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)

Thrombin stimulates polyphosphoinositide hydrolysis in embryonic chick heart cells and in 1321N1 astrocytoma cells and increases intracellular Ca2+ in the 1321N1 cells. The serine protease trypsin mimics these actions in a dose-dependent fashion, whereas the proteolytically inactive thrombin derivatives diisopropyl fluorophosphate-thrombin (DIP-thrombin) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin (PPACK-thrombin) are ineffective in this regard. The phosphoinositide responses to thrombin or trypsin and the muscarinic agonist carbachol are additive, but no additivity is observed between the responses to thrombin and trypsin. Unlike the response to carbachol, the phosphoinositide and Ca2+ responses to thrombin and trypsin desensitize, with no recovery of the calcium response even when Ca2+ stores are replenished. Cross-desensitization of phospholipase C activation and calcium mobilization between these proteases is also observed. In addition, PPACK-thrombin, which elicits no response itself, effectively inhibits trypsin-stimulated phosphoinositide hydrolysis. It is proposed that thrombin and trypsin act through the same receptor. Proteolysis appears to be important in the mechanism by which these agonists elicit phosphoinositide hydrolysis, calcium mobilization, and, perhaps, subsequent receptor desensitization.
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PMID:Thrombin and trypsin act at the same site to stimulate phosphoinositide hydrolysis and calcium mobilization. 254 47

The possibility that thrombin-induced platelet reactivity could occur via both a receptor-related and a proteolytic process was examined. Thrombin elicited the formation of considerably more [32P]phosphatidic acid (an index of phospholipase C catalysed phosphoinositide metabolism) than did platelet activating factor, 5-hydroxytryptamine, ADP, and the thromboxane A2 analogue EP171, when these agents were added either alone or in combination. Co-addition of thrombin and EP171 did not evoke significantly more [32P]phosphatide acid than did thrombin alone. The protease inhibitor leupeptin, decreased but did not abolish [32P]phosphatidic acid formation elicited by either thrombin alone or thrombin in combination with EP171. The serine protease, trypsin, stimulated an increase in [32P]phosphatidic acid and this effect was additive with that of EP171. This augmentation by trypsin of EP171-induced [32P]phosphatidic acid formation was inhibited by leupeptin. These results are consistent with the concept that thrombin-induced activation of phospholipase C occurs by two distinct mechanisms: one via proteolysis, which is sensitive to leupeptin, and the other via receptor activation, a process shared by EP171. The individual components of this dual mechanism can be mimicked by the co-addition of a receptor-directed agonist (EP171) and a proteolytic agent (trypsin).
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PMID:Evidence for two mechanisms of thrombin-induced platelet activation: one proteolytic, one receptor mediated. 255 47

Active site serine 195 of rat anionic trypsin was replaced with a cysteine by site-specific mutagenesis in order to determine if a thiol group could function as the catalytic nucleophile in serine protease active site environment. Two genetically modified rat thiol trypsins were generated; the first variant contained a single substitution of Ser195 with Cys (trypsin S195C) while the second variant contained the Ser195 to Cys as well as an Asp102 to Asn substitution (trypsin D102N,S195C) that more fully mimics the putative catalytic triad of papain. Both variants were expressed as his J signal peptide-trypsin fusion proteins to high levels under the control of the tac promoter. The mature forms of both variants were secreted into the periplasmic space of Escherichia coli. Trypsin S195C shows a low level of activity toward the activated ester substrate Z-Lys-pNP, while both trypsin S195C and trypsin D102N,S195C were active toward the fluorogenic tripeptide substrate Z-GPR-AMC. Esterase and peptidase activities of both thiol trypsin variants were inhibited by known Cys protease inhibitors as well as by specific trypsin inhibitors. The kcat of trypsin S195C was reduced by a factor of 6.4 x 10(5) relative to that of trypsin while the kcat of trypsin D102N,S195C was lowered by a factor of 3.4 x 10(7) with Z-GPR-AMC as substrate. Km values were unaffected. The loss of activity of trypsin D102N,S195C was partially attributed to an inappropriate Asn102-His57 interaction that precludes the formation of the catalytically competent His57-Cys195 ion pair although loss of the negative charge of D102 at the active site probably contributes to diminished activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Introduction of a cysteine protease active site into trypsin. 261 Dec 27

