Inhibition Of Β-galactosidase Research Articles

Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase: a cautionary note on use of xylE reporter fusions under aerobic conditions.

Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H(2)O(2)) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding beta-galactosidase) or -xylE fusion plasmids to H(2)O(2) stimulated beta-galactosidase activity, while there was little or no detectable C23O activity in these strains. More than 95% of C23O activity was lost after a 5-min exposure to equimolar H(2)O(2), while a 10,000-fold excess was required for similar inhibition of beta-galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H(2)O(2) nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity. Our results suggest using caution in interpreting data derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.

Rapid and cost-effective multiparameter toxicity tests for soil microorganisms

Three biochemical parameters, DNA quantification in soil samples and two enzymatic activities, β-galactosidase and dehydrogenase have been assessed as potential end-points for the use in cost-effective toxicity tests on soil microorganisms. The assessment included the development of a classical dose–response 24-h assay and the incorporation of measurements of the effects on microbial activities in soil column leaching studies and multispecies miniaturised terrestrial systems (MTS). Four different chemicals, copper, a new herbicide, thiabendazole and fenthion were studied. A rapid fluorescence DNA quantification technique did not produce adequate responses. The efforts to quantify DNA after extraction and clean-up procedures failed due to the presence of humic acids. From the protocol of the technique one could see that the technical procedure is time-consuming and expensive and, for this reason, not suitable for use as a parameter in rapid and cost-effective tests. However, the enzymatic activities showed their potential as toxicity end-points. Copper produced a concentration/response inhibition of β-galactosidase and dehydrogenase with EC 50 values of 78.39 and 24.77 mg Cu/kg soil, respectively. In the soil column study, these endpoints allowed the measurement of the microbial activities through the column. The effects of the new herbicide on β-galactosidase and dehydrogenase activities were statistically significant for the highest application dose (40 g/ha). Thiabendazole affected the microbial activity when mixed within the soil, but no effects were observed when this fungicide was applied on the soil surface. Fenthion produced effects when applied either in the soil or on the soil surface. These results can be explained by the low mobility of thiabendazole. The results show the capabilities of these biochemical parameters to be included as endpoints in cost-effective bioassays.

Solvent Deuterium Isotope Effect on the Binding of β-d-Galactopyranosyl Derivatives to β-Galactosidase (Escherichia coli, lac Z)

A value of 1.8 has been determined for (KI)HOH/(KI)DOD, the ratio of the values of KI for competitive inhibition of β-galactosidase by isopropyl β-d-thiogalactopyranoside in H2O and D2O. This is similar to the value of 1.7 for (Km)HOH/(Km)DOD, the ratio of the Michaelis constants determined for the β-galactosidase-catalyzed hydrolysis of 4-nitrophenyl β-d-galactopyranoside (Gal-OPNP) in H2O and D2O. The similarity of these solvent deuterium isotope effects suggests that the observed isotope effect on Km corresponds, mainly, to the isotope effect on the dissociation constant Kd for Gal-OPNP. The implications of these results for the interpretation of the solvent deuterium isotope effects on kcat and kcat/Km for β-galactosidase-catalyzed hydrolysis of Gal-OPNP is discussed.

Total Asymmetric Synthesis of Doubly Branched Carba-hexopyranoses and Amino Derivatives Starting from theDiels-Alder Adducts of Maleic Anhydride to Furfuryl Esters

