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)

The lectin-like activity of Escherichia coli K88, Salmonella choleraesuis, and Bifidobacteria pseudolongum of porcine gastrointestinal origin was studied by hemagglutination (HA) and HA inhibition assays. Although all the bacterial strains were able to agglutinate Porcine and Lagomorpna erythrocytes, much higher HA titers were consistently observed for B. pseudolongum than for E. coli K88 or S. choleraesuis. Proteinaceous components and glycoproteins were responsible for the HA of E. coli K88 and B. pseudolongum, respectively, because a remarkable reduction of HA titers occurred due to treatment of E. coli K88 with protease or trypsin and of B. pseudolongum with protease and periodate. Hemagglutination of E. coli K88, S. choleraesuis, and B. pseudolongum was strongly inhibited by galactosyl residue-containing glycoproteins, including porcine and bovine mucin, thyroglobulin, and fetuin. Some sugars, including lactose, galactose, xylose, and xylooligosaccharide (XOS), at a relatively high concentration (47 to 92 mg/mL) also exhibited an inhibitory activity for the HA of B. pseudolongum. This result, combined with the enhanced HA activity of the three bacterial strains by modification of Lagomorpna erythrocytes with neuraminidase, indicated that galactosyl residue-containing glycoproteins mediated the HA of E. coli K88, S. choleraesuis, and B. pseudolongum. Our study demonstrated that proteinaceous or glycoproteinaceous lectin-like substances that recognize galactosyl residue-containing molecules, especially intestinal mucin, exist on the surface of E. coli K88, S. choleraesuis, and B. pseudolongum.
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PMID:Lectin-like activity of Escherichia coli K88, Salmonella choleraesuis, and Bifidobacteria pseudolongum of porcine gastrointestinal origin. 949 65

Platelet-activating factor (PAF) acetylhydrolase from human serum/plasma was identified on a polyvinylidene difluoride (PVDF) membrane by electroblotting proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was detected in the 43 kDa region on the membrane as a decrease in the beta-radioluminescence of [3H]acetyl-PAF or by the convenient method for determining PAF acetylhydrolase activity (the TCA precipitation method). The enzyme activity on treatment with N-glycosidase F shifted to the 34 kDa region on the PVDF membrane. On the other hand, only one band was observed, corresponding to a molecular mass of 53 kDa, on analysis by SDS-PAGE with silver staining. Treatment of the 53 kDa protein with N-glycosidase F changed its molecular mass to 43 kDa (protein A). The NH2-terminal 32 amino acid sequence of protein A completely corresponds to that of the heterogenous enzyme with 54 amino acids deleted from the NH2 terminus reported by Tjoelker et al. (Nature 374, 549-553, 1995). Even after trypsin treatment of the N-glycosidase F-digested enzyme, its PAF-AH activity remained in the 34 kDa region, but the contaminating protein A disappeared, on the PVDF membrane. In addition, the majority of serum PAF-AH was retained on a Sambucus sieboldiana agglutinin (SSA)-agarose column and was eluted with the hapten sugar, lactose. These results indicate that PAF acetylhydrolase consisting of a 34 kDa protein and about 9 kDa asparagine-conjugated sugar chain(s) is a major enzyme in human serum/plasma.
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PMID:Identification of a major PAF acetylhydrolase in human serum/plasma as a 43 kDa glycoprotein containing about 9 kDa asparagine-conjugated sugar chain(s). 956 6

This study was carried out to determine the concentrations of eosinophil cationic protein (ECP) and tryptase in the nasal lavage fluid (NLF) of 24 children (C) with grass pollen rhinitis as well as rhinitis symptoms before and after nasal provocation tests with or without a levocabastine (anti-H1 topical antihistamine) pretreatment. All C were monosensitized to grasses only. Twelve patients (Active Group = AG) were tested with a nasal provocation test with grass pollen (NPT) carried out by the insufflation of increasingly higher doses of an allergenic extract powder, while the other 12 patients (Placebo Group = PG) underwent just a nasal provocation test with lactose (placebo) (NPTp). The prechallenge NLF, obtained both before (C) and after (AG) levocabastine pretreatment, was compared to that obtained after periods of 2 and 24 hours postchallenge. In the AG, before and after levocabastine pretreatment, the tryptase concentrations had not significantly increased, whereas the ECP concentrations were found to be significantly higher (p < 0.05) in just the 24-hour postchallenge samples. In the PG the rhinitis symptoms were not induced by the NPTp and there was no significant change in either ECP or tryptase concentrations. In the AG a levocabastine pretreatment induced a significant increase (p < 0.05) in the cumulative allergen doses administered by the NPT. There was a reduction of the nasal symptoms in 7 patients, while in 3 subjects there was only a slight improvement, but in 2 subjects no effect was encountered. In conclusion this study shows that a levocabastine pretreatment before an NPT in patients with grass pollen rhinitis, outside the grass pollen season, induces a significant increase in the cumulative allergen doses (administered by the NPT) which provoked rhinitis symptoms but is not able to demonstrate any significant reduction in the ECP concentrations of the NLF.
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PMID:Eosinophil cationic protein (ECP) and tryptase in the nasal lavage fluid (NLF) of children with grass pollen rhinitis: levocabastine effect. 957 15

