leupeptins has been researched along with benzamidine* in 9 studies
9 other study(ies) available for leupeptins and benzamidine
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Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands.
Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known. Topics: Benzamidines; Benzylamines; Binding, Competitive; Fluorine; Kinetics; Leupeptins; Ligands; Magnetic Resonance Spectroscopy; Monte Carlo Method; Trypsin | 2015 |
Tryptase increases proliferative activity of human conjunctival fibroblasts through protease-activated receptor-2.
Tryptase that is released by mast cell degranulation has recently been thought to play a key role in wound healing in allergic bronchitis. Conjunctival fibroblasts secrete mediators and extracellular matrices that could exacerbate inflammation and papillary formation in allergic conjunctivitis. This study was conducted to investigate the effect of tryptase on the proliferation of conjunctival fibroblasts and studied whether this effect was mediated by protease-activated receptor (PAR)-2.. Conjunctival fibroblasts were cultured with or without tryptase (0.1 ng/mL to 1.0 microg/mL), and the proliferation rate was assessed after 48 hours. The effects of tryptase inhibitors (leupeptin, benzamidine) and a PAR-2 agonist (SLIGKV) were examined. The existence of PAR-2 mRNA and protein in conjunctival fibroblasts was examined by RT-PCR and Western blot analysis, respectively. The existence of PAR-2 in cultured conjunctival fibroblasts and conjunctival papillae from patients with vernal keratoconjunctivitis, as well as conjunctival tissue from normal subjects was examined by immunohistochemistry.. Conjunctival fibroblast proliferation was upregulated by tryptase in a dose-dependent manner (P < 0.001). Leupeptin and benzamidine inhibited tryptase-induced fibroblast proliferation (P < 0.05), and SLIGKV mimicked tryptase's effect. PAR-2 mRNA and protein were detected in cultured conjunctival fibroblasts using RT-PCR and Western blot analysis. PAR-2 immunoreactivity in both the cultured conjunctival fibroblasts and in stromal cells in excised conjunctival tissues was observed.. Tryptase increased conjunctival fibroblast proliferation and this response appeared to be mediated by PAR-2. Mast cells are the most likely source of tryptase in the conjunctiva and may play an important role in chronic exacerbations with conjunctival papillary formation in allergic conjunctivitis. Topics: Benzamidines; Blotting, Western; Cell Proliferation; Cells, Cultured; Conjunctiva; Dose-Response Relationship, Drug; Fibroblasts; Humans; Immunoenzyme Techniques; Keratoconjunctivitis; Leupeptins; Mast Cells; Oligopeptides; Receptor, PAR-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine Endopeptidases; Serine Proteinase Inhibitors; Tryptases; Up-Regulation | 2005 |
Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme.
Atrial natriuretic peptide (ANP) is a cardiac hormone essential for the regulation of blood pressure. In cardiac myocytes, ANP is synthesized as a precursor, pro-ANP, that is converted to biologically active ANP by an unknown membrane-associated protease. Recently, we cloned a transmembrane serine protease, corin, that is highly expressed in the heart. In this study, we examine effects of corin on pro-ANP processing. Our results show that recombinant human corin converts pro-ANP to ANP and that the cleavage in pro-ANP by corin is highly sequence specific. Our findings suggest that corin is the long-sought pro-ANP-converting enzyme and that the corin-mediated pro-ANP activation may play a role in regulating blood pressure. Topics: Animals; Aprotinin; Atrial Natriuretic Factor; Benzamidines; Catalysis; Cell Line; COS Cells; Cricetinae; Gene Expression; Humans; Leupeptins; Membrane Proteins; Protein Precursors; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Serine Endopeptidases; Serine Proteinase Inhibitors; Structure-Activity Relationship; Trypsin Inhibitors | 2000 |
Submandibular salivary proteases: lack of a role in anti-HIV activity.
Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity. Topics: Anti-HIV Agents; Aprotinin; Benzamidines; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Edetic Acid; Egtazic Acid; Endopeptidases; Ethylmaleimide; HIV-1; Humans; Isoflurophate; Leupeptins; Metalloendopeptidases; Norleucine; Parotid Gland; Pepstatins; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Saliva; Serine Proteinase Inhibitors; Submandibular Gland; Trypsin Inhibitor, Bowman-Birk Soybean | 1998 |
Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases.
Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase. Topics: Amino Acid Sequence; Animals; Aprotinin; Base Sequence; Benzamidines; Cattle; DNA; Electrochemistry; Enzyme Activation; Enzyme Precursors; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Leupeptins; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligopeptides; Rats; Serine Endopeptidases; Serine Proteinase Inhibitors; Substrate Specificity; Thermodynamics; Trypsin; Trypsin Inhibitors | 1996 |
Evidence that acrosin activity is important for the development of fusibility of mammalian spermatozoa with the oolemma: inhibitor studies using the golden hamster.