The Diels-Alder adducts of maleic anhydride to furfuryl esters were reduced into 7-oxabicyclo[2.2.1]hept-5-ene-1,2-exo,3-exo-trimethanol (±)-15 and enantiomerically pure (−)-15 (Scheme 1). The tripivalate of (±)-15 was converted into (1RS,2RS,3RS,4RS,5SR,6SR)-1,5,6-tris(hydroxymethyl)cyclohexane-1,2,3,4-tetrol ((±)-23; Scheme 2). Reaction of BBr3 with the triacetate (±)-30 of (±)-15 gave (1RS,2RS,5RS,6RS)-5-bromo-6-hydroxycyclohex-3-ene-1,2,3-trimethyl triacetate ((±)-31) at −78°, and (1RS,2RS,5SR,8SR)-2-endo-hydroxy-6-oxabicylo[3.2.1]oct-3-ene-5,8-dimethyl diacetate ((±)-32) at 0° (Scheme 3). Single-crystal X-ray diffraction of (1RS,2RS,5SR,8SR)-2-acetoxy-6-oxabicyclo[3.2.1]oct-3-ene-5,8-dimethyl diacetate ((±)-33) was carried out. Displacement of bromide (+)-31 (derived from (−)-15) with azide anion gave (+)-38 which was transformed into (+)-(1R,2R,5S,6S)-5-amino-6-hydroxycyclohex-3-ene-1,2,3-trimethanol ((+)-40) (Scheme 4). Reaction of (±)-31 with BBr3 at 0°, followed by azide disubstitution led to (1RS,2RS,5SR,6SR)-5-amino-3-(aminomethyl)-6-hydroxycyclohex-3-ene-1,2-dimethanol ((±)-45). Dihydroxylation of (±)-38 and further transformations gave (1RS,2RS,3SR,4RS,5SR,6RS)-5-amino-1,4,6-trihydroxycyclohexane-1,2,3-trimethanol ((±)-49) and (1RS,2RS,3SR,4RS,5SR,6RS)-2,3-dihydroxy-7-oxabicyclo[4.1.0]heptane-2,3,4-trimethanol ((±)-55) (Schemes 5 and 6). Expoxidation of the 4-nitrobenzoate (±)-61 of (±)-38 allowed the preparation of (1RS,2RS,3SR,4RS,5RS)-5-amino-1,4-dihydroxycyclohexane-1,2,3-trimethanol ((±)-65) and of (1RS,2RS,3SR,4RS,5SR,6RS)-5-amino-4-hydroxy-7-oxabicyclo[4.1.0]heptane-1,2,3-trimethanol ((±)-67) (Scheme 7). The new unprotected polyols and aminopolyols were tested for their inhibitory activity toward commercially available glycohydrolases. At 1 mM concentration, 34, 30, and 31% inhibition of β-galactosidase from bovine liver was observed for (+)-40, (±)-65, and (±)-67, respectively.

Synthesis of α-C(1→3)-mannopyranoside of N-acetylgalactosamine, a new β-galactosidase inhibitor

Radical C-glycosidation of a 3-methylidene-7-oxabicyclo[2.2.1]heptan-2-one derivative with acetobromomannose gave a α-C-mannopyranoside that was converted into α-D-Man pCH 2(1→3)-D-GalNAc, a C-disaccharide that inhibits β-galactosidase from jack bean with IC 50 = 9.4 μM K i = 7.5 μM (mixed mode of inhibition).

Inhibition of Glycosidases by Lactam Oximes: Influence of the Aglycon in Disaccharide Analogues

