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)

The protease isolated jawasee shrub was found to hydrolyze egg albumin, casein, haemoglobin and gelatin optimally near neutral pH. Fibrin, bovin serum albumin, skin albumin and skin mucoids were hydrolyzed at slightly alkaline pH, while skin globulins were hydrolyzed at slightly acidic pH. The enzyme had no effect of fibrous collagen. The optimum conditions for the hydrolysis of 50 mg of egg albumin were found to be 50 mg of alhagain at pH 6.0 and 45 degrees C for 30 minutes. A Km value of 4.4 X 10(-3) M was obtained from the Lineweaver-Burk plot for the hydrolysis of egg albumin. The enzyme was found to be comparatively thermostable and was most stable at pH 4.7. Ultraviolet irradiation exhibited no appreciable effect on the enzyme activity. The ultraviolet absorption spectrum of alhagain in bi-distilled water resembles those of bromelain and trypsin. The sugar-containing enzyme was found to have a molecular weight of 20,650. The enzymeconsists of 189 amino acid residues per molecule, neutral and acidic amino acids being present in high concentrations. The partial specific volume of alhagain was calculated to be 0.743 ml/g from its amino acid composition. Phenylalnine and arginine formed the amino terminal amino acids of alhagain, while aspartic acid and serine were identified as its carboxy terminal amino acids. Results are discussed with relation to other plant proteases.
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PMID:Studies on the physico-chemical properties of alhagain. 2 Nov 47

Ca(2+)-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial-Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or alpha(1)-casein were hydrolysed with a maximum rate at 30 degrees C, pH7.5, and with 5mm-CaCl(2), but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, alpha-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150-2158]. The Ca(2+)-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca(2+)-activated neutral proteinase.
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PMID:Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle. 53 1

Enzymatic hydrolysates of various cottonseed flours were prepared with the proteolytic enzymes bromelain, HT-200, Pronase, and trypsin. The growth of various aerobic bacteria of clinical significance in these hydrolysates was compared to that obtained with a standard casein-soybean peptone culture medium, Trypticase soy. The generation times of the majority of bacteria grown in the bromelain cottonseed flour hydrolysate were shorter than that obtained with the standard control broth. A bromelain cottonseed flour hydrolysate agar preparation supported the growth of the bacteria comparably to that of the casein-soybean agar substrate. All the bacterial colonies were larger on the bromelain cottonseed flour hydrolysate blood agar medium than those grown on the control agar. The peptones derived from the enzymatic hydrolysis of cottonseed flour are sufficient to promote the rapid and luxuriant growth of a wide spectrum of aerobic bacteria without the addition of peptone from other sources. It is suggested that cottonseed flour peptones be utilized as a nutrient source in general-purpose media for the clinical microbiology laboratory.
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PMID:Growth potential of cottonseed culture media for various clinically significant aerobic bacteria. 110 Jun 68

Pineapple stem bromelain was photooxidized in the presence of Methylene Blue used as a sensitizer. The essential sulfhydryl group of the enzyme protein rapidly became inaccessible to react with 5,5'-dithiobis(nitrobenzoic acid), but the reactivity was readily regained to the original level upon treatment with dithiothreitol. Even after such reduction, the photooxidized enzyme showed a markedly decreased hydrolytic activity on casein. Spectral examination revealed that the oxidized enzyme had tyrosine residues intact. Amino acid analysis showed significant decreases in histidine, ethionine, and tryptophan residues. Photoinactivation occurred in a similar manner also in the presence of tetrathionate which reversibly blocked the essential sulfhydryl group. It is concluded that the irreversible photoinactivation of stem bromelain must be related to the oxidation of histidine, methionine, and tryptophan residues. When the photooxidation was carried out a different pH values ranging from 4.0 to 8.3, the inactivation and the decrease in histidine content were found to be markedly pH dependent. Thus, the photooxidation experiment provided a method for directly measuring the apparent pKa of the ionization of the single histidine residue in stem bromelain. Apparent pKa values of 6.4 and 7.1 were obtained for the histidine imidazole in the absence and in presence of tetrathionate, respectively. In view of these normal pKa values for an imidazole, a mechanism of ionization of the active-site group in a plant thiol proteinase is proposed, in which the validity of mechanism involving a close electronic interaction between histidine and cysteine residues is seriously questioned.
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PMID:Photosensitized inactivation of stem bromelain. Oxidation of histidine, methionine, and tryptophan residues. 112 Jan

