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)

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.
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PMID:Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. 753 May 43

A protease-inhibitor was isolated from mature ovaries of Schistocerca gregaria by a combination of trypsin-affinity chromatography and reverse-phase high performance liquid chromatography. It was characterized by aminoterminal amino acid sequencing using Edman degradation based automated microsequencing and by MALDI-TOF mass spectrometry. The N-terminal sequence (Y)XAEXDELA(A)EEY(Y)Q(Q)X(I)(L)M (X being a Cys, an irregular or modified amino acid) revealed no similarities with any other protease inhibitors isolated from invertebrate or vertebrate source. The 14 kDa inhibitor was found to be heat-stable. It shows potent inhibitory activity toward bovine trypsin and chymotrypsin, but not toward pancreatic elastase. It is likely that the characterized inhibitor will serve as an important tool for understanding its role in insect development.
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PMID:Purification of a novel, heat-stable serine protease inhibitor protein from ovaries of the desert locust, Schistocerca gregaria. 929 12

Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s (ST-PHPMA) with different functional end groups, namely carboxyl, methyl ester, hydrazide, and amino groups, were prepared by chain transfer free-radical polymerization. 2,2'-Azobisisobutyronitrile (AIBN) was used as an initiator and 3-mercaptopropionic acid, methyl 3-mercaptopropionate, 3-mercaptopropionic hydrazide, and 2-mercaptoethylamine were used as chain-transfer agents. The semitelechelic polymers have been characterized by end-group analysis, size-exclusion chromatography (SEC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The effects of the concentrations of the mercaptans and the initiator on the molecular weight of the polymers have been investigated. The higher the concentration of mercaptan, the lower the molecular weight of ST-PHPMA. The concentration of initiator did not have a significant effect on the molecular weight of the semitelechelic polymers. The end groups of the ST polymers can be readily transformed by polymeranalogous reactions. A model protein, alpha-chymotrypsin, has been modified with ST-PHPMA-CONHNH2 and ST-PHPMA-COOSu and the conjugates characterized by MALDI-TOF MS. The activity of modified chymotrypsins toward a high molecular weight substrate, P-Gly-Leu-Phe-NAp (where P is the HPMA copolymer backbone, and NAp is p-nitroanilide), was slightly lower than the activity of the native enzyme. The cleavage of a low molecular weight substrate, Z-Gly-Leu-Phe-NAp, by modified chymotrypsins was dependent on their structure. Whereas the activity of the amino group modified chymotrypsins was higher than that of the native enzyme, the activity of carboxyl-modified chymotrypsins was lower than that of the native enzyme. In summary, the data seem to indicate that ST-PHPMA is an effective protein-modifying agent.
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PMID:Functionalized semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] for protein modification. 981 74

Sieve tubes mediate the long-distance transport of nutrients and signals between source and sink organs of plants. To detect mobile phloem proteins that are differentially distributed in source and sink organs of Cucurbita maxima, we used both one-dimensional gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques revealed that phloem protein patterns depend on the sampling site: whilst several proteins were consistently observed in all phloem samples studied others appeared to occur in a organ-specific manner. For a characterization and identification of distinct phloem polypeptides, two approaches were chosen. First, protein bands resolved by SDS-PAGE were eluted from the polyacrylamide gel and the masses of the proteins were then determined by MALDI-TOF MS. Second, proteins resolved by SDS-PAGE were subjected to proteolytic degradation and the resulting peptides were analyzed by MALDI-TOF MS: the masses of the proteolytic peptides were used for a database search. By the latter approach, three mobile phloem compounds were identified as the phloem-specific protein PP2 (D.E. Bostwick et al., 1992, The Plant Cell 4, 1539-1548) a chymotrypsin and an aspartic proteinase inhibitor. None of the other polypeptides studied corresponded to any of the protein sequences present in the database. Furthermore, MALDI-TOF MS analyses indicated that some of the mobile phloem proteins occur in a covalently modified form and that the extent of the modification depends upon the plant organ.
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PMID:Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. by matrix-assisted laser desorption/ionization time of flight mass spectroscopy combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis. 1009