The influence of a substituent at the hydroximo function of the lactam analogue 1 on the inhibition of β- and α-glucosidases is evaluated. In contrast to 1, the O-alkyl oximes 5, 6, 9, and 10 are selective inhibitors of β-glucosidases. Alkylation of the D-gluconohydroximo-1,5-lactam 19 with the triflate 12, or condensation of the thiogluconolactam 20 with the hydroxylamines 14 or 18 afforded the benzylated cellobioside analogues 21 and 23, respectively. The O-alkyl oximes 33 and 39 were prepared similarly (Scheme 3). Deprotection afforded the cellobioside analogues 5 and 6, and the O-alkyl oximes 9 and 10. The lactam O-alkyl oximes 5, 6, 9, and 10 are strong inhibitors of the β-glucosidase from C. saccharolyticum (IC50=0.3 – 8 μM) and, with exception of the dodecyl analogue 9 (IC50=2 μM), moderate-to-weak inhibitors of β-glucosidases from sweet almond (IC50=60 – 1000 μM; see Table). In contrast to the strong inhibition of α-glucosidase from brewer's yeast by 1 (Ki=2.9 μM), the ethers 5, 6, and 10 are weak inhibitors of this enzyme (IC50 between 2500 and >5000 μM). Similarly, the D-galactohydroximo-1,5-lactam 7 is a potent inhibitor of the α-galactosidase from coffee beans and of the β-galactosidases from bovine liver and E. coli (Ki=5, 10, and 0.1 μM, resp.), while the lactoside analogue 8 is a strong inhibitor of the E. coliβ-galactosidase (Ki=0.1 μM), but a moderate-to-weak inhibitor of coffee-bean α-galactosidase and bovine-liver β-galactosidase (Ki=250 μM and IC50=2500 μM, resp.). The galacto-configured lactam oximes 7 and 8 are good inhibitors of the β-glucosidase isolated from C. saccharolyticum (Ki=2.5 and 3.3 μM, resp.).

Galactose competitive inhibition of β-galactosidase ( Aspergillus oryzae) immobilized on chitosan and nylon supports

Contrasting results have been published about the nature of galactose inhibition of β-galactosidase. In view of lactose hydrolysis in milk, we studied the inhibition of β-galactosidase ( Aspergillus oryzae) immobilized on chitosan beads or a nylon membrane (Immunodyne). In both cases, competitive inhibition has been found. The K i value for the β-galactosidase/chitosan system was higher than the one for the β-galactosidase/Immunodyne, showing that the former system is more appropriate to perform lactose hydrolysis. A new technology based on the use of nonisothermal bioreactors is suggested to overcome the inhibition problems.

Escherichia coli β-Galactosidase Recognizes a High-Energy Conformation of C-Lactose, a Nonhydrolizable Substrate Analogue. NMR and Modeling Studies of the Molecular Complex

The enzyme-bound conformation of C-lactose, an Escherichia coli β-galactosidase inhibitor has been determined by NMR spectroscopy. It is demonstrated that the enzyme selects a high-energy conformation of this closely related structural analogue of the natural substrate, lactose. In addition, a molecular modeling protocol has been performed in order to obtain a detailed three-dimensional structure of the complex that can explain, in structural terms, the role that the key amino acid residues play in the catalytic mechanism. The implications of the recognition of a high-energy conformation of the analogue are also outlined.

Galactocerebroside and not glucocerebroside or ceramide stimulate epidermal β-glucocerebrosidase activity

Glycosphingolipids including glucocerebroside (GluCer) and galactocerebroside (GalCer) have been recognized as bioreguratory lipids by our group and others. In addition, our recent study demonstrated that GalCer corrects dry skin conditions in humans. The processing of stratum corneum lipids, which occurs when β-glucocerebrosidase (β-GluCer'ase) changes GluCer to ceramide (Cer), is required to form the epidermal permeability barrier. We herein investigated the effects of GluCer, GalCer and Cer on the processing of GluCer to Cer by assaying epidermal β-CluCer'ase in mice (155%, P < 0.01) when compared to vehicle treated controls, while neither GluCer nor Cer had this effect. Studies using inhibitors of β-GluCer'ase or β-galactosidase and measuring the optimum pH of the enzyme verified that GalCer specifically activated β-GluCer'ase. We confirmed that GalCer significantly increased β-GluCer'ase activity in the outer epidermal fraction (172%, P < 0.01) and that the activation of β-GluCer'ase is not due to a direct activating effect of GalCer on the enzyme. Furthermore, the induction of β-GluCer'ase activity by GalCer was also observed in cultured normal human deratinocytes (123%, P < 0.01). Finally, acylceramide content in stratum corneum was increased in mice treated with GalCer (194%, P < 0.0005). These results indicate that GalCer appears to affect the Cer construct in the stratum corneum by the activation of β-GluCer'ase, which ultimately contribute to an enhancement of barrier formation.