Because substrate specificities differ between proteolytic enzymes and because knowledge of the optimal enzyme activity levels is necessary in order to standardize procedures used in antibody screening, a study was made of the best common assay method for the routinely employed enzymes bromelain, papain and ficin. Casein degradation was found better suited to this purpose than azoalbumin. With standardization achieved, a useful two-phase bromelain inhibitor technique was devised using bromelain at 20 casein units of activity. This method improved upon the one-stage bromelain technique in terms of sensitivity, freedom from false positive reactions and it compared well with the two-phase papain inhibitor technique.
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PMID:Differences in substrate specificities for cysteine proteinases used in blood group serology, and the use of bromelain in a two-phase inhibitor technique. 267 50

To check whether crude stem and fruit bromelains can be fractionated further or not, systematic separation procedures were applied to both enzymes. Six proteolytically active components, which were designated as SBB 1-5 and SBA, were fractionated from crude stem bromelain by successive use of gel filtration on Sephadex G-75, and chromatographies on CM-Sephadex and DEAE-Sephacel. One main and one minor active components, designated as FBA and FBB, respectively, were also separated from crude fruit bromelain by chromatographies on DEAE-Sephacel and then CM-Sephadex. Some of the physico-chemical and enzymatic properties of these eight components were compared. Each component migrated as a single band on SDS-polyacrylamide gel electrophoresis. Molecular weights determined by the same electrophoresis were about 27,000 for SBB 1-3 and FBB, and about 23,000 for the other four components. In terms of amino acid composition, FBB resembled SBB 1-3, which were remarkably similar to each other. FBA was also similar to SBA in amino acid composition, and contained much less basic amino acids than SBB 1 through 5. The principal amino-terminal residues determined by the cyanate method were valine in SBB 1-5 and SBA, and alanine in FBA and FBB. The principal carboxyl-terminal residues determined by the hydrazinolysis method were glycine in SBB 1-3, SBA and FBA, and serine in SBB 4-5 and FBB. However, fractional amounts of a few other amino- and carboxyl-terminal residues were also detected. As regards enzymatic activities, FBA and SBB 4 and 5 were much more active than the other five components against casein and some synthetic substrates [Bz-Arg-amide (at pH 6.1), Z-Gly-X, and Z-Ala-X (at pH 3.5)] with the notable exception that FBA was much less active than SBB 4 and 5 toward tripeptides (X-Gly-Gly).
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PMID:Reinvestigation of fractionation and some properties of the proteolytically active components of stem and fruit bromelains. 404 51

The polyclonal B-cell response to Escherichia coli lipopolysaccharide was studied in C57BL/6 mice maintained after weaning on either a moderate protein-restricted diet with 8% casein or a normal diet. After in vitro or in vivo stimulation with the endotoxin, autoreactive and anti-hapten antibody-producing cells were quantitated by direct plaque assay, using bromelain-treated mouse erythrocytes and trinitrophenylated sheep erythrocytes as targets. Larger numbers of plaque-forming cells were generated in cultures of spleen cells from dietary-restricted than from normal mice stimulated with various doses of lipopolysaccharide. The number of background plaque-forming cells was also higher in nonstimulated spleen cell cultures from restricted animals. After injection of lipopolysaccharide in vivo, the number of cells producing antibodies to bromelain-treated mouse erythrocytes per 10(7) spleen cells was significantly increased in dietary-deficient mice. The results are discussed in relation to the different sensitivities of lymphocyte populations to protein deficiency and to the possible presence of high levels of endogenous polyclonal B-cell activators in the restricted mice.
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PMID:Polyclonal B-cell response to stimulation with Escherichia coli lipopolysaccharide in dietary protein restriction. 675 15