We have determined the three-dimensional structures of engineered rat trypsins which mimic the active sites of two classes of cysteine proteases. The catalytic serine was replaced with cysteine (S195C) to test the ability of sulfur to function as a nucleophile in a serine protease environment. This variant mimics the cysteine trypsin class of thiol proteases. An additional mutation of the active site aspartate to an asparagine (D102N) created the catalytic triad of the papain-type cysteine proteases. Rat trypsins S195C and D102N,S195C were solved to 2.5 and 2.0 A, respectively. The refined structures were analyzed to determine the structural basis for the 10(6)-fold loss of activity of trypsin S195C and the 10(8)-fold loss of activity of trypsin D102N,S195C, relative to rat trypsin. The active site thiols were found in a reduced state in contrast to the oxidized thiols found in previous thiol protease structures. These are the first reported structures of serine proteases with the catalytic centers of sulfhydryl proteases. Structure analysis revealed only subtle global changes in enzyme conformation. The substrate binding pocket is unaltered, and active site amino acid 102 forms hydrogen bonds to H57 and S214 as well as to the backbone amides of A56 and H57. In trypsin S195C, D102 is a hydrogen-bond acceptor for H57 which allows the other imidazole nitrogen to function as a base during catalysis. In trypsin D102N,S195C, the asparagine at position 102 is a hydrogen-bond donor to H57 which places a proton on the imidazole nitrogen proximal to the nucleophile. This tautomer of H57 is unable to act as a base in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Crystal structures of two engineered thiol trypsins. 261 Dec 28

The crystal structure of bovine pancreatic beta-trypsin (BPT) has been determined from a novel orthorhombic crystal form which contains substantially more solvent (filling 57% of the volume of the unit cell) than previously determined orthorhombic (44%) and trigonal (37%) BPT structures. The native and benzamidine-inhibited crystal structures of BPT in ammonium sulphate at pH 5.3 have been determined for the new form by molecular replacement techniques. The structures have been refined at 1.5 A resolution with final R-values of 16.7% and 16.9%, respectively. Comparison with the previously refined old orthorhombic forms shows that the overall conformation of the protein backbone is highly conserved. A great number of previously undefined side-chains have been located in density. At the C terminus an extra ion pair involving lysines 87 and 107 has been revealed. A far more detailed picture of the ordered solvent structure has been derived. Thirty water clusters have been identified. A large water network extends from the calcium binding site to the activation area and the autolysis loop. There is evidence for a water channel reaching from the depth of the specificity pocket to the nearby protein surface which might be involved in the displacement of water molecules upon substrate binding. A sulphate anion which forms hydrogen bonds to the active site residues His57, Ser195 and Gly193 was for the first time positioned in clearly defined electron density. Interaction with the sulphate ion may explain the increase in the pKa value of His57 at high sulphate concentrations which was observed by nuclear magnetic resonance studies of a bacterial serine protease both in crystalline form and in solution. Thus, a His-Ser hydrogen bond will not exist in solvents containing sulphate at low pH (up to at least 6.8) where the imidazole of His57 is protonated. The new crystal form is of considerable interest for substrate binding studies. Wide solvent channels should allow diffusion of large substrates (comparable in size to, e.g. pancreatic trypsin inhibitor) into the enzyme crystal. The active site is accessible; intermolecular contact areas are further remote from the active site than in the old orthorhombic form.
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PMID:Crystal structure of bovine beta-trypsin at 1.5 A resolution in a crystal form with low molecular packing density. Active site geometry, ion pairs and solvent structure. 261 45

The eukaryotic serine protease, rat anionic trypsin, and various mutants created by site-directed mutagenesis have been heterologously expressed in Escherichia coli. The bacterial alkaline phosphatase (phoA) promoter was used to control the expression of the enzymes in an induced or constitutive fashion. The DNA coding for the eukaryotic signal peptide of pretrypsinogen was replaced with DNA coding for the phoA signal peptide. The phoA signal peptide successfully directs the secretion of the mammalian trypsinogen to the periplasmic space of E. coli. Active trypsin was expressed in the periplasm of E. coli by deleting the DNA coding for the activation hexapeptide of the zymogen. The activity of trypsin in the periplasm suggests that the enzyme is correctly activated and has folded such that the 12 cysteine residues involved in the six disulfide bonds of rat anionic trypsin have paired correctly. A transcription terminator increased the level of expression by a factor of two. However, increasing the copy number of the plasmid decreased the levels of expression. Localization of the active enzyme in the periplasm allows rapid screening of modified trypsin activities and facilitates the purification of protein to homogeneity and subsequently to crystallinity.
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PMID:An expression system for trypsin. 265 64

The amino acid sequence of a protease, protein C activator, from Agkistrodon contortrix contortrix venom was determined. Peptide fragments obtained by chemical or enzymatic cleavage of the S-carboxymethylated protein were purified by gel filtration and reverse-phase high-performance liquid chromatography. The present study demonstrates that protein C activator from A. contortrix contortrix venom is a trypsin-type serine protease that is composed of 231 residues with a molecular weight of 25,095 for the polypeptide portion of the molecule. By analogy to the mammalian serine proteases, the catalytic triad in venom protein C activator consists of His-40, Asp-85, and Ser-177. The protein also contains three N-linked glycosylation sites at Asn-21, Asn-78, and Asn-129. The amino acid sequence of protein C activator exhibits a high degree of sequence identity with other snake venom proteases: 73% with batroxobin, 68% with flavoxobin, and 55% with Russell's viper venom factor V activator.
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PMID:Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom. 265 26

