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)

Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism. Several proteins with proteolytic activity were detected in culture filtrates from T. harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity. The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7-4.9. The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity. Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1. The protein sequence showed <43% identity to mammal trypsins and 47-57% to other fungal trypsin-like proteins described thus far. Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media. The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations. This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T. harzianum CECT 2413. A role for PRA1 in the protection of plants against pests and pathogens provided by T. harzianum CECT 2413 is proposed.
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PMID:Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. 1522 Dec 28

Identification of molecular markers of monoterpenoid indole alkaloid (MIA) accumulation in cell-suspension cultures of Madagascar periwinkle (Catharanthus roseus (L.) G. Don) was performed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the protein patterns from alkaloid-producing and non-producing cells showed the specific occurrence of a 28 kDa polypeptide restricted to cells accumulating MIAs. The polypeptide was purified by preparative two-dimensional gel electrophoresis, digested with trypsin, and microsequenced by the Edman degradation method. Cloning of the corresponding cDNA revealed that the protein which has been named CrPS (Catharanthus roseus Protein S) is a member of the alpha/beta hydrolase superfamily. Time-course expression studies by northern blot analysis confirmed that CrPS gene expression was associated with MIA accumulation in cell suspension cultures. In the whole plant, multicellular compartmentation is required for alkaloid biosynthesis. In situ mRNA hybridization on developing leaves revealed that CrPS mRNA and transcripts encoding the first enzymes of the MIA pathway were co-localized in internal phloem parenchyma cells. The possible implication of the alkaloid-accumulation associated protein CrPS in the signal transduction pathway leading to MIA production is discussed.
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PMID:Purification, molecular cloning, and cell-specific gene expression of the alkaloid-accumulation associated protein CrPS in Catharanthus roseus. 1573 82

Fat-free bovine milk fermented by 12 kinds of lactic acid bacteria and yeast enhanced monoclonal antibody production of human hybridoma HB4C5 cells 2.8-fold in serum-free medium. Immunoglobulin production of human peripheral blood lymphocytes (PBL) was also stimulated in vitro. IgM and IgG production of human PBL was accelerated up to 2.8-fold and 5.4-fold, respectively. Interferon-gamma production of human PBL was also accelerated 6.0-fold by 50 microg/ml of the fermented milk. However, interleukin-4 production of PBL was not affected, and tumor necrosis factor-alpha production was suppressed. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 degrees C and was completely lost by trypsin digestion. The findings suggested that the active substance in the fermented milk was heat stable protein. Gel-filtration and the SDS-PAGE analysis revealed that the molecular weight of the active substance was estimated as 19.0 kDa, which was not detected in fat-free bovine milk before fermentation. N-terminal amino acid sequence of the 19.0 kDa protein was highly homologous to proteose-peptone component 3 (PP3). Since molecular weight of PP3 is 28 kDa, it is suggested that the 19.0 kDa protein is derived from degradation of PP3 during fermentation of fat-free milk. Moreover, PP3 purified from fat-free milk also enhanced IgM production of HB4C5 cells.
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PMID:Immunostimulation effects of proteose-peptone component 3 fragment on human hybridomas and peripheral blood lymphocytes. 1597 34

Current control of the sheep blowfly (Lucilia cuprina) relies on chemical insecticides, however, with the development of resistance and increasing concerns about human health and environmental residues, alternative strategies to control this economically important pest are required. In this study, we have identified several isolates of Bacillus thuringiensis (Bt), collected from various Australian soil samples, that produce crystals containing 130 and 28 kDa proteins. These isolates were highly toxic to feeding larvae in both in vitro bioassays and in vivo on sheep. By N-terminal amino acid sequencing, we identified the smaller crystal band (28 kDa) as a cytological (Cyt) protein. Upon solubilization and proteolytic processing by trypsin, the 130 kDa crystal protein yielded among others, a truncated 55-60 kDa toxin moiety which exhibited larvicidal activity against sheep blowfly. The amino-terminal sequence of the trypsin-resistant protein band revealed that this Bt endotoxin was encoded by a new cry gene. The novel cry protein was present in all the strains that were highly toxic in the larval assay. We have also identified from one of the isolates, a novel secretory toxin with larvicidal activity.
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PMID:Identification and characterization of proteins from Bacillus thuringiensis with high toxic activity against the sheep blowfly, Lucilia cuprina. 1609 86