Thermal treatment of milk leads to non-enzymatic glycosylation of proteins through Maillard reaction. Free NH2 groups of basic amino acids react with the reducing carbonyl group of lactose forming the so-called Amadori products. Electrospray mass spectrometry analysis shows that beta-lactoglobulin (beta-LG), the major whey protein, undergoes lactosylation under industrial thermal treatment. In order to investigate the specificity of reactive sites for lactose binding the analysis of trypsin hydrolysates of beta-LG isolated from different industrial milks was performed. Results demonstrate that Lys-100 is a preferential lactosylation site of beta-LG during industrial milk treatment. These results were confirmed by an analysis of the three-dimensional model of the protein which showed that Lys-100 had the highest solvent accessibility and proximity to another amino group making Lys-100 the best candidate to lactosylation. Lys-47, previously identified by other authors, showed a good proximity to another Lys residue, but an intermediate level of exposition to solvent.
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PMID:Identification of a beta-lactoglobulin lactosylation site. 985 53

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 6 microM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112-129) and, to a lesser extent, FGF-2(130-155), whereas peptides FGF-2(10-33), FGF-2(39-59), FGF-2(86-96), and the basic peptide HIV-1 Tat(41-60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.
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PMID:Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: biochemical characterization and biological consequences in endothelial cell cultures. 995 Jun 79

In the present study trypsin mixed with various carbohydrates, i.e. lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin, was spray-dried in order to investigate the effects of spray-drying on this enzyme, with particular emphasis on the effects of interactions between trypsin and the surface formed during spray-drying. The protein was strongly over-represented at the surface of the powder particles, the surface coverage ranging from 10 to 65%, depending on the amount of trypsin in the solids (0.2-5%). This indicates that the protein adsorbs at the air/liquid interface of the spray-droplets, and that this surface is also largely preserved after drying. The surface concentration of protein in the spray-dried powders could be controlled by adding a surfactant to the mixture before drying, since the surfactant adsorbs preferentially at the air/liquid interface of the spray droplets, thus expelling protein from the surface. In general, the residual activity of trypsin in these non-optimised formulations was 90% or higher, and in no case less than 82%. It was found that the loss of activity could partly be explained by inactivation of the protein adsorbed at the surface. For mannitol and sucrose, however, the level of inactivation was higher than could be explained by surface inactivation alone, and additional mechanisms must also be considered.
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PMID:Spray-drying of trypsin - surface characterisation and activity preservation. 1051 79

Amino acid alterations were designed at the C terminus of the hinge segment (amino acids approximately 51-59) that links two functional domains within lactose repressor protein (LacI). Gly was introduced between Gly(58) and Lys(59) to generate Gly(58+1); Gln(60) was changed to Gly or Pro, and up to three additional glycines were inserted following Gln(60) --> Gly. All mutant proteins exhibited purification behavior, CD spectra, assembly state, and inducer binding properties similar to wild-type LacI and only small differences in trypsin proteolysis patterns. In contrast, significant differences were observed in DNA binding properties. Gly(58+1) exhibited a decrease of approximately 100-fold in affinity for O(1) operator, and sequential Gly insertion C-terminal to Gln(60) --> Gly resulted in progressively decreased affinity for O(1) operator, approaching nonspecific levels for insertion of >/=2 glycines. Where sufficient affinity for O(1) operator existed, decreased binding to O(1) in the presence of inducer indicated no disruption in the allosteric response for these proteins. Collectively, these results indicate that flexibility and/or spacing between the core and N-terminal domains did not significantly affect folding or assembly, but these alterations in the hinge domain profoundly altered affinity of the lactose repressor protein for its wild-type target sequence.
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PMID:Glycine insertion in the hinge region of lactose repressor protein alters DNA binding. 1052 77

