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)

The natural product lysobactin is a cyclic peptide with greater antibiotic activity than vancomycin against Gram-positive aerobic and anaerobic bacteria. Although the potency of orally administered lysobactin has not been reported, lysobactin hydrolysis in the digestive tract following oral administration would be difficult to predict because of the cyclic structure and presence of beta-hydroxy and D-amino acids. Therefore, lysobactin served as a model compound for study using an in vitro assay to predict if hydrolysis in the digestive tract might severely limit its bioavailability. The in vitro immobilized digestive enzyme assay consisted of a gastric digester containing immobilized pepsin, and an intestinal digester consisting of immobilized trypsin, chymotrypsin, and mucosal peptidases. In order to determine the sites of lysobactin hydrolysis by each enzyme, lysobactin was incubated separately with immobilized pepsin, trypsin, chymotrypsin, or mucosal peptidases. Lysobactin was also incubated with a mixture of intestinal enzymes. The hydrolysis products of lysobactin in each incubation mixture were rapidly identified using reversed-phase HPLC, continuous-flow FAB/LC-MS, and tandem MS. Lysobactin was essentially stable at low pH and was not hydrolyzed by immobilized pepsin. Above pH 7, nonenzymatic hydrolysis of the lactone moiety occurred followed by proteolysis by all intestinal enzymes investigated. Lysobactin was not directly hydrolyzed by immobilized pepsin, chymotrypsin, trypsin, or mucosal peptidases. Hydrolysis rates of lysobactin at pH 7.5 with active or inactivated chymotrypsin were measured and were compared with the rate of chymotrypsin hydrolysis of the open-chain form following hydrolysis of the lactone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Application of an immobilized digestive enzyme assay to measure chemical and enzymatic hydrolysis of the cyclic peptide antibiotic lysobactin. 810 15

Zona pellucida glycoproteins play an important role in fertilization. In this study, attempts have been made to identify and define epitopes of monoclonal antibodies (mAbs) possessing contraceptive efficacy in vitro. The porcine zona glycoprotein pZPC, a homologue of mouse/human ZP3, was reduced and alkylated and subsequently digested with trypsin. Reverse-phase HPLC of the tryptic digest yielded twenty two peaks (T1-T22). When tested against mAbs reactive against sequential determinants on pZPC, T11 was immunoreactive with two mAbs, mAb-455 and mAb-467, as shown by antigen inhibition ELISA. IC50 values of 3.1 nM and 8.6 nM were recorded versus mAb-455 and mAb-467 respectively, and approximated the IC50 values obtained with intact pZPC. Amino acid analysis, Edman degradation, and FAB-MS identified T11 as the N-blocked decapeptide pyro-Gln-Pro-Val-Trp-Gln-Asp-Glu-Gly-Gln-Arg derived from the N-terminus of pZPC. Synthesis of overlapping octapeptides further identified VWQDE and WQDE as the minimum motifs with antigenic activity for mAb-455 and mAb-467, respectively. Glycine replacement peptides confirmed residues W,Q,E as critical for binding mAb-455 and W,Q,D,E as critical for binding mAb-467. Both mAbs inhibited binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of porcine zona glycoprotein, will assist in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZPC or its homologues in other species.
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PMID:Localization of epitopes for monoclonal antibodies at the N-terminus of the porcine zona pellucida glycoprotein pZPC. 856 67

Incubation of the C225S mutant of the R1 subunit of ribonucleotide reductase from Escherichia coli with the R2 subunit and nucleoside diphosphates leads to fragmentation of the polypeptide backbone of R1 [Mao, S. S., Holler, T. P., Bollinger, J.M., Jr., Yu, G. X., Johnston, M.I., & Stubbe, J. (1992) Biochemistry 31, 9744--9751]. The 26 and 60 kDa cleavage fragments were purified to homogeneity. The 26 kDa polypeptide was digested with Lys-C, and the peptides were partially purified by RP-HPLC. Mass spectrometric analysis (MALDI-TOF) of the HPLC fractions allowed the identification of the C-terminal peptide. The molecular mass of this peptide (2176) revealed that serine-224 constitutes its C-terminus, and further analysis of the distribution of its monoisotopic masses by FAB-MS indicated that Ser224 possesses a carboxamide rather than a carboxylate group. Treatment of the 60 kDa cleavage fragment with cyanogen bromide and subsequent MALDI-TOF analysis of the partially RP-HPLC purified peptides yielded a fraction containing its N-terminal peptide. This peptide was digested with trypsin, and the digestion mixture was purified by HPLC. Analysis of the fractions by MALDI-TOF identified the N-terminal peptide and determined a mass of 2222. This mass suggested valine 226 was the N-terminal residue (modified by an adduct of 28 mass units). Larger amounts of the C-terminal tetrapeptide of the 60 kDa fragment (V226LIE229) were obtained by complete digestion of the crude reaction mixture with endoproteinase Glu-C. The peptide mixture was then purified on an immunoadsorbent column containing immobilized antibodies raised against a synthetic peptide with the sequence KVLIE. After elution of the affinity-bound peptide, it was analyzed by CID-MS verifying that an adduct of 28 mass units was attached to valine 226. These results indicated that the amino group of Val226 is formylated. The localization of the residues at the cleavage site of C225SR1 provides a biochemical identification of the active site region of the R1 subunit of RDPR from E.coli. The details of the mechanism of cleavage remain to be elucidated.
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PMID:Identification of an active site residue of the R1 subunit of ribonucleotide reductase from Escherichia coli: characterization of substrate-induced polypeptide cleavage by C225SR1. 875 68

