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)

Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca2+, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IXEagle Rock (IXER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IXN) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of gamma-carboxyglutamic acid residues, and beta-OH aspartic acid content, and in its binding to an anti-IXN monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. Further, IXER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca2+ at a rate similar to that observed for IXN. However, in contrast to IXaN, IXaER does not bind to antithrombin-III (specific inhibitor of IXaN) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IXER, all eight exons of IXN and IXER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G----T) which results in the replacement of Val for Gly363 in the catalytic domain of IXER was identified. Gly363 in factor IXa corresponds to the universally conserved Gly193 in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly363----Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.
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PMID:Experimental and theoretical evidence supporting the role of Gly363 in blood coagulation factor IXa (Gly193 in chymotrypsin) for proper activation of the proenzyme. 230 34

A peptide having enzyme-like catalytic activity has been designed and synthesized. Computer modeling was used to design a bundle of four short parallel amphipathic helical peptides bearing the serine protease catalytic site residues serine, histidine, and aspartic acid at the amino end of the bundle in the same spatial arrangement as in chymotrypsin (ChTr). The necessary "oxyanion hole" and substrate binding pocket for acetyltyrosine ethyl ester, a classical ChTr substrate, were included in the design. The four chains were linked covalently at their carboxyl ends. The peptide has affinity for ChTr ester substrates similar to that of ChTr and hydrolyzes them at rates approximately 0.01 that of ChTr; total turnovers greater than 100 have been observed. The peptide is inhibited by ChTr specific inhibitors and is inactive toward benzoyl arginine ethyl ester, a trypsin substrate. The peptide is inactivated by heating above 60 degrees C, but recovers full catalytic activity upon cooling and lyophilization from acetic acid.
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PMID:Design and synthesis of a peptide having chymotrypsin-like esterase activity. 236 48

A new serine protease was encoded by a clone isolated from a murine cytotoxic T-lymphocyte complementary DNA library by an RNA-hybridization competition protocol. Complementary transcripts were detected in cytotoxic T lymphocytes, spleen cells from nude mice, a rat natural killer cell leukemia, and in two of eight T-helper clones (both cytotoxic), but not in normal mouse kidney, liver, spleen, or thymus, nor in several tested T- and B-cell tumors. T-cell activation with concanavalin A plus interleukin-2 induced spleen cells to express this gene with kinetics correlating with the acquisition of cytolytic capacity. The nucleotide sequence of this gene encoded an amino acid sequence of approximately 25,700 daltons, with 25 to 35 percent identity to members of the serine protease family. The active site "charge-relay" residues (His57, Asp102, and Ser195 of the chymotrypsin numbering system) are conserved, as well as the trypsin-specific Asp (position 189 in trypsin). A Southern blot analysis indicated that this gene is conserved in humans, mouse, and chicken. This serine protease may have a role in lymphocyte lysis and a "lytic cascade."
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PMID:Cloning of a cDNA for a T cell-specific serine protease from a cytotoxic T lymphocyte. 242 55

A trypsin-like protease(s) was found in the extracts from tuberculin reaction sites in the guinea pig skin. Extractable enzyme activities were chronologically paralleled with the gross appearance of the inflammation, and the maximum activity from 24-hour old inflamed sites was about 12 times stronger than that from control skin, suggesting a potential role in the pathogenesis of the delayed hypersensitivity reaction (DHR). The enzyme suitably hydrolyzed t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide, and was partially purified by gel filtration. It showed an apparent molecular weight of 700,000 with optimal pH of 9.0-9.5. The activity was inhibited by various serine protease inhibitors but not by thiol or metal protease inhibitors. It was relatively heat-stable. These findings suggest that the protease is similar to a trypsin-like protease from DHR to bovine gamma-globulin. Intradermal injection of the enzyme into normal guinea pigs induced a mixed neutrophil-mononuclear infiltrate. The reaction was reduced in intensity with the diisopropylfluorophosphate-inhibited enzyme preparation. The enzyme did not induce any increased vascular permeability. These findings suggest that the protease may be present generally in the DHR sites and probably participates in the cellular response of DHR.
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PMID:Inflammatory activity of a trypsin-like protease in the tuberculin reaction sites in guinea pig skin. 242 50

