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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein A (PA) of Staphylococcus aureus has an array of biological functions, such as antitumor, antitoxic, anticarcinogenic, immunomodulatory, antifungal, and antiparasitic properties. We have already established that a theoretical trypsin-digested peptide fragment of protein A (20-mer) mimics immunomodulatory and IgG binding property of PA. In the present report we have concentrated on a 16-mer chymotryptic fragment of protein A, which has a sequence of 13 amino acids in common with the previously studied 20-mer peptide. Molecular modeling study qualitatively predicted that both 20-mer and 16-mer peptides retain Fc binding ability from an interaction energy point of view. In the present study our aim was to understand whether this theoretically predicted 16-mer chymotryptic fragment could be formed in a real experiment and also to understand its biological activities. Chymotrypsin cleavage of PA at 37 degrees C for 24 h produced four major fragments on reverse-phase HPLC. The amino acid analyses of each fragment show the absence of cysteine residue from all fragments, which justifies the absence of cysteine in PA. We also observed high content of aspartic acid and glutamic acid residues in all fragments. On gel-filtration chromatography the chymotrypsin cleavage of PA shows five peaks, one of which overlaps with our theoretically selected 16-mer peptide on superimposition. We verified the IgG binding capacity of 16-mer peptide by capillary electrophoresis. The 16-mer peptide also induces the production of TNFalpha and IL-1alpha in serum of mice. The above observations suggest that the 16-mer peptide may be produced by chymotrypsin cleavage and also that this peptide possesses some of the major biological properties of PA, such as IgG binding, TNFalpha and IL-1alpha elicitation, etc.
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PMID:Functional mimicry of protein A of Staphylococcus aureus by a proteolytically cleaved fragment. 1038 52

Trichloroethylene (TCE) shows several types of toxicities, some of which may be the result of bioactivation. Oxidation by P450s yields the electrophile TCE oxide. We previously analyzed N(6)-acyllysine adducts formed from the reaction of TCE oxide with proteins [Cai, H., and Guengerich, F. P. (2000) Chem. Res. Toxicol. 13, 327-335]; however, we had been unable to measure ester adducts under the prolonged conditions of proteolysis and derivatization. Protein amino acid adducts were directly observed by mass spectrometry during the reaction of TCE oxide with the model polypeptides insulin and adrenocorticotropic hormone (ACTH, residues 1-24). The majority (80%) of the protein adducts were unstable under physiological conditions and had a collective t(1/2) of approximately 1 h, suggesting that they are ester type adducts formed from reactions of Cys, Ser, Tyr, or Thr residues with intermediates formed in TCE oxide hydrolysis. Synthetic O-acetyl-L-Ser and O-acetyl-L-Tyr had half-lives of 1 h and 10 min at pH 8.0, respectively, similar to the stabilities of the protein adducts. The effects of TCE oxide adduct formation on catalytic activities were examined with five model enzymes. No recovery of catalytic activity was observed during the reaction of TCE oxide with two model enzymes for which the literature suggests roles of a Lys, rabbit muscle aldolase and glucose-6-phosphate dehydrogenase. However, in the cases of papain (essential Cys residue in the active site), alpha-chymotrypsin (critical Ser residue), and D-amino acid oxidase (essential Cys and Tyr residues), time-dependent recoveries of enzyme activity were observed following reaction with TCE oxide or either of two model nucleophiles (dichloroacetyl chloride and acetic formic anhydride), paralleling the kinetics of removal of adducts from insulin and ACTH. Formation of adducts ( approximately 2%) was detected in the direct reaction of TCE oxide with 2'-deoxyguanosine, but not with the other three nucleosides found in DNA. During the reaction of TCE oxide with a synthetic 8-mer oligonucleotide, formation of adducts was observed by mass spectrometry. However, the adducts had a t(1/2) of 30 min at pH 8.5. These results indicate the transient nature of the adducts formed from the reaction of TCE oxide with macromolecules and their biological effects.
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PMID:Reaction of trichloroethylene oxide with proteins and dna: instability of adducts and modulation of functions. 1117 May 8

Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition. Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50-130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.
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PMID:Plasmodium vivax Duffy binding protein peptides specifically bind to reticulocytes. 1181 13

A 35-mer polypeptide isolated from the hemolymph of desert locust Schistocerca gregaria (SG) proved to be a canonical inhibitor of bovine trypsin (K(i) = 0.2 microM). Despite having a trypsin-specific arginine at the primary specificity P(1) site, it inhibits bovine chymotrypsin almost as well (K(i) = 2 microM). Furthermore, while the latter reactivity improves 10(4)-fold by the single replacement of P(1) Arg by Leu, changing P(1)' from Lys to Met only moderately improves trypsin affinity (K(i) = 30 nM). The apparent low compatibility to trypsin, however, is not observed vs two arthropodal trypsins: SG peptides with P(1) Arg inhibit crayfish and shrimp trypsins with K(i) values in the picomolar range. This unprecedented high discrimination between orthologous enzymes is postulated to derive from flexibility differences in the protein-protein interaction. The more than four orders of magnitude phylum selectivity makes these peptides prospective candidates for agricultural use.
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PMID:Remarkable phylum selectivity of a Schistocerca gregaria trypsin inhibitor: the possible role of enzyme-inhibitor flexibility. 1183 48

