Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.32 (bromelain)
 1,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur to pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5.0-induced conformation indicates that aggregation is a property of the BHA2 component and that the conformation change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.
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PMID:Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. 695 Nov 81

The capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were approximately 31-32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0 degrees C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection.
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PMID:Assaying for structural variation in the parvovirus capsid and its role in infection. 977 Apr 25

In this study, we investigated the presystemic metabolism of trypsin and bromelain and the influence of these proteolytic enzymes on the mucus layer covering the gastrointestinal (GI) epithelia. In vitro studies demonstrated that 77.3% +/- 4.0% (mean +/- SD, n = 3) of trypsin is autodegraded within 2 hr, whereas autodegradation of bromelain was negligible. In contrast to the metabolization of bromelain by all pancreatic serine proteases, trypsin is only degraded to some extent by elastase. Both therapeutically used enzymes remained stable after incubation with an excised porcine mucosa, demonstrating that proteolysis caused by brush border membrane-bound enzymes is negligible. Trypsin and bromelain were highly mucolytic active, thereby reducing the diffusion barrier based on the mucus gel layer. Strategies to improve the galenic of dosage forms for trypsin and bromelain include the use of bioadhesive polymers such as hydroxyethylcellulose or slightly modified chitosan-EDTA, providing strongly improved stability of these enzymes toward proteolytic degradation in vitro. The given information represents a good starting point to improve the galenic of dosage forms for orally administered proteolytic enzymes.
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PMID:Peroral administration of enzymes: strategies to improve the galenic of dosage forms for trypsin and bromelain. 1069 48

This study had the purpose to improve the paracellular uptake of drugs by combining the thiomer/reduced glutathione (GSH) permeation-enhancing system with a proteolytic enzyme. Due to the covalent binding of 2-iminothiolane to chitosan the thiomer chitosan-TBA (chitosan-4-thiobutylamidine) was obtained. Permeation studies were performed with freshly excised intestinal mucosa of guinea pigs mounted in Ussing-type chambers using on the one hand the low-molecular size marker flurescein (Na-Flu) and on the other hand the high-molecular size marker FITC-dextran. Apparent permeability coefficient (P(app)) as well as enhancement ratios (=P(app) permeation-enhancing system/P(app) control) were calculated. Trypsin, papain and bromelain displayed a permeation-enhancing effect for Na-Flu on the small intestinal mucosa. Enhancement ratios of 1.84, 1.63 and 1.78 were identified for 2% trypsin, 0.5% papain and 2% bromelain solutions, respectively. However, only bromelain could guarantee a significant permeation enhancement of FITC-dextran with a P(app) of 4.45+/-0.44 x 10(-6) cm/s representing an enhancement ratio of 1.57. A similar enhancement of FITC-dextran permeation was reached by the use of the chitosan-TBA (0.5%)/GSH (5%) system. Moreover, an additive permeation-enhancing effect of the chitosan-TBA/GSH system in combination with bromelain (2%) was observed, leading to a maximum P(app) of 5.91+/-0.51 x 10(-6) cm/s, which corresponds to an enhancement ratio of 2.1. According to these results, the combination of the thiomer/GSH system with bromelain might represent a new promising strategy in order to raise the in vivo efficacy of non-invasive administered hydrophilic macromolecular drugs.
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PMID:Improved paracellular uptake by the combination of different types of permeation enhancers. 1560 66

Influenza A virus hemagglutinin (HA) is a major envelope glycoprotein mediating viral and cell membrane fusion. HA is anchored in the viral envelope by a light HA(2) chain containing one transmembrane domain and a cytoplasmic tail. Three cysteine residues in the C-terminal region, one in the transmembrane domain and two in the cytoplasmic tail, are highly conserved and potentially palmitoylated in all HA subtypes. The HA(2) C- terminal anchoring segments were extracted to organic phase from the bromelain-digested viruses (subviral particles) of three strains: A/X-31 (H3 subtype), A/Puerto Rico/8/34 (H1 subtype) and A/FPV/Weybridge/34 (H7 subtype). Their primary structures were assessed by matrix-assisted laser desorption/ionization time-of-flight time-of- flight mass spectrometry (MALDI-ToF-ToF MS). Trypsin-type protease-cleaved peptides prevailed over bromelain- cleaved ones in the peptide mixtures. All of them included transmembrane domains. Several distinctive features of the C-terminal HA(2) peptides acylation character were discovered by MALDI-ToF MS: 1) the peptides isolated from the viruses, which were digested by bromelain in the absence of beta-mercaptoethanol, were predominantly triply acylated; 2) the peptides were acylated not only by palmitic, but also by stearic acid residues; 3) the palmitate/stearate ratio was different for the three strains studied; 4) the A/FPV/Weybridge/34 strain has a priority to stearate binding. This fatty acid residue was discovered at the first of three conservative cysteine residues located in the transmembrane domain. It was found that presence of thiol reagent during preparation of subviral particles led to the appearence of the C-terminal HA(2) peptides acylated to different degrees. Triply, doubly, mono- and even unacylated peptides were detected. It was demonstrated that the thioester bond in the isolated acylpeptides was extremely sensitive to thiol reagents.
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PMID:Mass spectrometric sequencing and acylation character analysis of C-terminal anchoring segment from Influenza A hemagglutinin. 1653 51

Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. epsilon-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield, e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes.
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PMID:Separation and purification of enzymes by continuous pH-parametric pumping. 1855 91

In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC-MS(n). Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS-PAGE and LC-MS(n). Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC-MS(n) profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research.
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PMID:Development of continuous microwave-assisted protein digestion with immobilized enzyme. 2453 Mar 98