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)

Human kidney gamma-glutamyl transpeptidase has been purified by a procedure involving Lubrol extraction, acetone precipitation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-150. The final preparation is a glycoprotein (molecular weight of approximately 84,000) composed of two nonidentical glycopeptides (molecular weights of 62,000 and 22,000). The isozymic forms, separable by isoelectric focusing, have different contents of sialic acid. The utilization of L-glutamine (which is both a gamma-glutamyl donor and acceptor) is stimulated about 3-fold by maleate in contrast to 10-fold stimulation of glutamine utilization by the rat kidney enzyme. The gamma-glutamyl analogs, 6-diazo-5-oxo-L-norleucine (DON) and L-azaserine inactivate the human kidney enzyme with respect to its transpeptidase and hydrolase activities. Inactivation is prevented by gamma-glutamyl substrates (but not by acceptor substrates) and is accelerated by maleate. [14C]DON reacts covalently and stoichiometrically at the gamma-glutamyl site, which was localized to the light subunit of the enzyme. The light subunit of human transpeptidase closely resembles that of rat kidney enzyme in having the gamma-glutamyl binding site, and similar molecular weight and amino acid composition. The heavy subunits of the two enzymes are markedly different in both molecular weight and amino acid content; this may account for differences observed in acceptor amino acid specificity and in the magnitude of the maleate effect.
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PMID:Human kidney gamma-glutamyl transpeptidase. Catalytic properties, subunit structure, and localization of the gamma-glutamyl binding site on the light subunit. 1 63

Gamma-Glutamyl transpeptidase was purified from rat kidney by a procedure involving Lubrol extraction, acetone precipitation, ammonium sulfate fractionation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-100. The final preparation (enzyme III), which exhibits a specific activity about 8-fold higher than that of the purified rat kidney transpeptidase previously obtained in this laboratory (enzyme I), was apparently homogeneous on polyacrylamide gel electrophoresis. Enzyme III is a glycoprotein containing 10% hexose, 7% aminohexose, and 1.5% sialic acid; a tentative molecular weight value of about 70,000 was obtained by gel filtration. Enzyme III has a much lower molecular weight and a different amino acid and carbohydrate content than the less active rat kidney transpeptidase preparation previously obtained, but obtained, but the catalytic properties of these preparations are virtually identical. It is suggested that bromelain treatment may liberate the transpeptidase from a brush border complex that contains other proteins. An improved method is described for the isolation of the higher molecular weight form of the enzyme (enzyme I) in which affinity chromatography on concanavalin A-Sephrose is employed. The purified transpeptidase (enzyme III) is similar to the phosphate-independent maleate-stimulated glutaminase preparation obtained from rat kidney by Katunuma and colleagues with respect to amino acid and carbohydrate content, apparent molecular weight, and relative transpeptidase and maleate-stimulated "glutaminase" activities. Both of these enzyme preparations are much more active in transpeptidation reactions with glutathione and related gamma-glutamyl compounds than with glutamine. In the absence of maleate, the enzyme catalyzes the utilization of glutamine (by conversion to gamma-glutamylglutamine, glutamate, and ammonia) at about 2% of the rate observed for catalysis of transpeptidation between glutathione and glycylglycine; the utilization of glutamine occurs about 8 times more rapidly in the presence of 0.1 M maleate. The transpeptidation and maleate-stimulated glutaminase reactions catalyzed by both enzyme preprations are inhibited by 5 mM L-serine in the presence of 5 mM sodium borate. Studies on gamma-glutamyl transpeptidase and maleate-stimulated glutaminase in the kidneys of fetal rats, newborn rats, and rats after weaning showed parallel development of these activities. The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase. The studies on the ontogeny of gamma-glutamyl transpeptidase and other data are considered in relation to the proposal that this enzyme is involved in amino acid and peptide transport. Its possible role in renal formation of ammonia is also discussed.
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PMID:Identity of maleate-stimulated glutaminase with gamma-glutamyl transpeptidase in rat kidney. 23 5

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

A kininogen-like protein was purified from Bothrops jararaca plasma by DEAE-Sephadex ion-exchange and carboxy-methyl-papain-Sepharose affinity chromatography. The molecular weight, estimated by SDS-gel electrophoresis, is about 100,000 and a species of about 75,000 is formed after incubation with horse urinary kallikrein. After incubation with trypsin, only traces of biological activity were detected in tests on guinea pig ileum. The purified protein inhibits papain and bromelain, does not correct the clotting time of a kininogen-depleted human plasma, and does not affect the clotting time of plasma from Waglerophis merremii, a nonpoisonous snake; the same type of inhibitor was found in this nonpoisonous snake. The dissociation constant (Ki) for the papain-inhibitor complex is approximately 1.6 nM.
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PMID:A Bothrops jararaca plasma cysteine-proteinase inhibitor related to mammalian kininogen. 263 47

