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)

Bovine pancreatic trypsin inhibitor (BPTI) was secreted by Aspergillus niger at yields of up to 23 mg l-1 using a protein fusion strategy. BPTI was linked to part of the fungal glucoamylase protein (GAM) with a dibasic amino acid (KEX2) processing site at the fusion junction. Electrospray ionisation mass spectrometry and N-terminal protein sequencing revealed that, although biologically active in vitro, the purified products from a number of independent transformants consisted of a mixture of BPTI molecules differing at the N-terminus. Approximately 35-60% of this mixture was processed correctly. Aberrant processing of the GAM-BPTI fusion protein by the A. niger KEX2-like endoprotease was the most likely cause of this variation although the involvement of other fungal endoproteases could not be ruled out. In vitro studies have highlighted a weak interaction between BPTI and the Saccharomyces cerevisiae KEX2 endoprotease, suggesting that BPTI is not a potent inhibitor of KEX2p. A small proportion of the recombinant BPTI (10%) showed 'nicking' of the K15-A16 bond, indicating an interaction with a fungal trypsin-like enzyme. Mutant BPTI homologues designed to have anti-elastase activity, BPTI(K15V), BPTI(K15V,P13I) and BPTI(K15V,G12A), have also been expressed and secreted by A. niger. They also showed a similar spectrum of aberrant N-terminal processing but no 'nicking' of the K15-V16 bond was observed. Comparison of A. niger with other expression systems showed that it is an effective system for producing BPTI and its homologues, although not all molecules were correctly processed. This variation in processing efficiency may be useful in understanding the important determinants of protein processing in this fungus.
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PMID:Aberrant processing of wild-type and mutant bovine pancreatic trypsin inhibitor secreted by Aspergillus niger. 977 52

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.
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PMID:Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry. 980 26

Multi-cycle replication and plaque formation of influenza A and B viruses and cleavage activation of their hemagglutinin (HA) by an endogenous protease(s) were examined in two MDCK cell lines, MDCK(-) and MDCK(+). No exogenous trypsin was required for multi-cycle replication and plaque formation of all the influenza A viruses tested in the MDCK(+) cell, while those of the viruses in the MDCK(-) cell were completely trypsin-dependent. In both cell lines, on the other hand, influenza B viruses grew well in the absence of trypsin. The capability of multiple replication and plaque formation of the influenza viruses correlated with cleavage of the HA precursor (HA0) to HA1 and HA2, indicating that both cell lines express an HA activating endoprotease(s); that of the MDCK(+) cell activates the HA of influenza A and B viruses, and that of the MDCK(-) cell does only the HA of influenza B virus. Furthermore, the protease of the MDCK(+) cell was strongly suggested to be present on the cell surface and a serine protease. The MDCK(+) cell would be useful for isolation of influenza viruses from clinical specimens and for screening of protease inhibitors for anti-influenza virus drugs.
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PMID:Endogenous protease-dependent replication of human influenza viruses in two MDCK cell lines. 985 79

The 20 S proteasome is an endoprotease complex that preferentially cleaves peptides C-terminal of hydrophobic, basic, and acidic residues. Recently, we showed that these specific activities, classified as chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide-hydrolyzing (PGPH) activity, are differently affected by Ritonavir, an inhibitor of human immunodeficiency virus-1 protease. Ritonavir competitively inhibited the chymotrypsin-like activity, whereas the trypsin-like activity was enhanced. Here we demonstrate that the Ritonavir-mediated up-regulation of the trypsin-like activity is not affected by specific active site inhibitors of the chymo-trypsin-like and PGPH activity. Moreover, we show that the mutual regulation of chymotrypsin-like and PGPH activities by their substrates as described previously by a "cyclical bite-chew" model is not affected by selective inhibitors of the respective active sites. These data challenge the bite-chew model and suggest that effectors of proteasome activity can act by binding to non-catalytic sites. Accordingly, we propose a kinetic "two-site modifier" model that assumes that the substrate (or effector) may bind to an active site as well as to a second non-catalytic modifier site. This model appears to be valid as it describes the complex kinetic effects of Ritonavir very well. Since Ritonavir partially inhibits major histocompatibility complex class I restricted antigen presentation, the postulated modifier site may be required to coordinate the active centers of the proteasome for the production of class I peptide ligands.
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PMID:Evidence for the existence of a non-catalytic modifier site of peptide hydrolysis by the 20 S proteasome. 1080 6

