Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.36 (caspase-1)
 6,285 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Moojeni protease A was purified from the venom of Bothrops moojeni by chromatography on Sephadex G-100, DEAE Sephadex A-50 and rechromatography on Sephadex G-100. The enzyme shows one protein band in polyacrylamide gel electrophoresis at pH 8.5 or at pH 4.3. The pI of moojeni protease A was approximately 7.7. In immunoelectrophoresis it migrates to the cathode. The enzyme was homogeneous by polyacrylamide gel electrophoresis, immunoelectrophoresis and analyses in the ultracentrifuge. The S20,w and D20,w are 2.68 S and 10.34 X 10(-7) cm2/sec, respectively. The molecular weight calculated by s/D ratio was 22,500 and a value of 22,800 was obtained by sedimentation equilibrium. In SDS-polyacrylamide gel electrophoresis the enzyme exhibits a single polypeptide chain of approximately 20,400 mol. wt under denaturating conditions. In water or low salt solution it undergoes denaturation and autolysis. The enzyme is also unstable at acidic pH and to heat treatment and precipitates in the presence of metal chelating compounds such as EDTA or 1,10 phenanthroline. Leucine, the NH2-terminal amino acid of moojeni protease A is blocked after EDTA treatment. The proteolytic activity of this enzyme increases about 20% in the presence of Ca2+; Mg2+ has no effect and other divalent cations cause inhibition. The removal of Ca2+ ions by oxalate causes about 20% inhibition; the activity was restored by addition of Ca2+.
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PMID:Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojeni (caissaca). 393 45

We report the preparation and characterization of interleukin-1beta converting enzyme (ICE) refolded from its p20 and p10 protein fragments. Refolded ICE heterodimer (p20p10) was catalytically active but unstable, and in size exclusion chromatography eluted at an apparent molecular mass of 30 kDa. The mechanisms of the observed instability were pH-dependent dissociation at low enzyme concentrations, and autolytic degradation of the p10 subunit at high concentrations. Binding and subsequent removal of a high affinity peptidic inhibitor increased the apparent molecular mass to 43 kDa (by size exclusion chromatography), and significantly increased its stability and specific activity. Chemical cross-linking and SDS-polyacrylamide gel electrophoresis analysis of the 43-kDa size exclusion chromatography conformer revealed a 60-kDa species, which was absent in the 30-kDa conformer, suggesting that inhibitor binding caused formation of a (p20p10)2 homodimer. The observation of a reversible equilibrium between ICE (p20p10) and (p20p10)2 suggests that analogous associations, possibly between ICE and ICE homologs, can occur in vivo, resulting in novel oligomeric protease species.
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PMID:Stability and oligomeric equilibria of refolded interleukin-1beta converting enzyme. 870 86

Dipeptides containing fluorescein or biotin have been incorporated into proteolytic substrate cleavage products of bovine serum albumin generated by human cathepsin S or neutrophil elastase and into a fragment of the 31-kDa interleukin 1beta precursor by human interleukin 1beta-converting enzyme. Incorporation of the nucleophile is blocked by prior inhibition of the enzymes, and is not seen when proteolysis occurs in the absence of label, and the protease is then inhibited before the addition of label. Labeling is dependent on the pH, the time of reaction, and the concentrations of the nucleophile and substrate. Labeling of proteins can be readily detected by SDS-polyacrylamide gel electrophoresis. The pattern of elastase-labeled bovine serum albumin bands differs among P1' Phe, Ala, and Gly, suggesting that nucleophilic attack on acyl enzyme intermediates derived from a large protein may differ from attack on small intermediates. The only observed labeled fragment catalyzed by interleukin 1beta-converting enzyme is fragment 28-116 from the interleukin 1beta precursor, suggesting that the cleavage between residues 27 and 28 is at least as efficient as between residues 116 and 117. This labeling method does not require organic solvent or nonphysiological pH values and thus may be useful for the discovery of novel protease substrates in cells or other in vivo systems or for diagnostic applications.
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PMID:Nucleophile labeling of cysteine and serine protease substrates. 891 Apr 64

The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1, -3, -6, -7, -8, and -10 with kass values from 1.2 x 10(3) to 7 x 10(5) (M-1 s-1), and with upper limits of Ki values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.
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PMID:Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex. 969 66

The processing and release of 31-kDa proIL-1 beta to the mature 17-kDa form of IL-1 beta are still poorly understood. To help elucidate the mechanisms involved in IL-1 beta processing and release, we measured IL-1 beta forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1 beta. Our studies demonstrate that in addition to the 17-kDa mature IL-1 beta, IL-1 beta is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1 beta represented 20-40% of the total released IL-1 beta, as measured by SDS-PAGE with densitometry. This released proIL-1 beta was susceptible to ICE processing; however, this proIL-1 beta was not detectable by either a mature or proIL-1 beta-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1 beta was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1 beta could complex the type II IL-1R, released proIL-1 beta did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1 beta binding to another protein. These findings imply that IL-1 beta is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1 beta.
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PMID:Endotoxin-stimulated monocytes release multiple forms of IL-1 beta, including a proIL-1 beta form whose detection is affected by export. 1020 30

