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)

Both the structural and chaperone-like properties of lens alpha-crystallins have been implicated in maintaining lens transparency. Modifications of lens alpha-crystallins may lead to formation of cataract by affecting the close-packing of the crystallins or by reducing the chaperone-like activity of the alpha-crystallins. A previously unreported modified alphaB-crystallin, whose molecular weight is 72 u greater than unmodified alphaB-crystallin, has been isolated from human lenses by size exclusion chromatography, reversed phase HPLC and ion exchange HPLC. Approximately one nanomole of this modified alphaB-crystallin was obtained from each of five human eye lenses. Molecular weight determinations of peptides produced by digestion with trypsin or endoproteinase Asp-N showed that the modification is in the C-terminal region of alphaB-crystallin. The fragmentation pattern of peptides from the C-terminal region, analysed by tandem mass spectrometry, located the modification of the epsilon-amino group of the C-terminal lysine. The elemental composition of this modification, determined from its exact mass, is C3H4O2. Because this modification decreases the net charge of alphaB-crystallin by one unit, and because the C-terminus has been implicated in the chaperone activity attributed to alphaB-crystallin, this modification at Lys 175 may have a significant role in cataractogenesis.
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PMID:In vivo modification of the C-terminal lysine of human lens alphaB-crystallin. 936 47

The complete amino acid sequence of [2Fe-2S] ferredoxin from Physalis alkekengi var. francheti has been determined by automated Edman degradation of the entire Cm-protein and of the peptides obtained by trypsin and endoproteinase Asp-N digestions. This ferredoxin exhibited ten, ten, and nine differences respectively in the amino acid sequence, when compared with the ferredoxins of Datura stramonium, D. metel, and D. arborea, but 21-28 differences for other angiosperms, and 34-37 differences for fern and horsetails. These results are in harmony with the taxonomic position for these plants.
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PMID:Amino acid sequence of ferredoxin from Physalis alkekengi var. francheti. 986 38

We have studied effects of the solvent composition on the activity and the conformation of human plasminogen activator inhibitor-1 (PAI-1) from HT-1080 fibrosarcoma cells. Non-ionic detergents, includine Triton X-100, reduced the inhibitory activity of PAI-1 more than 20-fold at 0 degrees C, but less than 2-fold at 37 degrees C, while glycerol partly prevented the detergent-induced activity-loss at 0 degrees C. The activity-loss was associated with an increase in PAI-1 substrate behaviour. Evaluating the PAI-1 conformation by proteolytic susceptibility of specific peptide bonds, we found that the V8-proteinase susceptibility of the Glu332-Ser333 (P17-P16) bond, part of the hinge between the reactive centre loop (RCL) and beta-strand 5A, and the endoproteinase Asp-N susceptibility of several bonds in the beta-strand 2A-alpha-helix E region were increased by detergents at both 0 and 37 degrees C. The susceptibility of the Gin321-Ala322 and the Lys325-Val326 bonds in beta-strand 5A to papain and trypsin, respectively, was increased by detergents at 0 degrees C, but not at 37 degrees C, showing a strict correlation between proteinase susceptibility of beta-strand 5A and activity-loss at 0 degrees C. Since the beta-strand 2A-alpha-helix E region also showed differential susceptibility to endoproteinase Asp-N in latent, active, and reactive centre-cleaved PAI-1, we propose that a detergent-induced conformational change of the beta-strand 2A-alpha-helix E region influences the movements of beta-sheet A, resulting in a cold-induced conformational change of beta-strand 5A and thereby an increased substrate behaviour at low temperatures. These results provide new information about the structural basis for serpin substrate behaviour.
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PMID:Solvent effects on activity and conformation of plasminogen activator inhibitor-1. 1010 70