Calystegine alkaloids from Duboisia leichhardtii

Pentahydroxy- nor-tropane alkaloids, calystegines C 1 and C 2, were isolated from the leaves of greenhouse-cultivated Duboisia leichhardtii, together with calystegines B 1, B 2 and B 4. Calystegine C 1 is a potent inhibitor of β-glucosidase and β-galactosidase, whereas calystegine C 2 exhibited no significant inhibitory activity against these enzymes. Only calystegine C 2, the 2-epimer of calystegine C 1, among calystegines isolated to date exhibited an inhibitory activity against all α-mannosidases tested.

A Solid-Phase Enzyme-Linked Assay for Ceramide Glycanase Using GM1 and a Novel β-Galactosidase Inhibitor

A Solid-Phase Enzyme-Linked Assay for Ceramide Glycanase Using GM1 and a Novel β-Galactosidase Inhibitor

Inhibition of Escherichia coli β-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl)ethyl, a photoreversible thiol label

1-Nitro-2-phenylethene (β-nitrostyrene, 1), which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817–818), was demonstrated in this work to be an irreversible inhibitor of β-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or though modifying a methionine residue at the active site. No reversal of the inhibition was observed upon subsequent incubation with mercaptoethanol or irradiation (350 nm). 1-(4,5-dimethoxy-2-nitrophenyl)-2-Nitroethene ( 2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. K cat values of β-galactosidase at pH 8.3 with o-nitrophenyl β- d-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 · 10 −4 M) ranged from 1.67 · 10 4 s −1 after 30 min of preincubation to < 0.07 · 10 4 s −1 after 180 min preincubation. For compound 2 (9.5 · 10 −5 M) K cat values ranged from 2.70 · 10 4 s −1 following 30 min preincubation to 1.15 · 10 4 s −1 after 180 min of preincubation; the changes in K m app, however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compound 2 and the inhibited enzyme are 2-nitrobenzyl derivatives, they are expected to be photosensitive and indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity.

P-461 Possible involvement of transforming growth factor-?1 in the immunosuppression in patients with advanced malignant diseases

A second indolizidine alkaloid, epimeric with castanospermine, has been isolated from seeds of the Australian tree Castanospermum australe. The structure was established as 6-epicastanospermine by proton and carbon-13 nuclear magnetic resonance spectroscopy and mass spectrometry. 6-Epicastanospermine was found to be a potent inhibitor of amyloglucosidase, (an exo-1,4,-α-glucosidase), a weak inhibitor of β-galactosidase, and not to inhibit β-glucosidase and α-mannosidase. These results indicate that glycosidase inhibitory activity cannot be predicted by comparison of the structure and stereochemistry with the appropriate sugars, since 6-epicastanospermine is an analog of mannose and not of glucose. The inhibition of amyloglucosidase was found to be competitive and to be more effective at higher pH values. Castanospermine and 6-epicastanospermine differed in their effect upon the mung bean processing enzymes, glucosidase I and II, in that the former is a potent inhibitor whereas the latter is a very poor inhibitor. Subtle alterations in stereochemistry of these alkaloids can therefore produce significant changes in their biological activity.

P-462 Changes of immunological parameters after hepatectomy for hepatocellular carcinoma and effects of ulinastatin

A second indolizidine alkaloid, epimeric with castanospermine, has been isolated from seeds of the Australian tree Castanospermum australe. The structure was established as 6-epicastanospermine by proton and carbon-13 nuclear magnetic resonance spectroscopy and mass spectrometry. 6-Epicastanospermine was found to be a potent inhibitor of amyloglucosidase, (an exo-1,4,-α-glucosidase), a weak inhibitor of β-galactosidase, and not to inhibit β-glucosidase and α-mannosidase. These results indicate that glycosidase inhibitory activity cannot be predicted by comparison of the structure and stereochemistry with the appropriate sugars, since 6-epicastanospermine is an analog of mannose and not of glucose. The inhibition of amyloglucosidase was found to be competitive and to be more effective at higher pH values. Castanospermine and 6-epicastanospermine differed in their effect upon the mung bean processing enzymes, glucosidase I and II, in that the former is a potent inhibitor whereas the latter is a very poor inhibitor. Subtle alterations in stereochemistry of these alkaloids can therefore produce significant changes in their biological activity.

