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)

This study evaluates the nature of glycated human insulin formed following exposure to hyperglycemic conditions in vitro. Glycated insulin was purified by RP-HPLC and its molecular mass (5971.3 Da) determined by plasma desorption mass spectrometry (MS). The difference in mass (163.7 Da) from nonglycated insulin (5807.6 Da) corresponds to a single reduced glucose (glucitol) residue. Following reduction of insulin disulfide bridges, MS confirmed that the B-chain was glycated. Enzymatic digestions with trypsin, endoproteinase Glu-C, and thermolysin, followed by RP-HPLC and identification of fragments by MS, localized glycation to the B-chain (1-5) region. Electrospray tandem MS identified the site of glycation as the B-chain NH2-terminal Phe1 residue. This was confirmed by automated Edman degradation with glycated human insulin.
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PMID:Identification of the site of glycation of human insulin. 897 27

The calcium-binding epidermal growth factor (cbEGF)-like domain is a structural motif that is present in many matrix proteins throughout the animal kingdom from invertebrates to mammals. This module has been demonstrated to bind calcium in the micromolar range. However, little is known about the functional consequences of calcium binding to proteins that contain this structural element. We used fibrillin-1, an extracellular matrix protein consisting of approximately 60% cbEGF-like motifs, as a model system to study stabilizing effects of calcium in protease degradation assays. Authentic human fibrillin-1 and recombinant human fibrillin-1 subdomains, spanning the whole molecule, showed significantly slower proteolytic degradation in the presence of CaCl2 than in the presence of EDTA, demonstrating that calcium stabilizes the structure of fibrillin-1 and protects the molecule against proteolytic degradation. Information about cleavage sites protected by calcium was obtained with a new recombinant subdomain, rF17 (Asp 952-Val 1527), comprising the longest stretch of cbEGF-like motifs in the center of the fibrillin-1 molecule. The most sensitive sites for trypsin and endoproteinase Glu-C were observed in cbEGF-like motifs 11 (Met 1034 and Asn 1046), 12 (Ser 1103), and 17 (Thr 1318). Since most of the currently known mutations in fibrillin-1 are found within cbEGF-like motifs and are predicted to disrupt calcium binding, we suggest that these mutations render fibrillin-1 more susceptible to proteolytic cleavage, and this might be one of the reasons why these mutations result in Marfan's syndrome.
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PMID:Calcium stabilizes fibrillin-1 against proteolytic degradation. 899 26

Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 degrees C, but not at 12 degrees C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.
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PMID:Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase. 900 4

In 2D-PAGE analysis of Bet v 1, the major birch pollen allergen, up to 12 isoforms can be demonstrated that differ in their isoelectric points from about pH 4.9 to pH 5.9. The molecular weights of these isoforms seem to be rather similar, but minor variations can also be seen. Preliminary experiments with birch leaves seem to indicate that in aging leaves some isoforms can be found that do not occur in pollen. In birch cells cultured in vitro, Bet v 1 isoforms can be induced by bacterial infection that do not occur in pollen (Swoboda et al. (1995), Pant, Cell and Environment 18, 865-874). In a recent paper (Swoboda et al (1995)., J. Biol. Chem. 270, 2607-2613) we show that in natural Bet v 1 from pollen the isoforms are due to different protein sequences. The derived protein sequences of 10 different isoforms (corresponding to 13 different cDNAs) were determined and confirmed by plasma desorption mass spectrometry of purified natural Bet v 1 after trypsin and endoproteinase Glu-C digestion. These experiments also showed that pollen Bet v 1 isoforms were reactive to patients' sera to different degrees and that common post-synthetic modifications (besides N-terminal methionine cleavage) did not occur on Bet v 1. Recombinant isoforms were produced in E. coli, purified and tested with selected patients allergic to birch pollen (Ferreira et al., J. Exp. Med., in the press). The pattern of IgE binding to Bet v 1 isoforms widely differs. Also, T-cell clones from individual patients in some cases are specific to peptides occurring only in certain isoforms. It was of particular interest that three of the naturally occurring pollen Bet v 1 isoforms do not or hardly bind IgE of untreated patients allergic to Bet v 1. However, a comparison of IgE reactivity in patients before and after conventional immunotherapy with natural pollen extract clearly showed that this form of immunotherapy induced IgE to the isoforms that had been unreactive in untreated patients. One of these, Bet v 1d, showed a particularly strong potency towards T-cell stimulation. The isoform(s) that do not bind IgE in untreated patients but still show T-cell reactivity could be potentially utilized for a new form of immunotherapy that avoids the risk of anaphylaxis.
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PMID:Biological and immunological importance of Bet v 1 isoforms. 909 31

We describe the use of an HPLC/MS technique for the characterization of nicked fragments of hCG beta-subunit. After reductive alkylation of the nicked hCG beta-subunit with vinylpyridine, endoproteinase Glu-C or trypsin was used to digest the protein to produce peptides that could be analyzed by HPLC/electrospray ionization MS. Human leukocyte elastase digestion was used to produce an experimentally nicked hCG. Two nicking sites were observed, between amino acids 42Thr and 43Arg and between 44Val and 45Leu. The former site has not been previously reported for elastase digestion. The structures of the fragments were confirmed by HPLC/MS after removal of the oligosaccharide by direct mass measurement and by mass determination of their proteolytic digests. Without the glycopeptidase treatment, the microheterogeneity of the two N-linked oligosaccharides could be deduced from the spectra of the proteolytic fragments. Nicking with elastase was found to alter the oligosaccharide structures. Nicked beta-subunit samples isolated from the urine of choriocarcinoma patients were also analyzed and the location of the nicking site(s) agreed with that determined by classical techniques. Important differences in the oligosaccharide structures were also observed in these samples, including the presence of triantennary oligosaccharides not found in hCG from healthy subjects. These findings demonstrate the potential of HPLC/MS for characterization of glycoprotein standard preparations.
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PMID:Mass spectrometric characterization of nicked fragments of the beta-subunit of human chorionic gonadotropin. 921 53