Incubation of purified human plasma prekallikrein with sulfatides or dextran sulfate resulted in spontaneous activation of prekallikrein as judged by the appearance of amidolytic activity toward the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide. The time course of generation of amidolytic activity was sigmoidal with an apparent lag phase that was followed by a relatively rapid activation until finally a plateau was reached. Soybean trypsin inhibitor completely blocked prekallikrein activation whereas corn, lima bean, and ovomucoid trypsin inhibitors did not. The Ki of the reversible inhibitor benzamidine for autoactivation (240 microM) was identical to the Ki of benzamidine for kallikrein. Thus, spontaneous prekallikrein activation and kallikrein showed the same specificity for a number of serine protease inhibitors. This indicates that prekallikrein is activated by its own enzymatically active form, kallikrein. Immunoblotting analysis of the time course of activation showed that, concomitant with the appearance of amidolytic activity, prekallikrein was cleaved. However, prekallikrein was not quantitatively converted into two-chain kallikrein since other polypeptide products were visible on the gels. This accounts for the observation that in amidolytic assays not all prekallikrein present in the reaction mixture was measured as active kallikrein. Kinetic analysis showed that prekallikrein activation can be described by a second-order reaction mechanism in which prekallikrein is activated by kallikrein. The apparent second-order rate constant was 2.7 X 10(4) M-1 s-1 (pH 7.2, 50 microM sulfatides, ionic strength I = 0.06, at 37 degrees C). Autocatalytic prekallikrein activation was strongly dependent on the ionic strength, since there was a considerable decrease in the second-order rate constant of the reaction at high salt concentrations. In support of the autoactivation mechanism it was found that increasing the amount of kallikrein initially present in the reaction mixture resulted in a significant reduction of the lag period and a rapid completion of the reaction while the second-order rate constant was not influenced. Our data support a prekallikrein autoactivation mechanism in which surface-bound kallikrein activates surface-bound prekallikrein.
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PMID:Autoactivation of human plasma prekallikrein. 244 Aug 89

Recent findings of the protease inhibitor domain in amyloid precursor protein of Alzheimer's disease (APPI) raised a novel hypothesis on the mechanism of amyloid deposition in the brain. APPI has significant amino acid sequence homology with Kunitz-type basic trypsin inhibitor super-family proteins, and the gene expression product showed real inhibitory activity. Since the three-dimensional model of APPI would help in understanding biological phenomena in molecular detail, we constructed an atomic model of APPI based on the structure of bovine pancreatic trypsin inhibitor (BPTI). The substitution of BPTI side chains by best-fitting corresponding amino acid structures was followed by the removal of van der Waals overlappings by molecular mechanics energy minimization with the AMBER force field, to give the feasible model of APPI. We also built serine protease models based on the structure of trypsin and investigated the target enzyme specificity of the inhibitory activity by the active-site mapping method. The models can explain the relative enzyme spectra of APPI and BPTI.
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PMID:Structure prediction of protease inhibitor region in amyloid precursor protein of Alzheimer's disease. 248 10