An extracellular 1,3-specific lipase with molecular weight of 35.5 kDa and an isoelectric point of 4.4 from Aspergillus niger has been purified 50-fold by pH precipitation followed by a series of chromatographic steps with an overall yield of 10%. The enzyme was homogeneous as judged by denaturing polyacrylamide gel electrophoresis and size-exclusion fast-performance liquid chromatography. It contained 2.8% sugar which was completely removed by endoglycosidase F treatment, and the deglycosylated enzyme retained full activity. The native lipase showed optimal activity between temperatures 35 and 55 degrees C and pH 5.0 and 6.0. The amino acid composition and the N-terminal sequence were found to be different from lipases previously purified from A. niger. The enzyme was resistant to trypsin, chymotrypsin, endoprotease Glu-C, thrombin, and papain under native conditions but was susceptible to cleavage by the same proteases when heat-denatured.
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PMID:Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger. 1090 84

Accurate definition of the carboxyl terminal of proteins is necessary for elucidating posttranslational processing at the C-terminal and more generally for characterizing protein primary structures. Here, we describe a strategy for isolating and characterizing the C-terminal peptide of a protein after proteolysis with endoprotease Lys-C. Isolation is achieved using anhydrotrypsin, a catalytically inert derivative of trypsin that binds peptides containing lysine or arginine residues at their C-termini without cleaving them. Rapid, accurate characterization of the isolated C-terminal peptide is achieved by mass spectrometry. Initial identification of the C-terminal peptide is obtained by comparing matrix-assisted laser desorption/ionization time-of-flight mass spectra of the digest prior to and after incubation with anhydrotrypsin. Characterization of the C-terminal sequence is achieved by capillary-HPLC electrospray ionization tandem mass spectrometry of the isolated peptide using a quadrupole ion trap mass spectrometer in the selective reaction monitoring mode. This strategy was successfully applied to the characterization of the C-terminal of proteins with molecular masses ranging up to 56 kDa.
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PMID:A method to define the carboxyl terminal of proteins. 1093 15

The role of the C-terminal domain of rhodopsin in the activation of transducin was studied. The treatment of photoreceptor membranes with trypsin, thermolysin, and Asp-N-endoprotease led to the respective rhodopsin species devoid of 9, 12-, or 19-aa C-terminal fragments. It was shown that the removal of 9-aa fragment by trypsin does not affect the catalytic activity of the receptor, whereas the thermolysin-induced truncation of the rhodopsin C-terminus by 12 aa about 1.5-fold enhances its activity. The Asp-N-endoprotease-assisted removal of 19 aa (i.e., the shortening by seven more C-terminal aa) virtually unchanges the rhodopsin catalytic activity compared to the preparation truncated with thermolysin. These results suggest that the part of the rhodopsin C-terminal fragment between the sites of its cleavage by trypsin and thermolysin (Val337-Ser338-Lys339) inhibits the signal transduction from rhodopsin to the next component of visual cascade. The English version of the paper.
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PMID:[The effect of proteolytic removal of the C-terminal fragment of rhodopsin on its ability to activate visual cascade]. 1187 69

The acetylation isoforms of histone H4 from butyrate-treated HeLa cells were separated by C(4) reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a "zip" model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is di-methylated in all modified isoforms, as well as the non-acetylated isoform of H4.
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PMID:Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry. 1223 78

Recently, proteolytic 18O labeling has been demonstrated as a promising strategy for comparative proteomic studies (Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-42). In this approach, protein mixtures are digested in parallel in H216O and H218O and the ratios of isotopically distinct peptide products are measured by mass spectrometry. In the initial report from this laboratory, trypsin was shown to catalyze incorporation of two 18O atoms into the carboxyl terminus of each new peptide formed by cleavage of the adenovirus proteome. In the present study, a second enzyme, endoprotease Glu-C, is evaluated as an agent for cleavage and labeling. Proteolytic 18O labeling by Glu-C is shown to occur readily with phosphorylated and glycosylated proteins and with cysteinealkylated and disulfide-linked proteins. A sequential double-labeling strategy is used to characterize N-linked glycopeptides. Labeled and unlabeled peptide pairs are found to coelute chromatographically, and measurements of isotope ratios by nanospray and capillary LC-MS are found to be accurate and precise.
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PMID:Proteolytic 18O labeling for comparative proteomics: evaluation of endoprotease Glu-C as the catalytic agent. 1264 23

Digestive endoprotease activities of the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Larvae of this species were found to use a complex proteolytic system that includes cathepsin D-, cathepsin B-, trypsin-, and chymotrypsin-like activities. Trypsin-like activity was evenly distributed among the anterior, middle, and posterior portions of the gut, whereas cathepsin B- and cathepsin D-like activities were mainly located in the anterior and middle sections, and the chymotrypsin-like activity was highest in the middle and posterior sections. Gelatin-containing native-PAGE gels indicated the presence of several aspartyl, cysteine, and serine protease forms and confirmed the spatial organization of the proteolytic digestive process.
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PMID:Proteolytic gut activities in the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae). 1270 Nov 11


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