preincubated with alpha 1-PI or when alpha 1-PI is modified with iodoacetamide prior to incubation with ECM, implying that a fraction of bound alpha 1-PI is covalently linked to ECM via disulfide bond formation. Moreover, high molecular weight complexes between [125I]alpha 1-PI and ECM components can be visualized by SDS-PAGE under nonreducing conditions but disappear upon reduction. Approximately 50% of the total alpha 1-PI bound covalently or noncovalently to ECM retains the ability to inhibit HLE-mediated ECM proteolysis. alpha 1-PI-HLE complexes bound to ECM can be visualized by SDS-PAGE following the addition of HLE to ECM that was pretreated with [125I]alpha 1-PI. alpha 1-PI from normal plasma or serum also binds to ECM with retention of immunoreactivity and partial retention of inhibitory activity. However, ECM pretreated with alpha 1-PI-deficient serum retains no HLE-inhibitory activity.
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PMID:Human alpha 1-proteinase inhibitor binds to extracellular matrix in vitro. 825 98

Eight new biotinylated, mechanism-based isocoumarin serine protease inhibitors have been designed and synthesized to detect, localize, and isolate serine proteases. Isocoumarins that contain a 4-chloro group, a biotinylated substituent at the 7-position, and different 3-alkoxy groups are inhibitors of various serine proteases including human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), trypsin, human recombinant granzyme A, chymotrypsin, and cathepsin G. Insertion of spacers between the isocoumarin moiety and the biotin moiety enhanced enzyme inhibitory potency and may also promote binding of the enzyme-inhibitor complex to avidin. The 3-alkoxy groups conferred selectivity toward different serine proteases with chymotrypsin being inhibited effectively by compounds with 3-phenylethoxy groups while derivatives with 3-methoxy, ethoxy, or propoxy groups were potent inhibitors of HLE and moderate inhibitors of PPE. Full enzymatic activity was regained after the immediate addition of hydroxylamine to the inactivated chymotrypsin and PPE derivatives, which indicated that a simple acyl enzyme derivative is formed initially in the inhibition reaction. Egg avidin did not effect the rate of spontaneous enzyme reactivation rate while streptavidin accelerated the reactivation reaction. PPE inhibited by 7-[[6-(biotinylamino)caproyl]amino]-4-chloro-3- ethoxyisocoumarin (BIC 5) or 7-[[6-[[6-(biotinylamino)caproyl]amino] caproyl]amino]-4-chloro-3-methoxyisocoumarin (BIC 7) was bound to immobilized avidin columns. Most of inhibited PPE could be eluted from the monomeric or tetrameric avidin columns but only a portion (40-70%) of the enzyme was active due to the partial formation of a stable alkylated enzyme derivative during the isolation process.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biotinylated isocoumarins, new inhibitors and reagents for detection, localization, and isolation of serine proteases. 830 26

Effects of heparin on the inhibitory activities of human urinary trypsin inhibitor (ulinastatin) on trypsin, chymotrypsin and leukocyte elastase were studied. Heparin per se neither influenced the enzymatic activities nor changed the mode of inhibition of ulinastatin on the enzymes. In the presence of heparin, inhibitory effects of ulinastatin on trypsin were enhanced, whereas its effects on chymotrypsin and elastase were attenuated. These results suggest that the two functional domains in ulinastatin are differently affected by heparin.
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PMID:Effects of heparin on the inhibitory activities of human urinary trypsin inhibitor (ulinastatin) on trypsin, chymotrypsin and leukocyte elastase. 834 Oct 25


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