The sperm plasma membrane over the equatorial segment of the acrosome gains the ability to fuse with the oolemma some time during, or after, the acrosome reaction. Since acrosin is a major component of the acrosome matrix that dissolves during the acrosome reaction, we sought to determine the effect of acrosin inhibitors on the sperm's ability to fuse with the oolemma. Five acrosin inhibitors (soybean trypsin inhibitor (SBTI), leupeptin, benzamidine, N-p-tosyl-1-lysin-chloromethyl ketone (TLCK) and phenylmethylsulphonyl fluoride (PMSF) and one non-acrosin inhibitor (N-p-tosyl-1-phenylalanine chloromethyl ketone (TPCK) were tested at non-toxic levels (below motility-disturbing concentrations). These inhibitors were added at three different times: (1) during the acrosome reaction of spermatozoa, (2) during sperm-oocyte contact and fusion, and (3) soon after sperm-oocyte fusion was completed. TLCK prevented sperm-oocyte fusion by inhibiting the acrosome reaction. PMSF inhibited gamete fusion, without inhibiting the acrosome reaction. SBTI, leupeptin and benzamidine also inhibited gamete fusion, but they had no effect if spermatozoa were allowed to acrosome-react in inhibitor-free medium. TPCK was without any inhibitory effects, suggesting that chymotrypsin-like enzymes are not involved in gamete fusion. Although acrosin inhibitors prevented acrosome-reacted spermatozoa from becoming fusion-competent, acrosin (and trypsin) alone could not make the plasma membrane of acrosome-intact spermatozoa fusion-competent. The data suggest that (1) the plasma membrane of the acrosomal region first undergoes dramatic changes immediately before or during the acrosome reaction and (2) acrosin released from the acrosome during the acrosome reaction further alters biophysical and biochemical characteristics of the plasma membrane over the equatorial segment. Such dual changes make the plasma membrane of this specialised region of the spermatozoon competent to fuse with the oolemma. Acrosin may not be the only acrosomal enzyme to participate in these changes. Topics: Acrosin; Acrosome; Animals; Benzamidines; Cell Membrane; Cricetinae; Female; In Vitro Techniques; Leupeptins; Male; Membrane Fusion; Mesocricetus; Microscopy, Electron; Oocytes; Protease Inhibitors; Sperm-Ovum Interactions; Spermatozoa; Trypsin | 1993 |
Successful 7-day perfusion preservation of the canine kidney.
Topics: Adenosine; Allopurinol; Animals; Benzamidines; Creatinine; Dogs; Female; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Leupeptins; Mersalyl; Mitochondria; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Perfusion; Quinacrine; Raffinose; Solutions; Time Factors | 1993 |
Effects of synthetic peptides and protease inhibitors on the interaction of a human ovarian cancer cell line (NIH:OVCAR-3) with a reconstituted basement membrane (Matrigel).
We have investigated the adhesive properties and invasiveness of cells of the human ovarian carcinoma line, NIH:OVCAR-3, in vitro. OVCAR-3 cells exhibited a similar rate of adhesion to all substrates tested including laminin, fibronectin, and collagens I and IV. The synthetic peptide YIGSR-NH2, which corresponds to an attachment site in laminin, inhibited the adhesion of the cells to laminin, but not to fibronectin. In contrast, a GRGDS-NH2 peptide blocked adhesion to fibronectin but not to laminin. OVCAR-3 cells invaded and formed branched colonies on Matrigel. Colony formation was retarded by both YIGSR-NH2 and GRGDS-NH2 peptides. Serine protease inhibitors and human recombinant TIMP, the tissue inhibitor of metalloproteases, inhibited ovarian tumor cell invasion while a synthetic collagenase IV inhibitor (SC-44463) had no effect. These studies suggest that metalloproteases other than collagenase IV may be important for the invasive activity of ovarian cancer cells. It is possible that synthetic peptides with antiadhesive cellular activity and certain antiproteases could be used to control the progressive colonization and invasion of peritoneal surfaces by malignant ovarian cancer cells. Topics: Amides; Amino Acid Sequence; Aminocaproates; Antineoplastic Agents; Benzamidines; Biocompatible Materials; Cell Adhesion; Cell Line; Chemotaxis; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Female; Glycoproteins; Humans; In Vitro Techniques; Laminin; Leupeptins; Metalloendopeptidases; Molecular Sequence Data; Neoplasm Invasiveness; Oligopeptides; Ovarian Neoplasms; Pepstatins; Polyamines; Protease Inhibitors; Proteoglycans; Tissue Inhibitor of Metalloproteinases; Tumor Cells, Cultured; Tyrosine | 1991 |
On the mechanisms of inhibition of insulin action by small-molecular-weight trypsin inhibitors.
Evidence from a number of laboratories has suggested that the mechanism of insulin action involves the release of an intracellular mediator polypeptide from the plasma membrane. It has been proposed that activation of a protease with trypsin-like specificity is involved in release of the putative mediator. In an effort to assess the potential role of such a protease in intact cells, the present study tested the effects of a variety of low-mol-wt protease inhibitors on insulin's metabolic action in isolated rat epididymal fat cells. The protease inhibitors studied included p-aminobenzamidine, benzamidine, phenylguanidine, diisopropylfluorophosphate, leupeptin, and the competitive substrate N-alpha-tosyl-L-arginine methylester. Leupeptin was devoid of activity. Most of the other inhibitors used were able to interfere with insulin-stimulated metabolism if used in sufficiently high concentrations, concentrations considerably higher than those required for inhibition of known proteases or inhibition of intracellular processes in a previously described system which involves a trypsin-like enzyme. Moreover, they displayed various activities unrelated to protease inhibition that could explain their effects on insulin action better than protease inhibition. While none of the data on individual inhibitors were by themselves convincing enough to either confirm or reject the hypothesis concerning the involvement of a protease with trypsin-like specificity in insulin action, taken together our results do weaken the hypothesis considerably and in particular render the involvement of an extracellular trypsin-like enzyme improbable. Topics: Adipose Tissue; Animals; Benzamidines; Glucose; Guanidines; In Vitro Techniques; Insulin; Insulin Antagonists; Isoflurophate; Leupeptins; Molecular Weight; Rats; Tosylarginine Methyl Ester; Trypsin Inhibitors | 1982 |