The complete amino acid sequence of Achromobacter lyticus protease I (EC 3.4.21.50), which specifically hydrolyzes lysyl peptide bonds, has been established. This has been achieved by sequence analysis of the reduced and S-carboxymethylated protease and of peptides obtained by enzymatic digestion with Achromobacter protease I itself and Staphylococcus aureus V8 protease and by chemical cleavage with cyanogen bromide. The protease consists of 268 residues with three disulfide bonds, which have been assigned to Cys6-Cys216, Cys12-Cys80, and Cys36-Cys58. Comparison of the amino acid sequence of Achromobacter protease and other serine proteases of bacterial and mammalian origins has revealed that Achromobacter protease I is a mammalian-type serine protease of which the catalytic triad comprises His57, Asp113, and Ser194. It has also been shown that the protease has 9- and 26-residue extensions of the peptide chain at the N and C termini, respectively, and overall sequence homology is as low as 20% with bovine trypsin. The presence of a disulfide bridge between the N-terminal extension Cys6 and Cys216 close to the putative active site in the C-terminal region is thought to be responsible for the generation of maximal proteolytic function in the pH range 8.5-10.7 and enhanced stability to denaturation.
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PMID:The primary structure and structural characteristics of Achromobacter lyticus protease I, a lysine-specific serine protease. 249 88

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.
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PMID:Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation. 250 77

Exposure of rat serosal mast cells (RSMC) to chymase, an endogenous secretory granule serine protease, at 37 degrees results in exocytosis, as determined by beta-hexosaminidase release. As the number of RSMC is increased with a set amount of chymase, the net percentage beta-hexosaminidase release decreases linearly, implying a finite set of cellular interactions per chymase unit. Pretreatment of RSMC with trypsin at 37 degrees renders them refractory to subsequent exocytosis mediated by chymase in a dose- and time-dependent fashion, with complete refractiveness occurring by 15 min at 37 degrees with 2.5 micrograms trypsin/ml. Anti-IgE-mediated coupled activation-secretion of RSMC is not affected by the same trypsin pretreatment. When RSMC are pretreated with trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree a progressive loss of sensitivity to activation by chymase at 37 degrees occurs. RSMC susceptibility to chymase-mediated degranulation after trypsin pretreatment can be partially regenerated by culturing the RSMC for about 24 hr in medium at 37 degrees. These findings suggest that a trypsin-sensitive constituent, possibly a receptor or substrate, is necessary for the functional interaction of chymase with RSMC. When added with diisopropyl fluorophosphate (DFP), chymase does not induce RSMC degranulation at 37 degrees. However, if the DFP is removed before addition of chymase at 37 degrees or is added after the chymase-priming event occurs at 1 degree, subsequent degranulation at 37 degrees is not inhibited. Thus, the induction and not the secretion phase is DFP-inhibitable in chymase-induced activation-secretion. In addition, the priming but not the exocytosis phase of chymase-initiated RSMC activation-secretion, which is not dependent on temperature and calcium ion concentration, involves a cellular trypsin-sensitive protein.
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PMID:Modulation of chymase-mediated rat serosal mast cell degranulation by trypsin or diisopropyl fluorophosphate. 252 9

Protein S is unique among the vitamin K-dependent proteins found in blood plasma because it is a cofactor rather than a zymogen of a serine protease. Instead of a trypsin-like domain, protein S contains a domain that has sequence homology with steroid binding proteins. In order to understand the function of this structural domain, peptides have been synthesized with amino acid sequences that are homologous between human protein S and rat androgen binding protein. Two peptides, corresponding to amino acids 400-407 (PINPRLDG) and 605-614 (GVQLDLDEAI) of the protein S sequence have been tested for their effects on protein S function. Neither peptide altered the clotting of bovine or human plasma. The peptide GVQLDLDEAI enhanced the anticoagulant activity of human-activated protein C in human plasma while the peptide PINPRLDG had no effect. The peptide GVQLDLDEAI was observed to inhibit the binding of protein S to C4b-binding protein in plasma, resulting in increased concentrations of free protein S. GVQLDLDEAI was also observed to enhance the disassociation of the protein S.C4b-binding protein complex when purified complex was used. Finally, C4b-binding protein was observed to bind to GVQLDLDEAI. These results suggest that the carboxyl-terminal region of protein S, which contains the sequence GVQLDLDEAI, is involved in the interaction between protein S and C4b-binding protein.
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PMID:Characterization of a synthetic peptide that inhibits the interaction between protein S and C4b-binding protein. 253 Feb 13


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