Lachesis muta snake venom induced aggregation of bromelain sensitized human erythrocytes at a concentration of 1 mg/ml. The hemagglutinating protein was purified by DEAE-Sephadex A-50 column chromatography. Polyacrylamide gel electrophoresis revealed at least three bands, whereas SDS electrophoresis in the presence of 2-mercaptoethanol showed a single one. Isoelectric focusing revealed hemagglutinating activity in the range of pH 3-8. The maximum peak (mutina) at pH 5.5. This fraction was active in agglutinating human RBC of types A, B, O Rh (+) and B, O Rh (-). One mM EDTA and 1 mM Ca++ did not alter the agglutinating time significantly. Lactose and inositol inhibited the agglutination of A, B, O Rh (+) and B, O Rh (-) human RBC. The present study showed the non specificity of the hemagglutinating activity of mutina. It was also shown that mutina is a non-mitogenic protein.
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PMID:Purification and some properties of hemagglutinating protein mutina from bushmaster Lachesis muta snake venom. 367 7

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

An endogenous inhibitor of calcium-activated neutral protease (CANP), which was isolated from rabbit skeletal muscle under mild conditions, comprised high- and low-molecular-weight components. The latter (LMW-inhibitor; Mr=50,000) was purified to homogeneity by means of chromatography on DEAE-cellulose and phenyl-Sepharose CL-4B and chromatofocusing. The purified inhibitor is a protein composed of two polypeptide chains with molecular weights of 26,000 and 24,000 daltons. It contains large amounts of glutamic acid, alanine, and serine, and small amounts of aromatic amino acids. It was specific for CANPs having low (m-type) and high (mu-type) Ca2+-sensitivity, had no effect on any other protease examined (trypsin, alpha-chymotrypsin, bromelain, ficin, papain, thermolysin, etc.), and inhibited rabbit mCANP more effectively than rabbit muCANP or chicken mCANP. It was demonstrated that the inhibition is due to the formation of a stoichiometric complex between two molecules of rabbit mCANP and one inhibitor molecule.
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PMID:Purification and characterization of an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle: purification of 50,000-dalton inhibitor. 609 76

An endogenous inhibitor of calcium-activated neutral protease was purified to homogeneity from rabbit skeletal muscle using ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex A-50 columns, chromatofocusing, and hydrophobic interaction chromatography on a phenyl-Sepharose CL-4B column. The purified inhibitor was shown to be a dimer of identical subunits and each subunit has a molecular weight of about 34,000. This inhibitor was remarkably thermo- and acid-stable. It was specific for calcium-activated neutral protease and had no effect on any other protease examined (trypsin, papain, alpha-chymotrypsin, bromelain, etc.). It is demonstrated that the inhibition is due to the formation of stoichiometric complex between two enzyme molecules and one inhibitor molecule.
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PMID:Purification and characterization of an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle. 627 75

The glycopeptidase preparation that has been isolated from almond emulsin and acts on beta-aspartylglycosylamine linkages in glycopeptides was separated into three active fractions by DEAE-cellulose column chromatography. The three discrete species of glycopeptidase (Groups A, B and C) have been purified 30-, 136-, and 99-fold, respectively. The optimum pH value of Group A was 6.0 and those of Groups B and C, 5.0. Isoelectric points of Groups A, B and C were pH 7.7, 8.6 and 8.7, respectively. All three glycopeptidases hydrolyzed quantitatively glycopeptides with 3-11 amino acid residues prepared from stem bromelain, ovalbumin and ovotransferrin. Group C preferred glycopeptides with shorter peptide chains, whereas Groups A and B preferred those with longer chains. Glycopeptidase Group A also hydrolyzed intact glycoproteins such as stem bromelain, ovalbumin, Taka-amylase A and desialylated human transferrin.
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PMID:Almond glycopeptidase acting on aspartylglycosylamine linkages. Multiplicity and substrate specificity. 721 57

The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAE-Sephadex, trypsin-Sepharose (removal of trypsin inhibitors), Mono Q and a reversed-phase C4 column. The primary structure is homologous to other plant Kunitz-type inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slow-tight binding mechanism with a low dissociation constant (Ki 1.2 nM). The studied Bauhinia protein also inhibits cruzain (Ki 0.3 nM), a C-terminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (Ki 0.22 nM), but not cathepsin B, papain, bromelain or ficin.
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PMID:Structure of cruzipain/cruzain inhibitors isolated from Bauhinia bauhinioides seeds. 1151 40


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