The serine protease enterokinase is the physiological activator of trypsinogen and has a specificity for the sequence (Asp)4-Lys-Ile. The enzyme consists of two subunits linked by a disulfide bond. The heavy chain achors enterokinase in the intestinal brush border membrane and the light chain is the catalytic subunit, which has the same mechanism of action as trypsin and chymotrypsin. Many properties of enterokinase resemble blood-clotting enzymes, suggesting that enterokinase lies on the same phylogenetic branch as the blood-clotting proteins.
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PMID:Enterokinase (enteropeptidase): comparative aspects. 265 18

A detergent-dispersed adenylate cyclase from rat brain was used to study the effects of ammonium salts and polyamines on the proteolytic activation of the enzyme by a sperm protease and on the sensitivity of adenylate cyclase to inhibition via its "P"-site. A purified preparation of a trypsin-like, serine protease from bovine sperm was used to activate solubilized adenylate cyclase in the presence of guanosine 5'-O-(3-thiotriphosphate (GTP gamma S). The proteolytically activated form of adenylate cyclase was found to be particularly sensitive to further activation by ammonium bicarbonate. The activation by NH4HCO3 was found to be due to the NH+4 cation and was characterized by an increased Vmax and by a decreased sensitivity of adenylate cyclase to inactivation by elevated concentrations of the sperm protease or by trypsin. NH4Cl and (NH4)2SO4 also caused biphasic effects on adenylate cyclase, which mimicked but were less effective than those caused by NH4HCO3. Consistent with observations of others, adenylate cyclase activity was enhanced by ammonium ions whether in the presence of reversible (Mn2+) or irreversible (GTP gamma S) activators. Mn2+- and GTP gamma S-stimulated activities were similarly optimally enhanced by 30 mM (NH4)2SO4 and by 30 to 150 mM NH4Cl or NH4HCO3. Ammonium ions did not increase the activity of the purified catalytic unit. Moreover, the effect of ammonium ions was not accompanied by an increased rate of activation by GTP gamma S, suggesting that the activation of Gs (guanine nucleotide-dependent stimulatory component) may not be the primary cause of stimulation by ammonium salts. Several polyamines at millimolar concentrations blocked the stimulatory effect of NH+4. This was observed when adenylate cyclase was activated by Mn2+, but not when it was activated by GTP gamma S or by the sperm protease + GTP gamma S. The inhibitory effect of polyamines was not due to the formation of a complex with ATP. Both the increase in Vmax of the Mn2+-stimulated enzyme by NH+4 and the decrease in Vmax caused by spermine were accompanied by an increase in the enzyme's Km MnATP app. Spermine increased the IC50 for inhibition of Mn2+-activated adenylate cyclase by 2',5'-dideoxyadenosine (2',5'-ddAdo) from 0.75 to 4.6 microM, consistent with the idea that increased sensitivity of P-site-mediated inhibition is associated with increased enzyme activity. In contrast, activation of Mn2+-stimulated adenylate cyclase by 30 mM (NH4)2SO4 also reduced sensitivity to inhibition by 2',5'-ddAdo(IC50 1.1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Ammonium ions enhance proteolytic activation of adenylate cyclase and decrease its sensitivity to inhibition by "P"-site agonists. 265 8

Serine proteases are one of the biologically most important and widely distributed families of enzymes. Isolation of serine protease genes from organisms of widely diverged phylogenetic groups would provide a basis for studying their biological function, the relationship between structure and function, and the molecular evolution of these enzymes. Serine proteases for which little structural information is known are those that are important in the pathogenesis of parasitic nematode and protozoan diseases. Identification and isolation of protease genes from these organisms is a critical first step in understanding their function for the parasite and possibly suggesting innovative approaches to arresting parasitic diseases. Serine protease gene fragments were isolated from genomic DNA of the parasitic nematode Anisakis simplex by using degenerate oligonucleotide primers and the polymerase chain reaction. Primers were designed based upon the consensus sequence of amino acids flanking the active site serine and histidine residues of eukaryotic serine proteases. Four serine protease gene fragments from this parasite were sequenced and one is 67% identical to the rat trypsin II gene. Alignment of these two genes revealed that the intron-exon junctions are conserved between nematode and rat suggesting that this Anisakis serine protease is structurally and functionally similar to rat trypsin. The generality of this approach to identify serine protease genes from genomic DNA of two very divergent species, a parasitic protozoan and a mammal, was also confirmed. Genes for other enzymes or any protein with conserved structural motifs can be identified and isolated using this technology. Using a similar strategy, a cathepsin B-like cysteine (thiol) protease gene fragment was isolated from Caenorhabditis elegans DNA.
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PMID:Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes. 266 85


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