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)

Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.
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PMID:Proline-valine pseudo peptide enol lactones. Effective and selective inhibitors of chymotrypsin and human leukocyte elastase. 198 87

Skin fibroblasts from patients with inherited adenomatosis of the large bowel (ACR-SF) possess alterations in actin microfilament (MF) organization which serve to distinguish "predisposed" cells from fibroblasts derived from normal individuals (NSF). MF bundle frequency and diameter were considerably reduced in ACR-SF compared to NSF. This deficit in MF density correlated with a 60% decline in cytoskeletal-associated actin half-life. Absence of a well-structured MF network in ACR-SF was reflected in relatively poor cell-to-substrate adhesion (as indicated by increased sensitivity to trypsin release) and extensive membrane ruffling. Unlike NSF, ACR-SF failed to develop well-defined vinculin-containing focal contacts although the cellular content of vinculin was approximately the same in both cell types. The relatively low substrate adhesivity and reduced incidence of adhesive structures (i.e., MF and associated focal contacts) which typify ACR-SF correlated with a sixfold increase in cellular plasminogen activator (PA) activity. This increased protease activity corresponded with a 50-70% reduction in the content of the PA inhibitor-like protein p50 in both the saponin-resistant undersurface matrix and the culture medium. Increased motility and reduced cell-to-substrate adhesion, involving several cellular structural elements, appear to be significant correlates of the "predisposed" phenotype in cultured fibroblasts.
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PMID:Analysis of actin microfilaments and cell-to-substrate adhesive structures in human fibroblasts from individuals genetically predisposed to colonic carcinoma. 207 Aug 20

A 77-kDa complex of thrombin and a protein secreted by activated platelets had little if any thrombin amidolytic activity, indicating that the secreted protein is an inhibitor. The molecular weight of the inhibitor before reaction with thrombin was approximately 50,000. The apparent second-order rate constant for complex formation was estimated to be 1.3 x 10(6) M-1 s-1 (mean of four measurements); it was not affected by heparin or heparinase. These properties distinguish this inhibitor from other protease inhibitors secreted by platelets. The inhibitor reacted with trypsin and possibly with urokinase but not with factor Xa.
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PMID:Characteristics of a thrombin inhibitor secreted by activated platelets. 210 89

Slow and fast contracting muscles differ in their innervation and electrophysiological properties as well as in their regenerating potentialities. The purpose of the present work was to investigate the expression of plasminogen activators and its possible relation to each type of muscle. Slow (Soleus) and fast (Extensor Digitorum Longus) muscles were obtained from white Wistar rats. Before sectioning the muscles, the euthanized rats were perfused with cold phosphate buffer saline to avoid interference by circulating proteases and inhibitors. Muscle extracts were pounded in an ice-cold Potter tube. Plasminogen activators (PAs) were assayed by fibrin zymography and by both liquid and solid-phase fibrin spectrophotometric assays for the detection of PAs activity. Both urokinase (uPA) and tissue-type plasminogen activator (tPA) activities corresponding to proteins of 38 kDa and 65 kDa molecular masses, were detected in the extracts. Slow muscles contained higher amounts of both activators than fast muscles, but the relative amount of uPA was higher in both types of muscles. In addition, the characteristics of each type of extracts differed somewhat: the fast muscle activity curve was typical of an accelerating process, while the slow muscle curve showed an activity probably related to already formed plasmin or to some other trypsin-like enzyme. These results suggest that the amount of plasminogen activators could be a new criterion of discrimination between slow and fast skeletal muscles.
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PMID:Slow and fast rat skeletal muscles differ in their plasminogen activator activities. 211 33

The correlation between urokinase-type plasminogen activator (uPA) expression and tumor cell invasion and metastasis has been well documented. Urokinase converts the zymogen plasminogen to plasmin, a trypsin-like enzyme with broad substrate specificities. Net uPA activity is determined not only by the amount of the enzyme itself, but also by its state of activation and the amount of specific plasminogen activator inhibitors (PAIs) present. Both uPA and its substrate, plasminogen, can bind to cells via specific membrane-associated receptors. Expression of uPA, uPA receptor (uPAR), and PAIs is regulated by growth factors, oncogenes, and other effector molecules. In the present review we discuss the interactions of uPA with its receptor, inhibitors, and substrate and how these interactions influence malignant behavior. We also review recent reports in which investigators have used anti-catalytic antibodies and/or gene transfection to demonstrate that uPA is directly involved in tumor cell invasion and metastasis.
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PMID:The role of urokinase-type plasminogen activator in aggressive tumor cell behavior. 212 23