From muscle extracts of the European hedgehog, Erinaceus europaeus, an antihemorrhagic factor, erinacin, was purified by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, hydroxylapatite and gel filtration columns. A purification of approx. 1400-fold was achieved with an overall yield of 21% in antihemorrhagic activity. The molecular weight of erinacin determined by gel filtration was approx. 1000 kDa. SDS-PAGE of erinacin under reducing conditions indicates that it consists of two types of subunits, alpha and beta, with molecular weights of 37 and 35 kDa, respectively, in a ratio of 1:2. In the presence of 6 M guanidine-HCl, erinacin dissociates into alpha-subunits and beta-subunit decamers. From these results the subunit assembling of erinacin has been formulated as alpha(10).2beta(10). The molecular weight of the subunits and of the beta-subunit decamer was confirmed by MALDI-TOF mass spectrometry. Erinacin inhibits the hemorrhagic and proteolytic activity of the major hemorrhagic metalloprotease from the venom of Bothrops jararaca. Complete inhibition was achieved in an equimolar mixture of inhibitor and enzyme suggesting an equimolar complex. Erinacin is not inhibiting serine proteases such as trypsin and chymotrypsin, it was characterized to be a metalloprotease inhibitor. In electronmicroscopy, flower bouquet-like structures characteristic for some animal lectins were observed. Amino acid sequence analysis indicated that both subunits are almost identical and are composed of common amino terminal, collagen- and fibrinogen-like domains homologous to proteins of the ficolin/opsonin P35 lectin family.
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PMID:The antihemorrhagic factor, erinacin, from the European hedgehog (Erinaceus europaeus), a metalloprotease inhibitor of large molecular size possessing ficolin/opsonin P35 lectin domains. 1077 56

Soluble proteins from porcine brain were divided into two packs: (1) proteins which pass freely through CM52-cellulose, and (2) proteins retained on CM52. Each of these two packs of proteins was fractionated on preparative flat-bed isoelectrofocusing gel in the range of pH 2-12. Native FKBP-25 and its truncated forms were found among other proteins retained on CM52-cellulose. Immunoblotting with anti-FKBP-25 showed two bands in the range 27-30 kDa, one due to unmodified FKBP-25 and other due to FKBP-25 mixed with high-mobility group II protein (HMG-II). Selective immunostaining with anti-FKBP-25 antibodies of proteins which were not retained on CM52-cellulose showed several bands within the range of pI 7-5 and mass of 23 +/- 2 kDa. These fractions of proteins were next resolved on two-dimensional gels and immunostained with anti-FKBP-25 antibodies. Six proteins in the pI range 7-5 were detected. Edman degradation of alpha-chymotrypsin digests of the major spot suggests that it contains the GTP-binding protein Rab5 co-migrating with guanylyl kinase, whereas MALDI-TOF showed that a residual content of FKBP-25 may be also associated with these two proteins. A residual quantity of FKBP-25 was also associated with the phosphatidylethanolamine-binding protein which is abundant in the brain.
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PMID:Mammalian FKBP-25 and its associated proteins. 1090 Jan 28

Three major serine proteinase inhibitors (SBI-1, -2, and -3) were purified from the seeds of white sword bean (Canavalia gladiata) by FPLC and reversed-phase HPLC. The sequences of these inhibitors were established by automatic Edman degradation and TOF-mass spectrometry. SBI-1, -2, and -3 consisted of 72, 73, and 75 amino acid residues, with molecular masses of 7806.5, 7919.8, and 8163.4, respectively. The sequences of SBI-1 and -2 coincided with those of CLT I and II [Terada et al. (1994) Biosci. Biotech. Biochem., 58, 376-379] except only N- or C-terminal amino acid residues. Analysis of the amino acid sequences showed that the active sites of the inhibitors contained a Lys21-Ser22 against trypsin and Leu48-Ser49 against chymotrypsin, respectively. Further, it became apparent that about seven disulfide bonds were present. These results suggest that sword bean inhibitors are members of the Bowman-Birk proteinase inhibitor family.
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PMID:Complete amino acid sequences of three proteinase inhibitors from white sword bean (Canavalia gladiata). 1112 13