The erythrocyte-binding antigen 140 (EBA140) sequence was chemically synthesized in 61 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte-binding assays. Peptides 26135, 26144, 26147, 26160, 26170 and 26177 presented high erythrocyte-binding activity, with affinity constants ranging from 350 to 750 nM. Critical erythrocyte-binding residues were determined by competition-binding assays with glycine analogous peptides. Cross-linking assays with SDS-PAGE from high erythrocyte membrane protein binding peptides showed that all these peptides bound specifically to 25, 52 and 75 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide-binding assays using enzyme-treated erythrocytes, showing that these protein receptors are susceptible to structural changes provoked by enzyme treatment (neuraminidase, trypsin or chymotrypsin). Inhibition invasion assays in 'in vitro' cultures showed that all specific high binding sequences were able to inhibit invasion by 11-69% at 200 microM concentration.
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PMID:Plasmodium falciparum EBA-140 kDa protein peptides that bind to human red blood cells. 1296 97

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K(i) values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.
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PMID:Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato. 1608 43

Transferrin exhibits heterogeneity in glycosylation characteristic of pathological changes in alcohol abuse and congenital disorders in glycosylation. This study investigated an alternative approach in the detection of carbohydrate-deficient transferrin based on the premise that glycosylation may afford some degree of protection to proteolytic action. Differential susceptibility to proteolysis by chymotrypsin was demonstrated for normal glycosylated and nonglycosylated recombinant human transferrin, using reverse-phase (RP) HPLC, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and LC-tandem mass spectrometry (MS/MS). Peptide fragmentation profiles were consistent with a predominantly high-specificity cleavage pattern of chymotrypsin. The observed peptide fragmentation profile showed that the C-lobe of recombinant full-length nonglycosylated transferrin (rhTf-NG) appeared to be preferentially cleaved, while cleavage of the N-lobe was restricted to the N-terminal and link sequence regions. Although chymotryptic cleavage sites abound in the N-lobe, their resistance to cleavage was independent of glycosylation. Compared to previous studies of lactoferrin, our data suggest disparity in the role by which glycosylation exerts a protective effect in the siderophilin family. It was clear from the transferrin digestions analyzed by HPLC that N-linked glycosylation did confer protection from proteolysis by chymotrypsin. After fragmentation, a range of peptides representing previously cryptic epitopes were identified as potential candidates for an immunological approach to differentiate between the different transferrin glycoforms. Based on its proximity to the Asn413 glycosylation site, a 15-mer peptide, m/z 1690.472 (NKSDNCEDTPEAGYF), was identified as a suitable candidate for raising anti-peptide antibodies for subsequent immunological detection. This novel approach could form the basis for an alternative assay or reference method for the detection of carbohydrate-deficient transferrin.
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PMID:Differential susceptibility of transferrin glycoforms to chymotrypsin: a proteomics approach to the detection of carbohydrate-deficient transferrin. 1633 60

Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity. This binding occurred in a length- and concentration-dependent manner. DF can mobilize proangiogenic factors such as bFGF from their depot or storage sites on bovine corneal endothelial matrix. However, these molecules do not interfere with high-affinity binding of bFGF to FGFR1 IIIc but can replace heparin as a required cofactor for binding and hence cellular mitogenesis. DF also protects bFGF against digestion by trypsin and chymotrypsin and from air oxidation. In addition, DF binds to collagen I with low nanomolar affinity and can promote human microvascular endothelial cell-1 (HMEC-1) cell mitogenesis and tubular morphogenesis in three-dimensional collagen I gels. Thus, our data suggest that DF may provide a stimulus to the sinusoidal endothelium of a liver that has suffered a severe angiotoxic event, thus helping to ameliorate the clinical sVOD/MOF syndrome.
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PMID:Angiogenesis alteration by defibrotide: implications for its mechanism of action in severe hepatic veno-occlusive disease. 1917 63

The membrane-associated histidine-rich protein-1 (MAHRP-1) is a Maurer's cleft-resident molecule that has been recently described as an important protein for the trafficking of PfEMP-1 to infected erythrocyte membrane, a major virulence factor. We have studied the specific interactions between 20-mer-long synthetic peptides spanning the complete MAHRP-1 sequence and erythrocytes. A high-activity binding peptide (HABP) with saturable binding to a 46-kDa erythrocyte membrane protein was identified and its binding was affected by chymotrypsin treatment. Random coil and alpha-helical features were found in the HABP's structure. Our results suggest that MAHRP-1 specifically interacts with erythrocyte membrane through a 20-mer-long amino acid region, raising questions about this region's potential as a therapeutic target against malaria.
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PMID:A Maurer's cleft-associated Plasmodium falciparum membrane-associated histidine-rich protein peptide specifically interacts with the erythrocyte membrane. 1916 14

The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (alphabetagamma). In humans an additional delta-subunit (delta-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of alphabetagamma- and deltabetagamma-hENaC and investigated whether deltabetagamma-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (DeltaI(ami)) was about 11-fold larger in oocytes expressing deltabetagamma-hENaC than in oocytes expressing alphabetagamma-hENaC. The 2-fold larger single-channel Na(+) conductance of deltabetagamma-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of deltabetagamma-hENaC must be higher than that of alphabetagamma-hENaC. Experiments exploiting the properties of the known betaS520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on deltabetagamma-hENaC whole-cell currents compared with its effect on alphabetagamma-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for deltabetagamma-hENaC than for alphabetagamma-hENaC. Proteolytic activation of deltabetagamma-hENaC was associated with the appearance of a delta-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human alpha-ENaC inhibited DeltaI(ami) in oocytes expressing alphabetagamma-hENaC but not in those expressing deltabetagamma-hENaC. We conclude that the delta-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.
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PMID:The delta-subunit of the epithelial sodium channel (ENaC) enhances channel activity and alters proteolytic ENaC activation. 1971 56


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