The in vitro effect of bongkrekic acid on stem bromelain, papain and ficin was studied. The hydrolysis of casein by these enzymes was inhibited by bongkrekic acid, but the inhibition was always incomplete even with a large excess of the effector. Using a fully activated specimen of stem bromelain, purified on an organomercurial agarose affinity column, the inhibition by bongkrekic acid was not stoichiometric. The SH group of cysteine remained intact after incubation with an excess of bongkrekic acid at 24 degrees C for 20 min. However, partial inhibition of stem bromelain by bongkrekic acid was reversed by incubation at 37 degrees C for 5 min with 5mM cysteine or 2-mercaptoethanol. Ethylene glycol and glycerol had no such restorative effect. These results indicate that molecules of bongkrekic acid are non-covalently bound to a thiol protease, only partially and reversibly shielding its essential SH group.
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PMID:Effect of bongkrekic acid, a product of Pseudomonas cocovenenans, on thiol proteases. 716 5

Young adult Sprague-Dawley rats were partially hepatectomized (two-thirds organ removal) and administered a basal diet supplemented with various animal- and plant-derived enzymes (trypsin, alpha-chymotrypsin, pepsin, lipase, alpha-amylase, malt diastase, ficin and bromelain) over a post-operative period of up to 10 days. Porcine or bovine dialyzed and lyophilized crystalline trypsin products containing 2400-3200 NF u/mg in addition to enteric-coated tablets with trypsin to chymotrypsin in a ratio of 6:1, were tested at supplementary levels of up to 4980 u/g ration. With the weight of tissue regenerated or the liver increment as indicator, trypsin in excess of 1000-1200 u/g ration proved inhibitory. This effect did not extend to alpha-chymotrypsin (levels of up to 4000 u/g diet) and the remaining 6 enzyme products specified above, nor to the s.c. injection of trypsin daily at 12,860 u/rat for the 1st 7 days. The last route promoted little change in increment with soy bean trypsin inhibitor (8.0 mg/rat daily for days 1 to 9). When a portion of the group fed a trypsin supplement of 2000 u/g was injected with phenobarbital i.p. at 80 mg/kg daily on each of the last 3 days, the resulting liver increment rose to the control range. As with lysine and arginine, acids of pertinence in tryptic proteolysis, no significant change was elicited by feeding a diet supplemented with peptone from tryptic digestion of casein. The enzyme-containing diets fed to sham-operated rats over a similar interval, did not affect the wet- or dry-liver weight per 100 g body weight. Microsomal parameters as total protein, cytochrome P-450 and the enzymes, aminopyrine demethylase and benzo[a]pyrene hydroxylase of livers from the partially hepatectomized or sham-operated rats fed trypsin and the other enzyme diets, presented no significant changes in the respective levels. The possible action of dietary trypsin in conjunction with inhibitors and growth factors controlling liver regeneration is discussed.
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PMID:Liver regeneration in trypsin-fed partially hepatectomized rats. 843 34

A new proteolytic assay is described involving Coomassie blue. Under specified conditions, the amount of Coomassie-stained casein protein hydrolyzed by several proteases was proportional to the amount of protease. Coomassie dye reaction was used directly to determine the change in protein concentration of the substrate casein during proteolysis by three proteases: stem bromelain, papain, and trypsin. This method can be used with 0.1- to 0.5-micrograms quantities of protease. The dye reagent was used directly on the protease protein in order to obtain an assay of autodigestion. Autodigestion of bromelain at 50 and 25 degrees C was followed by measuring the amount of residual protease protein with time.
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PMID:Protease activity and autodigestion (autolysis) assays using Coomassie blue dye binding. 848 11


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