Fragments of characteristic size retaining the ability of sequence-specific DNA binding were generated by partial proteolysis of transcription factor Stat3 with trypsin, chymotrypsin, or Staphylococcus V8 proteinase. The molecular masses of the smallest DNA-binding fragments were 75, 48, and 32 kDa after digestion with V8 proteinase, chymotrypsin, and trypsin, respectively. The fragments contained major parts of the domain controlling the sequence specificity of DNA binding (amino acids 406-514), the SH3 and SH2 domains, and the phosphorylated tyrosine residue Tyr-705, but not the C-terminal 20 amino acids. The N terminus of the 32-kDa tryptic fragment (ANCDASLIV) matched the sequence of amino acids 424-432 deduced from cDNA. The fragments were observed after proteolytic treatment of preformed complexes between DNA and native factors eluted from rat liver nuclei or recombinant, tyrosine-phosphorylated rat Stat3 from insect cells. It was possible to elute all three minimal fragments from their complexes with DNA and to obtain specific re-binding. The minimal fragments eluted from complexes with DNA still contained the phosphorylated Tyr-705 and the SH2 domain suggesting that they were probably bound to DNA as dimers. The DNA-binding domain of Stat3 identified by these experiments overlapped the domain previously identified by genetic experiments as the domain controlling the sequence specificity of DNA binding. The DNA-binding domain defined here by partial proteolysis probably represents an autonomously folding portion of Stat3.
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PMID:A 32-kDa proteolytic fragment of transcription factor Stat3 is capable of specific DNA binding. 926 55

Photoaffinity cross-linking with [azidobenzoyl-d-Lys6]GnRH leads to irreversible activation of the gonadotropin-releasing hormone (GnRH) receptor. In order to localize the cross-linking site, the disulfide bridge structure was initially probed by mutagenesis. A consistent pattern of changes in the ability of GnRH to stimulate signal transduction after Ser substitutions of extracellularly located Cys residues indicated that Cys14 in the N-terminal domain is connected to Cys200 in the second extracellular loop, while Cys196 in this loop is connected to the highly conserved Cys114 at the extracellular end of transmembrane helix 3. Protein chemical analysis of radioactive fragments of cross-linked GnRH receptor following deglycosylation and enzymatic digest with endoproteinase Glu-C and trypsin before and after introduction or elimination of potential protease cleavage sites indicated that 125I[azidobenzoyl-d-Lys6]GnRH cross-links to a segment comprising residues 12-18 of the N-terminal domain. The existence of the Cys114-Cys196 bridge was directly confirmed as a labeled fragment, including that Cys114 was resolvable only under reducing conditions. The observation that the cross-linked N-terminal enzymatic fragments had identical apparent size under non-reducing conditions shows that the cross-linking reaction disconnected the disulfide bridge between Cys14 and Cys200 and indicates that Cys14 is probably the residue involved in cross-linking of the ligand. It is concluded that covalent tethering of GnRH through a photoreactive side chain located at position 6 in the middle of the peptide leads to continued activation of the receptor presumably through covalent binding to Cys14 in the N-terminal domain of the receptor.
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PMID:Irreversible activation of the gonadotropin-releasing hormone receptor by photoaffinity cross-linking: localization of attachment site to Cys residue in N-terminal segment. 933 46

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.
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PMID:Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8 939 10

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.
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PMID:Expression, purification, and inhibitory properties of human proteinase inhibitor. 940 54

A synthetic peptide, VLSPADKTNWGHEYRMF(cmC)QIG, was reacted with 4-chlorobenzenediazonium hexafluorophosphate as a model for reactions of aromatic diazonium ions with proteins. At a ratio of diazonium ion to peptide of 0.8:1, three products could be seen by reversed-phase HPLC. Electrospray mass spectrometric analysis of the isolated products revealed that two of the products had the same mass of 2648 Da, being 138 Da higher than the parent peptide and corresponding to the addition of a 4-chlorobenzenediazo group. The third isolated product had a mass of 2787 Da which corresponded to the addition of two 4-chlorobenzenediazo groups (276 Da). Digestion of the monoadducted intact peptides with trypsin or endoproteinase Glu-C and HPLC separation of adduct oligopeptides followed by sequencing with electrospray ionization tandem mass spectrometry showed unambiguously that histidine and tyrosine residues were the major sites of modification. Incubation of human serum albumin with 4-chlorobenzenediazonium hexafluorophosphate at molar ratios of 1:1, 1:2, and 1:10 resulted in adduct formation as detected by shifts in the HPLC retention time of the protein and also by an increase in mass as determined by electrospray mass spectrometry.
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PMID:Characterization of azo coupling adducts of benzenediazonium ions with aromatic amino acids in peptides and proteins. 943 29


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