The lipoprotein Lp(a) is a cholesterol-rich plasma lipoprotein from the density fraction 1.06-1.21 g/ml. Numerous clinical and epidemiological studies have revealed a strong correlation between high plasma Lp(a) concentrations and the incidence of coronary heart disease. Furthermore, the Lp(a)-specific protein apo(a) has been detected in atherosclerotic lesions. Lp(a) is essentially an LDL-like lipoprotein particle to which the glycoprotein apo(a) is attached through a disulfide bridge with apo B-100. The elucidation of the amino acid sequence of apo(a) revealed a high homology to specific regions of human plasminogen. The latter consists of five tandemly arranged kringle domains followed by a C-terminal trypsin-like protease region. Apo(a) is composed of a large number of kringle domains, all highly homologous to kringle IV of plasminogen, followed by a kringle V-like protease-domain. The lipoprotein Lp(a), therefore, combines structural elements of both the lipoprotein and coagulation systems. In contrast to plasminogen, Lp(a) cannot be activated by TPA, streptokinase or urokinase to give proteolytic activity. However, in vitro studies have shown that Lp(a) can both inhibit endothelial cell induced fibrinolysis and can also bind to plasmin modified fibrin. These findings provide a pathobiochemical basis for the involvement of Lp(a) in atherosclerotic and thrombotic processes. The function of this lipoprotein is, however, still unclear.
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PMID:[Lipoprotein(a): characteristics of a special lipoprotein and its potential clinical significance]. 214 32

Cultured human endothelial cells synthesize and secrete two types of plasminogen activator, tissue plasminogen activator (t-PA) and urokinase (u-PA). Previous work from this laboratory (Hajjar, K.A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719) has demonstrated dose-dependent, saturable, and high affinity binding of t-PA to two sites associated with cultural endothelial cell monolayers. We now report that an isolated plasma membrane-enriched endothelial cell fraction specifically binds 125I-t-PA at a single saturable site (Kd 9.1 nM; Bmax 3.1 pmol/mg membrane protein). Ligand blotting experiments demonstrated that both single and double-chain t-PA specifically bound to a Mr 40,000 membrane protein present in detergent extracts of isolated membranes, while high molecular weight, low molecular weight, and single-chain u-PA associated with a Mr 48,000 protein. Both binding interactions were reversible and cell-specific and were inhibitable by pretreatment of intact cells with nanomolar concentrations of trypsin. The relevant binding proteins were not found in subendothelial cell matrix, failed to react with antibodies to plasminogen activator inhibitor type 1 and interacted with their respective ligands in an active site-independent manner. The isolated t-PA binding site was resistant to reduction and preserved the capacity for plasmin generation. In contrast, the isolated u-PA binding protein was sensitive to reduction, and did not maintain the catalytic activity of the ligand on the blot. The results suggest that in addition to sharing a matrix-associated binding site (plasminogen activator inhibitor type 1), both t-PA and u-PA have unique membrane binding sites which may regulate their function. The results also provide further support for the hypothesis that plasminogen and t-PA can assemble on the endothelial cell surface in a manner which enhances cell surface generation of plasmin.
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PMID:Identification and characterization of human endothelial cell membrane binding sites for tissue plasminogen activator and urokinase. 215 65

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.
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PMID:Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I. 218 Apr 85

Polymorphonuclear leucocyte (PMN) accumulation is associated with damage to airways epithelial cells in bronchitis, bronchiectasis and some forms of asthma. PMNs release several molecules which may mediate this damage, particularly proteases and oxidants. Using an in vitro model of intact human amnionic epithelial cells (EC) attached to native basement membrane (BM), we evaluated the capacity of several proteases and oxidants to induce detachment of EC from the BM. Maximum desquamation was observed with collagenase, elastase and trypsin, with minimum effective concentrations required to produce 50% EC-desquamation (MEC50) for highly purified collagenase, pancreatic elastase, human leucocyte elastase, human leucocyte cathepsin-G (Cath-G), trypsin, and kallikrein being 3616 +/- 989 U/mL, 32.3 +/- 14.7 U/mL, 85.8 +/- 26.7 U/mL, 360 +/- 20 U/mL, 340 +/- 49 BAEE U/mL and 300 +/- 23 U/mL, respectively. Urokinase (20 U/mL) and plasmin (500 U/mL) produced no desquamation in this system. Relatively high concentrations of oxidants also produced detachment (MEC50 for H2O2 and HOCl being 0.59 +/- 0.006 mol/L and 0.015 +/- 0.009 mol/L, respectively) and pretreatment of EC membranes with non-detaching concentrations of H2O2 rendered them 10-fold more susceptible to protease-induced desquamation, suggesting synergism. Reduced glutathione (GSH), N-acetyl cysteine (NAC), ethylenediamine tetra-acetic acid (EDTA) and 1,10 phenanthroline ablated collagenase induced EC-detachment. Elastase induced detachment was sensitive to inhibition by phenyl methyl sulfonyl fluoride (PMSF) and alpha 1-anti-proteinase (alpha 1-AP) and, to a lesser extent by aprotinin; trypsin-induced detachment was ablated by PMSF, alpha 1-AP and soybean trypsin inhibitor (SBTI) but not by 1,10 phenanthroline or EDTA. Cath-G induced detachment was profoundly inhibited by SBTI, GSH and NAC. These data demonstrate that human EC can be detached from intact BM by several PMN products, including collagenase, Cath-G and elastase, and that PMN-mediated detachment can be prevented by Cath-G and collagenase inhibitors. The data suggest a role for proteases, particularly Cath-G and collagenase, plus oxidants in synergism with proteases, in mediating PMN-induced EC detachment.
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PMID:Study of human epithelial cell detachment and damage: effects of proteases and oxidants. 220 Jul 49

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.
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PMID:Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils. 221 72


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