The solubility of lactose-beta-lactoglobulin conjugates at pH 4.6, after heating near neutral pH in phosphate buffer/0.116 M NaCI, was investigated by size exclusion chromatography and compared with unmodified protein. Heated conjugates in the temperature range 65-90 degrees C showed greater solubility at pH 4.6. The proportion of soluble protein increased with the number of bound lactose molecules. Total solubility was obtained for conjugates with nine lactose residues attached per monomer of beta-lactoglobulin. The protective effect of bound sugar toward precipitation was associated with the formation of soluble disulfide cross-linked dimers, highly accessible to trypsin digestion. These results suggested that bound lactose, through steric hindrance and high surface hydrophilicity, prevents the thiol-disulfide exchange reactions of the polymerization-aggregation process of lactose-beta-lactoglobulin conjugates.
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PMID:Formation of stable covalent dimer explains the high solubility at pH 4.6 of lactose-beta-lactoglobulin conjugates heated near neutral pH. 1056 4

In the present investigation freeze-drying of proteins (BSA or trypsin) together with various carbohydrates, i.e. lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin, has been studied with particular emphasis on the surface composition of the freeze-dried powders. The proteins were found to be over-represented on the powder surface as compared to the bulk concentration of protein. The mechanism behind the surface accumulation is believed to be that proteins adsorb preferentially over carbohydrates to the ice/liquid interface in the frozen sample. The degree of surface accumulation depended on the carbohydrate used, and was increased in annealed samples compared to reference samples. The activity of trypsin was fairly well preserved (58-90%) in the freeze-dried powders, but depended on the carbohydrate excipient, whilst the surface composition had little effect on the activity. The activity preservation was improved when the protein concentration was raised from 1 to 10% in the solids. The surface composition of powders containing mixtures of mannitol and dextrin as excipients depended on the ratio between the two carbohydrates, with the lowest surface coverage of protein obtained in 50/50 mixtures.
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PMID:Surface characterisation of freeze-dried protein/carbohydrate mixtures. 1056 37

This study evaluated tableting compression by using internal and external lubricant addition. The effect of lubricant addition on the enzymatic activity of trypsin, which was used as a model drug during the tableting compression process, was also investigated. The powder mixture (2% crystalline trypsin, 58% crystalline lactose, and 40% microcrystalline cellulose) was kneaded with 5% hydroxypropyl cellulose aqueous solution and then granulated using an extruding granulator equipped with a 0.5-mm mesh screen at 20 rpm. After drying, the sample granules were passed through a 10-mesh screen (1680 microm). A 200-mg sample was compressed by using 8-mm punches and dies at 49, 98, 196, or 388 MPa (Mega Pascal) at a speed of 25 mm/min. The external lubricant compression was performed using granules without lubricant in the punches and dies. The granules were already dry coated by the lubricant. In contrast, the internal lubricant compression was performed using sample granules (without dry coating) containing 0.5% lubricant. At 98 MPa, for example, the compression level using the external lubricant addition method was about 13% higher than that for internal addition. The significantly higher compressing energy was also observed at other MPas. By comparison, the friction energy for the external addition method calculated based on upper and lower compression forces was only slightly larger. The hardness of tablets prepared using the internal addition method was 34% to 48% lower than that for the external addition method. The total pore volume of the tablet prepared using the external addition method was significantly higher. The maximum ejection pressure using the no-addition method (ie, the tablet was prepared using neither dry-coated granules nor added lubricant) was significantly higher than that of other addition methods. The order was as follows: no addition, external addition, and then internal addition. The ejection energy (EE) for internal addition was the lowest; for no addition, EE was the highest. In the dissolution test, the tablets obtained using external addition immediately disintegrated and showed faster drug release than those prepared using internal addition. This result occurred because the water penetration rate of the tablet using the external addition was much higher. The trypsin activity in tablets prepared using the external addition method was significantly higher than that produced using the internal addition method at the same pressure. All these results suggest that the external addition method might produce a fast-dissolution tablet. Because the drug will be compressed using low pressure only, an unstable bulk drug may be tableted without losing potency.
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PMID:Comparative evaluation of tableting compression behaviors by methods of internal and external lubricant addition: inhibition of enzymatic activity of trypsin preparation by using external lubricant addition during the tableting compression process. 1174 Dec 71


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