Monoclonal Abs 21 and 132 were raised against human functionally inactive rIL-18, and plasma IL-18 levels were determined by the sandwich ELISA established with these mABS: Plasma IL-18, designated type 2, was detected by this ELISA, and the levels found were not consistent with those obtained with the commercially available kit for determination of functionally active IL-18 (type 1). Type 1 was detected in all volunteers, whereas type 2 was detected in approximately 30% of healthy subjects, and the levels of type 2 in their blood plasma were high (25-100 ng/ml) compared with those of type 1 (0.05-0.3 ng/ml). We purified IL-18 type 2 from blood plasma of volunteers with high IL-18 type 2 concentrations, and its M(r) was determined to be 800 kDa by SDS-PAGE and molecular sieve HPLC. The purified 800-kDa protein, either caspase-1-treated or untreated, expressed no or marginal IL-18 function in terms of potentiation of NK-mediated cytolysis and IFN-gamma induction, and it barely bound IL-18R-positive cells. N-terminal amino acid analysis indicated that the purified protein was IgM containing a minimal amount of IL-18 proform and its fragment. Again, the purified IgM from IL-18 type2-positive volunteers exhibited cross-reaction with mAb 21 against IL-18. This band was not detected with 125-2H, an mAb against functionally active IL-18. Hence, human IgM carries functionally inactive IL-18 forming a disulfide-bridged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type 1 IL-18 in blood circulation in approximately 30% normal subjects.
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PMID:An alternative form of IL-18 in human blood plasma: complex formation with IgM defined by monoclonal antibodies. 1135 22

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.
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PMID:The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. 1174 53

A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS-PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40 degrees C to 50 degrees C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.
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PMID:Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus. 1200 50

Bothrops protease A (BPA) is a serine peptidase isolated from the venom of Bothrops jararaca. Unlike many venom enzymes, it is stable at pHs between 3 and 9 and resists heating at 86 degrees C for 10 min. Mature snake venom serine peptidases of the chymotrypsin family are in general glycoproteins composed of around 232 amino acids and their molecular masses vary between 25 and 40 kDa. BPA is a glycosylated protein that migrates on SDS-polyacrylamide gel electrophoresis (PAGE) as a single band of 67 kDa. In order to find out whether BPA has the typical serine peptidase primary structure or if it is composed of a longer amino acid sequence, we cloned a cDNA encoding BPA. Its deduced amino acid sequence showed that BPA is composed of 234 residues with a calculated molecular mass of 25,409 Da implying that approximately 62% of its molecular mass assessed by SDS-PAGE is due to carbohydrate moieties. Eight putative N-glycosylation and two putative O-glycosylation sites were found in BPA amino acid sequence. Deglycosylation experiments indicated that all 10 potential glycosylation sites in BPA are utilized. Complete N- and O-deglycosylation was only achieved under denaturing conditions and generated main products of 25 and 55 kDa, respectively, which were enzymatically inactive. N-deglycosylation under non-denaturing conditions was only partial and gave a main product of 50 kDa and fragments ranging from 25 to approximately 10 kDa. Kinetic parameters K(m) and V(max) of partially N-deglycosylated BPA upon substrate Bz-Arg-pNA were similar to the native form. However, when partially N-deglycosylated BPA was submitted to pH 3 and pH 10, it appeared to be unstable as it underwent hydrolysis, as shown by the presence of two main products of 30 and 12 kDa while the 50 kDa protein band disappeared. These changes also had effects on V(max) upon Bz-Arg-pNA which dropped to approximately 45%, while K(m) values remained unchanged. Fluorescence emission spectroscopy indicated that in partially N-deglycosylated BPA, tryptophan residues are more exposed to a polar environment than in the fully glycosylated protein. Taken together, these studies indicate that glycosylation has a stabilizing effect on BPA.
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PMID:The unusual high molecular mass of Bothrops protease A, a trypsin-like serine peptidase from the venom of Bothrops jararaca, is due to its high carbohydrate content. 1458 Sep 91

Active caspases are generally composed of two catalytic domains, each containing a large (p20) and a small (p10) subunit so that a fully active caspase has the organization (p20-p10)(2). The cowpox serpin crmA suppresses host apoptosis and inflammation by inhibiting endogenous caspases. We report on the mechanism crmA uses to inhibit caspases 1, 6, and 8. Native PAGE showed formation of tight crmA-caspase complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided evidence for a covalent crmA-p20 thioester linkage. SDS-PAGE of isolated complexes showed near complete loss of the p10 subunit from initiator caspases 1 and 8 but not from the executioner caspase-6. This was confirmed for caspase-1 by sequencing and Western blotting. Size exclusion chromatography indicated a size of approximately 60 kDa for complexes with caspases 1 and 8, consistent with a crmA.p20 species, suggesting that the p20-p10 interface and possibly the p10-p10 interface had been disrupted. In contrast, crmA.caspase-6 complex behaved as an equilibrium mixture of crmA(2).(p20-p10)(2) and crmA.(p20-p10). Complex deacylation rates were all slow, suggesting effective kinetic trapping of the covalent thioacyl intermediate. These results suggest a novel serpin inhibition mechanism through which crmA down-regulates apoptosis and inflammation. This involves (i) rapid formation of covalent complex with initiator caspases 8 or 1, (ii) very slow deacylation, and (iii) loss of the caspase p10 subunit for initiator but not for executioner caspases, so that any free p20 formed by deacylation of initiator caspases cannot reassociate to active heterotetramer, thus resulting in irreversible inhibition of apoptosis and inflammation.
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PMID:Cytokine response modifier a inhibition of initiator caspases results in covalent complex formation and dissociation of the caspase tetramer. 1705 83


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