Preparations and reactions of acylated and partially acylated glycosyl fluorides

Partial acylation of glycosyl fluorides in the galacto (2 and 3) and gluco series (8–10) is readily achieved. Attempts to further alkylate these acyl derivatives failed. Treatment of 2 with Lewis acid, however, provided a facile route to the 1,4-anhydro derivative 5. Lewis acid-mediated glycosylation of 2-acetamido-3,4,6-tri- O-acetyl-α- d-glucopyranosyl fluoride 11 led to the simple glycosides and thioglycosides 12–16. Similarly, in the galacto series (17) an advantageous route to the β-galactosidase inhibitor 19 could be elaborated.

Nocompetitive inhibition of β-galactosidase ( A. oryzae) by galactose

Inhibition of β-Galactosidase (EC 3.2.1.23, β-galactoside galactohydrolase) by galactose was sutdied using a wide range of substrate and inhibitor concentrations. Contrary to the published reports, a noncompetitive inhibition was observed. The importance of inhibition type is discussed in relation to enzyme reactor modeling.

Synthwsis of (+)-Galactostatin

The chiral synthesis of (+)-galactostatin, a new β-galactosidase inhibitor, has been achieved, in which the key step involved a diastereoselective epoxidation of the allylic alcohol derived from L-tartaric acid.

The Structures of aβ-Galactosidase Inhibitor, Galactostatin, and Its Derivatives

The structure of the β-galactosidase inhibitor, galactostatin, isolated from Streptomyces lydicus PA-5726 was determined to be 5-amino-5-deoxygalactopyranose, in which the ring oxygen of galactose has been replaced by nitrogen. For this study, two derivatives of galactostatin were prepared by oxidation and reduction, respectively. The oxidation product of galactostatin was 5-amino-5-deoxygalactonic-δ-lactam (galactostatin-lactam) and the reduction product was 5-amino-1,5-dideoxygalactopyranose (1-deoxygalactostatin).

Isolation and properties of a new .BETA.-galactosidase inhibitor, galactostatin, from Streptomyces lydicus.

A new β-galactosidase inhibitor was found with a newly developed rapid and practical screening method for β-galactosidase inhibitors. Several strains of Actinomycetes that produced β-galactosidase inhibitors were isolated from soil samples using this method, and Streptomyces lydicus PA-5726 was found to produce a high concentration of new inhibitor in the culture medium. Improvement of the medium led to 20~25-fold augmentation (45 to 900~1,100 μg/ml) of the production. This new inhibitor, which was named galactostatin, was isolated by adsorption on Dowex-50W × 8 (H+), crystallization from the bisulfite adduct, development on Dowex-2 × 8 (OH−) and precipitation with EtOH. Galactostatin displayed strong inhibitory activity toward several β-galactosidases in acidic and neutral media but low acute toxicity in mice (LD50 1016 mg/kg, i.v.).

The structures of a .BETA.-galactosidase inhibitor, galactostatin, and its derivatives.

The structure of the β-galactosidase inhibitor, galactostatin, isolated from Streptomyces lydicus PA-5726 was determined to be 5-amino-5-deoxygalactopyranose, in which the ring oxygen of galactose has been replaced by nitrogen. For this study, two derivatives of galactostatin were prepared by oxidation and reduction, respectively. The oxidation product of galactostatin was 5-amino-5-deoxygalactonic-δ-lactam (galactostatin-lactam) and the reduction product was 5-amino-1,5-dideoxygalactopyranose (1-deoxygalactostatin).