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)

Loop residues in domain II of Bacillus thuringiensis Cry delta-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops beta6-beta7 (S(387)SPS(390)), beta8-beta9 (S(410), N(411), T(413), T(415), E(417) and G(418)) and beta10-beta11 (D(454)YNS(457)) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S(410) and E(417) in the beta8-beta9 loop, and Y(455) and N(456) in the beta10-beta11 loop are involved in larvicidal activity of the Cry4Ba toxin.
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PMID:Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity. 1562 55

Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.
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PMID:Proteomic mapping of brain plasma membrane proteins. 1568 8

Both the disulphide bond (Cys192-Cys199) and the proline-rich motif (Pro193ProAsnPro196) in the long loop connecting the alpha4-alpha5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry delta-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes aegypti larvae were determined, Escherichia coli cells expressing each mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A mutants. Increasing the flexibility of the alpha4-alpha5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations significantly decreased the larvicidal activity. Similar to the wild-type protoxin, all mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These findings are the first biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the alpha4-alpha5 loop of the Cry4Aa toxin.
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PMID:Structural requirements of the unique disulphide bond and the proline-rich motif within the alpha4-alpha5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa delta-endotoxin. 1579 13

The substrate specificity of alpha-chymotrypsin and other serine proteases, trypsin, elastase, proteinase K and subtilisin, towards hydrolysis of various polyesters was examined using poly(L-lactide) (PLA), poly(beta-hydroxybutyrate) (PHB), poly(ethylene succinate) (PES), poly(ethylene adipate) (PEA), poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBS/A), poly[oligo(tetramethylene succinate)-co-(tetramethylane carbonate)] (PBS/C), and poly(epsilon-caprolactone) (PCL). alpha-Chymotrypsin could degrade PLA and PEA with a lower activity on PBS/A. Proteinase K and subtilisin degraded almost all substrates other than PHB. Trypsin and elastase had similar substrate specificities to alpha-chymotrypsin.
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PMID:Hydrolysis of polyesters by serine proteases. 1592 50

Tryptic cleavage has been a potential method for studying the structure and mechanism of many membrane transport proteins. Here, we report tight association of trypsin to pig kidney plasma membranes enriched in Na,K-ATPase. Trypsin also associated with protein-free vesicles prepared from plasma membrane lipids. Membrane-associated trypsin was found to be highly resistant to autolysis and insensitive to inhibition by PMSF. Na,K-ATPase substrate ions differentially influenced the level of trypsin membrane association. Thus, NaCl significantly increased trypsin membrane association compared to KCl. The ions seem to exert direct effects on the membrane independent of their effects on protein conformation. Bicarbonate anions, which detach peripheral membrane proteins, efficiently released trypsin from the membrane. Trypsin membrane association was found to enhance the cleavage of the Na,K-ATPase gamma-subunit. Comparison between membranes from shark rectal gland and pig kidney showed that trypsin association was significantly higher in the former. This was found to be partly due to the presence of higher cholesterol levels in the membrane. In conclusion, the differential membrane association of trypsin may affect the outcome of proteolytic cleavage of membrane-bound proteins.
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PMID:Stabilization of trypsin by association to plasma membranes: implications for tryptic cleavage of membrane-bound Na,K-ATPase. 1635 71

A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065 kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, alpha-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (Ki) of 0.52 and 3.27 nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known.
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PMID:A five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities. 1651 41

Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol that includes sodium carbonate to prevent cytoplasmic contamination. These proteins were analyzed by SDS-PAGE, trypsin digestion and LC-MS/MS for peptide identification. Elongation factors, mitochondrial membrane proteins and a mannosyltransferase involved in cell wall mannan biosynthesis were also identified in this fraction.
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PMID:Genetic and proteomic evidences support the localization of yeast enolase in the cell surface. 1654 86

This study investigated the effects of chemical treatments of salseed meal (SSM) on nutrient digestibility and digestive enzymes in colostomized hens and intact broilers. Finely ground SSM was treated (820 mL/ kg of SSM DM) with distilled water (pH 5.3), 0.67 M acetic acid (pH 2.4), or 0.67 M sodium hydrogen carbonate (pH 8.2), and incubated for 12 h at 37 degrees C. Five isonitrogenous and isocaloric diets each for colostomized hens (25.6 g of N/kg of DM and 12.5 MJ/kg of DM) and for broilers (33.6 g of N/kg of DM and 12.3 MJ/kg of DM) were formulated. For each group, 1 of these diets was wheat-based (control) whereas the other 4 were SSM-based diets (untreated SSM or SSM treated with water, acetic acid, and sodium bicarbonate). Inclusion of SSM in diets markedly reduced apparent protein and starch digestibility in hens and broilers. Chemical treatments of SSM improved the protein and starch digestibilities in colostomized hens and broilers. Treatment of SSM with alkali reduced the pancreatic hypertrophy in broilers that was observed when SSM was included in the diet. Inclusion of SSM in broiler diets did not affect pancreatic trypsinogen activity; however, chymotrypsinogen and alpha-amylase activities were depressed with its inclusion. Activities of these enzymes were improved by all chemical treatments of SSM. Dietary inclusion of SSM in broiler diets depressed the activities of trypsin, chymotrypsin, and alpha-amylase in the jejunal digesta. Alkali treatment proved to be the most effective in reducing the adverse effects of SSM upon trypsin and chymotrypsin activities. The hens receiving SSM in their diets produced eggs with discolored yolks (dirty greenish-yellow). In conclusion, nutrient digestibility, and pancreatic and intestinal enzymes were markedly depressed with the inclusion of SSM in the diets of the fowl. Bicarbonate was the most effective treatment to improve nutrient digestibility and mitigate, to some extent, the poor digestion of SSM.
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PMID:Chemical treatments to reduce antinutritional factors in salseed (Shorea robusta) meal: effect on nutrient digestibility in colostomized hens and intact broilers. 1713 78

Poly(2-ethyl-2-oxazoline) (POZ) was synthesized by living cationic ring-opening polymerization under microwave irradiation yielding polymers with low polydispersity indices (PDI, 1.15). The polymerization was quenched with sodium carbonate yielding a hydroxyl end-group with a high degree of functionality. The hydroxyl group was converted to carboxylate and the polymer was purified by ionic exchange chromatography. Following activation to succinimidyl ester, POZ-conjugates to high and low molecular weight biomolecules, trypsin and Ara-C, were obtained. The properties of the conjugates were compared to those of the corresponding conjugates with poly(ethylene glycol) (PEG) of similar size. The coupling of POZ to trypsin did not affect the enzymatic activity towards low mass substrates but, on the contrary, reduced the activity on the higher mass ones. Furthermore, the POZ-protein conjugates showed hydrodynamic volumes and protein rejecting properties similar to those of PEG-conjugates. Similarly, the POZ-Ara-C conjugate revealed a drug release profile, stability towards the degrading enzyme cytidine deaminase and in vitro cytotoxicity comparable to what has already been described for the PEG derivative. These data support the potential of POZ as a versatile alternative to the well-known and widely used PEG for protein and drug conjugation and delivery.
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PMID:Synthesis and characterization of poly(2-ethyl 2-oxazoline)-conjugates with proteins and drugs: suitable alternatives to PEG-conjugates? 1803 60

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.
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PMID:Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies. 1809 73


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