Isolation of proteinase inhibitors from the sea anemone Stichodactyla helianthus was achieved by trichloroacetic acid treatment of the aqueous extract followed by affinity chromatography on trypsin-Sepharose and ion-exchange chromatography on CM-cellulose. The average molecular mass of the major inhibitor (ShPI-I) obtained by fast atom bombardment mass spectrometry (FAB-MS) was 6110.6 Da. The amino acid sequence was determined by FAB-MS combined with manual Edman degradation, digestions with endopeptidases and exopeptidases and automatic sequencing. The sequence of ShPI-I (55 amino acids) was compared with those reported in the SwissProt database for several proteinase inhibitors and significant similarity to inhibitors belonging to the Kunitz family was observed. ShPI-I exhibits a broad specificity for serine, cysteine and aspartic proteinases. The dissociation constants of the complexes formed with different enzymes were determined. The affinity-purified fraction (PI) was immobilized on Sepharose and used in the purification of different classes of proteinases.
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PMID:Purification, characterization and immobilization of proteinase inhibitors from Stichodactyla helianthus. 902 93

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.
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PMID:Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties. 955 45

A subset of patients with systemic mastocytosis (SM) develop acute myeloid leukaemia (AML). However, little is known about the biology of such leukaemias and their relationship to the mast cell (MC) lineage. We report on two female patients who suffered from SM and AML. According to FAB criteria, the leukaemias were classified as AML-M4 (patient 1) and AML-MO (patient 2). The coexistence of the two distinct neoplasms (AML and SM) was demonstrable by immunostaining of serial bone marrow (BM) sections with monoclonal antibodies (mAb). In particular, the MC infiltrates were found to react with mAb against MC-tryptase and MC growth factor receptor c-kit (CD117), but not with mAb to CD15 or CD34. In contrast, the AML blasts were immunoreactive for CD15 (patient 1) or CD34 (patient 2), but did not express tryptase. The c-kit point mutation Asp-->Val at codon 816, considered to play a role in the transformation of MC progenitors, was detected in patient 1 in a BM cell fraction containing 4% MC. However, no c-kit mutation was found in pure AML blasts (<1% MC). These findings argue against an evolution of the AML clone from neoplastic MC or MC-committed progenitors.
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PMID:Systemic mastocytosis associated with acute myeloid leukaemia: report of two cases and detection of the c-kit mutation Asp-816 to Val. 985 25

The chemical assessment of the complete disulphide bridge pattern in the beta-chain of human recombinant follicotropin (betaFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native betaFSH using the broad-specificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FAB-MS, leading to the assignment of the first three disulphide bridges. The remaining S-S bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in betaFSH was determined as: Cys3-Cys5l, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confirming the arrangement inferred from the crystal structure of the homologous betaCG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.
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PMID:Assignment of the complete disulphide bridge pattern in the human recombinant follitropin beta-chain. 1150 62

A number of autocrine and paracrine growth regulators are considered to be involved in the survival and proliferation of blast cells in acute myeloid leukemia (AML). We have recently shown that blast cells in a group of patients with AML produce and secrete the mitogenic enzyme tryptase. In the present study, we examined functional effects of tryptase in the context of AML. As assessed by 3H-thymidine uptake experiments, tryptase-containing serum from patients with AML as well as heparin-complexed recombinant tryptase were found to promote the proliferation of cultured bone marrow- and lung fibroblasts in a dose-dependent manner. A neutralizing antibody against human beta-tryptase was found to diminish these growth-stimulatory effects of serum-tryptase in all patients examined. Tryptase also induced the expression of mRNA for GM-CSF and SCF, two cytokines known to promote growth of AML cells, in cultured bone marrow fibroblasts. Neither recombinant tryptase nor tryptase-rich serum of AML patients, showed an effect on the growth of leukemic blast cells irrespective of the FAB category or expression of protease-activated receptor (PAR)-2, a putative molecular target of tryptase. Together, tryptase is secreted from AML blasts as a biologically active molecule that may exhibit paracrine rather than autocrine effects in AML.
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PMID:Evaluation of biologic activity of tryptase secreted from blast cells in acute myeloid leukemia. 1675 62


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