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent molecular weights of 15.0 kDa and 14.5 kDa under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for dabocetin alpha and beta subunits were isolated and sequenced. The deduced protein sequences of both subunits were confirmed by N-terminal amino acid sequencing and trypsin-digested peptide mass fingerprinting. Dabocetin did not induce platelet aggregation in platelet-rich plasma. It also had little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin. Whereas, dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC50 value of 0.35 microM. Flow cytometry analysis showed that dabocetin significantly inhibited mAb SZ2 binding to platelet membrane glycoprotein Ib alpha, indicating that platelet membrane glycoprotein Ib is involved in the inhibitory effect of dabocetin on ristocetin-induced platelet agglutination.
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PMID:Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom. 1633 60

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-DL-arginine-p-nitroanilide (DL-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors - soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 degrees C and was stable from pH 4.0 to pH 10.0 when incubated at 20 degrees C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of DL-BAPNA by the chinook salmon enzyme was 60 degrees C, however, the enzyme was unstable at temperatures above 40 degrees C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS-PAGE.
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PMID:Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha). 1645 61

A monomeric glycoprotein with a molecular mass of 28 kDa in SDS-PAGE was isolated from the Withania somnifera root tubers. The protein designated WSG (Withania somnifera glycoprotein) demonstrated potent antimicrobial activity against the phytopathogenic fungi and bacteria tested. Antifungal effect has been demonstrated in that WSG exerts a fungistastic effect by inhibiting spore germination and hyphal growth in the tested fungi. WSG showed potent antifungal activity against Aspergillus flavus, Fusarium oxysporum, F. verticilloides and antibacterial activity against Clvibacter michiganensis subsp. michiganensis. WSG is an acidic, non-toxic (trypsin-chymotrypsin) protease inhibitor. These results encourage further studies of WSG as a potential therapeutic agent for its antifungal activity.
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PMID:Antimicrobial properties of a non-toxic glycoprotein (WSG) from Withania somnifera (Ashwagandha). 1700 92

The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for approximately 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver-Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.
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PMID:Biochemical characterization and molecular cloning of a plasminogen activator proteinase (LV-PA) from bushmaster snake venom. 1703 51

Lumbrokinase gene F238 was amplified by RT-PCR from the total RNA of earthworm (Eisenia fetida). The gene including signal peptide sequence was inserted into pUCm-T vector to construct pUCm-T-F238. The product was sequenced. The GenBank accession number was DQ202401. Lumbrokinase F238 comprised 738bp and included an open reading frame that encoded a polypeptide of 245 amino acid residues, containing a signal peptide of 7 amino acid residues and a mature peptide of 238 amino acid residues. Both nucleotide and amino acid sequences homologies were 99% after the sequence was compared with Lumbricus rubellus F-III-2. There were two base pair mutations, which subsequently caused two amino acid mutations. The characteristics and structure of F238 was analysed and predicted with biology softwares and databases. The pl of F238 was 4.61. It had eleven Cysteines, which formed three disulfide bonds. Its secondary structure mainly consisted of beta-sheet. Lumbrokinase F238 had serine active center. It was a protease in trypsin family of serine protease superfamily. Lumbrokinase gene F238-m without signal peptide sequence was obtained by PCR using pUCm-T-F238 as template. The expression vector pPIC9-F238-m was constructed by inserting gene F238-m into yeast expression and secretion plasmid pPIC9. Plasmid pPIC9-F238-m was linearized with BgIII and then transformed into Pichia pastoris strain GS115 cell by electroporation method. Phenotypes of transformants were screened in MM and MD plates to ensure the integration of lumbrokinase gene F238-m into yeast chromosome DNA. Methanol was added to a final concentration of 0.5% for the expression of recombination protein every 24h to maintain induction. The result of SDS-PAGE showed that the molecular weight of the expression product was about 28 kDa, in correspondence with the theoretical molecular weight. After the induction of expression, the fibrinolytic activity of the supernatant was measured using artificial fibrin plates. Then the engineering strain of high activity was obtained, and the fibrinolytic activity was up to 100 U/mL.
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PMID:[Cloning and expression of lumbrokinase gene in Pichia pastoris]. 1703 59

A myofibril-bound serine proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by serine proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type serine proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of serine proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of serine proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other serine proteinases, especially in well-conserved regions.
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PMID:Purification, characterization, and cDNA cloning of a myofibril-bound serine proteinase from the skeletal muscle of crucian carp (Carassius auratus). 1725 17


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