Divercin V41 (DV41) is a class IIa bacteriocin produced by Carnobacterium divergens V41. This antilisterial peptide is homologous to pediocin PA-1 and contains two disulfide bonds. To establish the structure-activity relationships of this specific family of bacteriocin, chemical modifications and enzymatic hydrolysis were performed on DV41. Alteration of the net charge of this cationic bacteriocin by succinylation and acetylation revealed that, in a certain range, the electrostatic interactions were surprisingly not necessary for the activity of DV41. Cleavage of DV41 by endoproteinase Asp-N released two fragments N1[1-17] and N2[18-43] corresponding to the conserved hydrophilic N-terminal and the variable hydrophobic C-terminal sequences, respectively. Inhibitory assays showed that only the C-terminal fragment was active, and after trypsin cleavage at Lys42 or disulfide reduction it lost its inhibitory activity. These results suggested that both hydrophobicity and folding imposed by the Cys25-Cys43 disulfide bond were essential for antilisterial activity of the C-terminal hydrophobic peptide. Chemical oxidation of tryptophan residues by N-bromosuccinimide demonstrated that these residues were crucial for inhibitory activity since modification of any one of them rendered DV41 inactive. On the contrary, only the modification of all the three tyrosine residues caused a total loss of antilisterial activity. These latter results strengthened previous results suggesting that the N-terminal domain containing the YGNGV consensus sequence was not involved in the binding of DV41 to a potential specific receptor on listerial cells.
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PMID:Delineation of key amino acid side chains and peptide domains for antimicrobial properties of divercin V41, a pediocin-like bacteriocin secreted by Carnobacterium divergens V41. 1038 80

mAb CB2, directed against outer surface protein B (OspB), causes bacteriolysis of Borrelia burgdorferi in the absence of complement. How this happens is unknown. We examined the effect of mAb binding on OspB tertiary structure by using limited proteolysis to probe changes in protein conformation. Truncated OspB (tOspB) that lacked N-terminal lipid was cleaved by four enzymes: trypsin, endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C. CB2 affected the cleavage by trypsin and Arg-C, but not by AspN or Glu-C. None of the enzymes cleaved CB2 under these conditions. Both trypsin and Arg-C cleaved tOspB near the N-terminus; CB2 slowed the rate of cleavage, but did not affect the identity of the sites cleaved. Irrelevant mAb had no effect, indicating that the effect was specific. CB2 was active against tOspB of strain B31, but not against tOspB of strain BEP4, to which it does not bind, suggesting that binding was required to elicit the effect on cleavage. With trypsin, CB2 showed a maximal effect at 8 mol of tOspB to 1 mol of mAb. At this ratio, not enough CB2 was present to bind all the tOspB; therefore, either CB2 shows turnover or CB2 acts by binding tOspB and effecting a change in this tOspB such that it, in turn, propagates the effect in other molecules of tOspB. Regardless of the mechanism, these data show that CB2 elicits a change in tOspB that can be measured by its reduced susceptibility to protease cleavage.
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PMID:A bactericidal monoclonal antibody elicits a change in its antigen, OspB of Borrelia burgdorferi, that can be detected by limited proteolysis. 1064 Jul 58

The hydrophobicity of human recombinant interleukin 11 (rhIL-11) with an oxidized Met58 residue is nearly identical to the hydrophobicity of native rhIL-11. Consequently, separation of these species using standard gradient elution or isocratic elution is very difficult. Using an optimized, shallow gradient RP-HPLC method. Met58 oxidized rhIL-11 could be separated sufficiently from native rhIL-11. The identity of the oxidized form detected with this method was confirmed by peptide mapping with trypsin and endoproteinase Asp-N, N-terminal sequencing and mass spectrometric analysis. This method was employed to determine the effect of disposable laboratory plastic tubes for the oxidation. The amounts of Met58 oxidized rhIL-11 were increased when rhIL-11 samples were stored in plastic tubes at 37 degrees C in the dark. Samples stored in polypropylene tubes were oxidized much more than samples stored in polystyrene tubes. Additionally, the oxidation was greatly enhanced when samples were stored in polypropylene tubes exposed to light before rhIL-11 sample storage. The extent of the oxidation was also affected by the sources of polypropylene tubes. A maximum increase in Met58 oxidized rhIL-11 was more than 30% when samples were stored at 37 degrees C for 14 days in polypropylene tubes exposed to a daylight fluorescent lamp for 25 days. Consequently, these results indicate that attention should be paid for selection of suitable plastic tubes used for storage of protein samples, and for protection of the plastic tubes themselves from extended exposure to light while in storage.
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PMID:Reversed phase HPLC of Met58 oxidized rhIL-11: oxidation enhanced by plastic tubes. 1113 Feb 10

CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys6-Cys21, Cys13-Cys30, Cys20-Cys48, Cys32-Cys46). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (omega-AGA-IVA, omega-AGA-IVB, mu-AGA-I and mu-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail.
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PMID:CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure. 1169 32

The purification and unique carbohydrate binding properties, including blood group B-specific agglutination and preferential binding to Galalpha1,3Gal-containing sugar epitopes, of the Marasmius oreades agglutinin (MOA) are reported in an accompanying paper (Winter, H. C., Mostafapour, K., and Goldstein, I. J. (2002) J. Biol. Chem. 277, 14996-15001). Here we describe the cloning, characterization, and expression of MOA. MOA was digested with trypsin and endoproteinase Asp-N, and the peptide fragments were purified by high performance liquid chromatography. Amino acid sequence data were obtained for eight peptides. Using oligonucleotides deduced from the peptide sequences for a reverse transcriptase-PCR, a 41-base pair cDNA was obtained. The 41-base pair fragment allowed the generation a full-length cDNA using 5' and 3' rapid amplification of cDNA ends. MOA cDNA encodes a protein of 293 amino acids that contains a ricin domain. These carbohydrate binding domains were first described in subunits of bacterial toxins and are also commonly found in polysaccharide-degrading enzymes. Whereas these proteins are known to display a variety of sugar binding specificities, none to date are known to share MOA's high affinity for Galalpha1,3Gal and Galalpha1,3Galbeta1,4GlcNAc. Recombinantly expressed and purified MOA retains the specificity and affinity observed with the native protein. This study provides the basis for analyzing the underlying cause for the unusual binding specificity of MOA.
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PMID:Cloning, expression, and characterization of the Galalpha 1,3Gal high affinity lectin from the mushroom Marasmius oreades. 1183 54

Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with extracellular deposition of wild-type TTR, its variants or fragments as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and identification of TTR variants. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), in combination with trypsin digestion, have been shown to be powerful tools in characterizing TTR variants. Typically, TTR or its tryptic digest is analyzed by MALDI-TOF MS, liquid chromatography ESI MS, or both. Analysis of tryptic digests by MALDI-TOF MS does not provide enough sequence coverage in TTR to identify all possible modifications. To improve sequence coverage, aliquots of immunoprecipitated TTR samples were digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. Identification of the peptides from each digest by MALDI-TOF MS provided preliminary information about the sites and mass shifts due to amino acid substitutions from genetic mutations and to posttranslational modifications. The location and identity of the modifications in the variant proteins were then confirmed by tandem mass spectrometry, accurate mass measurements, and direct DNA sequence analysis. Using these methodologies, we achieved 100% sequence coverage. The detection of two nonpathologic variants (Thr119Met and Gly6Ser) and four pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser, and previously unreported Trp41Leu) are described as illustrations of this approach.
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PMID:Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis. 1186 53

The amino acid sequence of a thrombin like enzyme , named elegaxobin II, isolated from the venom of Trimeresurus elegans (Sakishima-habu) was determined by Edman sequencing of the peptides which was derived from digests with cyanogen bromide, achromobacter protease I, trypsin, endoproteinase Asp-N, and chymotrypsin. Elegaxobin II consisted of 233 amino acids and showed conservation of the catalytic amino acid residues (His(57), Asp(102), and Ser(195)) of chymotrypsin family serine protease in its amino acid sequence. The carboxyterminal amino acid, Leu, was determined using carboxypeptidase Y. This enzyme contains glucosamine and an N-linked glycosylation site. Elegaxobin II was 91% homologous in sequence to elegaxobin and protease I from the same snake venom, and it was 67, 75, 31 and 26% homologous in sequences to flavoxobin, KN-BJ 2, human kallikrein and bovine thrombin, respectively. Elegaxobin II lacked thrombin's ETW (146-148) loop, as well as its functionally important YPPW (60-insertion loop).
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PMID:Amino acid sequence of a thrombin like enzyme, elegaxobin II, from the venom of Trimeresurus elegans (Sakishima-Habu). 1550 Aug 47


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