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)

Ultraviolet light-inactivated, non-infectious influenza virus is pyrogenic; virion components are probably responsible for this pyrogenicity. To try to identify the pyrogenic component, influenza virions were disrupted with either bromelain or sodium deoxycholate (DOC). Treatment of infectious virions with bromelain, under conditions that removed the surface glycoproteins (spikes), destroyed their pyrogenicity. The supernatant, containing non-aggregated and modified glycoproteins, was also non-pyrogenic. Disruption of virions with DOC considerably reduced pyrogenicity; however, some was retained by the sub-viral cores. Viral nucleoprotein and matrix protein, purified from the supernatant, were non-pyrogenic. Aggregated stellate clusters of surface glycoproteins separated on sucrose gradients were pyrogenic in half of numerous tests performed with different batches of material. Treatment of virus with ether resulted in complete loss of pyrogenicity. Liposomes made from extracted viral lipid were non-pyrogenic. In contrast, virosomes made from the viral lipid and the aggregated stellate clusters of surface glycoproteins were pyrogenic. Hence, optimum pyrogenicity depends upon the integrity of the virus particle, but haemagglutinin and/or neuraminidase appear essential, and lipid may be involved.
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PMID:Influenza virus pyrogenicity: central role of structural orientation of virion components and involvement of viral lipid and glycoproteins. 160 57

A431 human epidermoid carcinoma cells monophenotypically express the placental alkaline phosphatase (PLAP)-like enzyme shown by its catalytic and antigenic characteristics, properties which are shared by the Nagao isozyme. More specifically, it is L-leucine sensitive just as is the rare placental D-variant of PLAP and the testicular heat-stable enzyme. Collectively, these are all referred to as PLAP-like enzymes. The enzyme was localized to the surface of the plasma membrane since it was released in an active form by bromelain treatment of cells. The number of molecules per A431 cell was estimated by radioimmunoassay at 7.5 X 10(5), a value significantly higher than that observed for HeLa TCRC-1 cells (5 X 10(4) which express the S-variant of PLAP, also referred to as the Regan isozyme. The quantity of the enzyme was increased significantly (10-fold) by treating the cells with modulating agents including sodium butyrate, prednisolone, and hyperosmolar sodium chloride. The identification of a cell line such as A431 with enhanced expression in the amount of the PLAP-like enzyme and which can be further enhanced by modulating agents will facilitate studies of the differences and the similarities between this protein and other variants of PLAP. The A431 cell line now takes its place with other cell lines which are phenotypically restricted in their expression of alkaline phosphatase. Finally, the A431 cell line is also shown here to be a suitable model system for in vivo tumor studies such as immunolocalization.
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PMID:Characterization of the placental alkaline phosphatase-like (Nagao) isozyme on the surface of A431 human epidermoid carcinoma cells. 257 98

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.
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PMID:The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis. 284 68

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.
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PMID:Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H. 293 33

Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either 3H-fatty acids or [3H]ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the 3H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of [3H]ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from 3H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the 3H-fatty acid and the [3H]ethanolamine label from the purified alkaline phosphatase. Subtilisin was also able to remove the [3H]ethanolamine-labeled from purified alkaline phosphatase. The 3H radioactivity in alkaline phosphatase purified from [3H]ethanolamine-labeled cells comigrated with authentic [3H]ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the 3H-fatty acid and [3H]ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.
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PMID:Phosphatidylinositol anchor of HeLa cell alkaline phosphatase. 367 79

1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N(2)-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500+/-1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500+/-1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400+/-1400. 7. The N-terminal amino acid composition is 0.64+/-0.04mol of valine and 0.36+/-0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis.
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PMID:The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-L-phenylalanyl-L-serine methyl ester. 446 42

Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-(14)C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and alpha-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin.
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PMID:The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain. 542 46

Recent evidence suggests that gamma-glutamyl transpeptidase may be involved in the transport of amino acids into the lactating mammary gland. The enzyme also is secreted in milk and is associated mainly with milk membranes. The objective of this study was to purify and characterize gamma-glutamyl transpeptidase from milk membranes. The enzyme has been purified from milk membranes by solubilization with Lubrol WX; treatment with acetone, deoxylcholate, and bromelain; and chromatography on ion exchange and molecular-sieving resins. gamma-Glutamyl transpeptidase was purified over 11,000-fold from milk. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicates that the enzyme is composed of two subunits with molecular weights of 57,000 and 25,500. Both subunits are glycoproteins and have been identified in the sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of whole milk membrane. Kinetic characteristics of the purified enzyme are similar to those determined for intact milk membranes and lactating mammary tissue indicating that the purified enzyme has not been modified functionally by the purification procedure.
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PMID:Purification and identification of gamma-glutamyl transpeptidase of milk membranes. 610 10

gamma-Glutamyltransferase was solubilized from human hepatoma tissues by bromelain treatment, and some of its properties were compared with those of the normal adult liver enzyme. An electrophoretic study showed a slightly different mobility between the two enzymes before and after neuraminidase treatment. The hepatoma tissue enzyme was distinguished from the normal liver enzyme by decreased affinity to Con A. However, the enzymes from the two sources were found to be very similar or identical with respect to molecular weight, Michaelis constant, pH optimum, thermostability, effect of various L-amino acids as acceptors, behavior to divalent cations or ethylenediaminetetraacetate, inhibition by urea or sodium dodecyl sulfate, and immunological properties. These results suggest that the hepatoma tissue gamma-glutamyltransferase is largely due to altered glycosylation of this glycoprotein in hepatoma cells.
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PMID:gamma-Glutamyltransferase from human hepatoma tissue in comparison with normal liver enzyme. 611 82

By digestion of detergent-solubilized gamma-glutamyltransferase (GGT), isolated from bovine kidney with bromelain, the liberation of 4 protein fragments was demonstrated. The fragment migrating most quickly in gel electrophoresis showed gamma-glutamyltranspeptidase activity and the most slowly migrating fragment showed peptidase activity. Protease-solubilized GGT is a sialoprotein with a molecular weight of 95,000. After treatment with sodium dodecylsulfate it was separated into two unequal subunits with molecular weights of 26,000 and 69,000. Sugar components were found only in the heavy subunit. Some catalytic differences were found between the two solubilized GGT forms. The immunoprecipitate obtained from detergent-solubilized GGT retained about 50% of the initial enzyme activity. The enzyme is inactivated with phenylmethanesulfonyl fluoride in the presence of maleate and with 6-diazo-5-oxo-L-norleucine.
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PMID:Bovine kidney gamma-glutamyltransferase. Solubilized forms, biochemical and immunochemical properties. 615 97


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