Whey proteins were modified by reaction with selected phenolic compounds (ferulic-, chlorogenic-, caffeic- and gallic acid) and related substances (quinic acid and p-quinone) as well as with extracts from coffee, tea, potato and pear at pH 9. The derivatives formed were characterized in terms of their physicochemical and digestion properties. The derivatization was accompanied by a reaction at the lysine and tryptophan side chains, whereby their content was decreased in comparison to that in the control whey proteins. Moreover, the solubility of the derivatives decreased over a broad pH range and the derivatization influenced the hydrophobe-hydrophile character of the whey proteins. The isoelectric points were shifted to lower pH values in the order of reactivity as follows: gallic acid > p-quinone > caffeic acid > chlorogenic acid. The other derivatives showed no or few changes compared to the control whey proteins. The formation of high molecular fractions was documented with SDS-PAGE. Especially the derivatives of chlorogenic-, caffeic-, gallic acid and p-quinone showed an increase in molecular weight of beta-lactoglobulin fraction from 18,300 to 20,000 Da. A dimer formation in molecular range 40,000 was also registered. MALDI-TOF-MS was applied to characterize the binding of the individual phenolic compounds or their oxidation products to the whey protein fractions, alpha-lactalbumin and beta-lactoglobulin. In vitro experiments showed that the digestion of the derivatized whey proteins with the enzymes of the gastrointestinal tract (trypsin, chymotrypsin, pepsin and pancreatin) was adversely effected. Similar results with regard to physicochemical characterization and digestion properties of the whey proteins treated with the applied extracts from plant beverages, fruit and vegetable were also documented. Coffee and tee were comparatively the most reactive extracts.
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PMID:Model studies on reactions of plant phenols with whey proteins. 1137 91

The whey protein pattern of milk from animals affected by mastitic inflammation was resolved by two-dimensional gel electrophoresis (2D-PAGE) and compared to milk from unaffected cows. Inflammation caused the appearance of four spots aligned at a molecular weight level of 26 kDa and over a pH-region of 5.0 to 6.4. The spots excised from 2D gels were treated with chymotrypsin and the resulting peptides analyzed by MALDI-TOF mass spectrometry and RP-HPLC. All four spots yielded highly similar chymotryptic peptide mass fingerprints as well as chromatographic peak patterns. A database search could identify the four spots as isoforms of the bovine prostaglandin D synthase (PGD-S). In one of the isoforms a defined cysteine residue was shown to be oxidized to a sulfonic acid.
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PMID:Lipocalin-type prostaglandin D synthase in milk: a new biomarker for bovine mastitis. 1178 99

The electrospray ionization (ESI)-tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF) mass spectrometer combined with the nano-HPLC system was utilized to determine the glycosylation site and the glycan structure in glycoprotein TIME-EA4 (EA4) from Bombyx diapause eggs. LC-MS analysis of EA4 and deglycosylated EA4 indicated that the carbohydrate moiety of EA4 has the mass of 730.58 Da. Then, EA4 was digested with trypsin and chymotrypsin to identify the glycosylated peptide. The peptide fragment from G1y21 to Phe25 was found to carry the carbohydrate moiety. LC-MS/MS analysis of this peptide fragment revealed the sequence of the attached oligosaccharide and the glycosylation site at the same time. The present methodology utilizing the combination of the nano-HPLC system and a highly sensitive Q-TOF mass spectrometer is demonstrated to be quite effective for analyses of glycoproteins of relatively low purity and limited availability from natural sources.
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PMID:Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS. 1193 29


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