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PubMed1984

Isolation of a homogeneous glucosidase II from pig kidney microsomes.

The processing of the oligosaccharide precursor chain, (GlcNAc)2(Man)9(Glc)3, of N-glycosylated glycoproteins starts with the action of glucosidase I which excises the terminal (alpha 1-2)-linked glucose residue. Glucosidase II removes the two inner (alpha 1-3)-linked glucose residues. We have purified glucosidase II to homogeneity from pig kidney microsomes. The enzyme is a glycoprotein and contains a single type of subunit of molecular mass approximately equal to 100 kDa. The native enzyme is probably a tetramer. It cleaves glucosidic alpha 1-3 and alpha 1-4, but not alpha 1-1, alpha 1-2 or alpha 1-6 bonds and lacks alpha-mannosidase and glucosidase I activity. The pH optimum is between 6.0 and 7.5. Specific antibodies against the native enzyme and the denatured subunit were prepared. By activity measurements and immune blotting, a similar enzyme was found in rat liver. In the fractionated rat liver, the enzyme was localized in the lumen of the endoplasmic reticulum, probably loosely bound to the inner face of the membrane. Purified Golgi fractions contained only low levels of the enzyme.

Animals↗

PubMed1984

Rutin-induced beta-glucosidase activity in Streptococcus faecium VGH-1 and Streptococcus sp. strain FRP-17 isolated from human feces: formation of the mutagen, quercetin, from rutin.

A fecal isolate, Streptococcus sp. strain FRP-17, and strain VGH-1 of Streptococcus faecium were shown to contain beta-glucosidases which converted rutin (quercetin-3-O-beta-D-glucose-alpha-L-rhamnose) to quercetin and were active against o-nitrophenyl-beta-D-glucose. The activity against rutin could be measured by increased mutagenicity in the Ames assay or visualized on thin-layer chromatography plates. In both organisms, the beta-glucosidase activities were inducible by the addition of rutin to the growth media. Several closely related strains of Streptococcus spp. lacked any beta-glucosidase activity. In cell preparations of the active organisms, activities with rutin and o-nitrophenyl-beta-D-glucose were optimal at pH 6.8 and could be enhanced by increasing the ionic strength of the assay system. At low ionic strengths, both quercetin and a new product (intermediate between the polarities of rutin and quercetin) were formed by the incubation of rutin with cell preparations of either active organism. This product disappeared with increased ionic strength, suggesting that it may be a reaction intermediate, quercetin-3-O-beta-D-glucose. These results suggest that the beta-glucosidase active against rutin and that active against o-nitrophenyl-beta-D-glucose are the same.

Bacterial Proteins↗

PubMed1984

Enzymatic profiles of Enterobacter sakazakii and related species with special reference to the alpha-glucosidase reaction and reproducibility of the test system.

The enzymatic profiles of Enterobacter sakazakii, Enterobacter cloacae, Enterobacter aerogenes, and Enterobacter agglomerans were determined with the API ZYM system (API System S.A., La Balme Les Grottes, France). Each assay was performed three times. A simple formula was derived and applied to assess the reproducibility of the API ZYM tests. In addition, a separate alpha-glucosidase test was performed. All E. sakazakii isolates produced alpha-glucosidase, in contrast to the other Enterobacter isolates. No phosphoamidase activity was detected in any of the E. sakazakii isolates, whereas it was present in 72% of E. cloacae, 89% of E. agglomerans, and 100% of E. aerogenes isolates. It was concluded that detection of alpha-glucosidase permits rapid and reliable differentiation between E. sakazakii and other Enterobacter species. The reproducibilities of alpha-glucosidase and phosphoamidase reactions were estimated to be 89 and 81%, respectively.

Enterobacter↗

PubMed1978

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.

Basidiomycota↗

PubMed1988

A macrophage receptor for liver lysosomal beta-glucosidase.

beta-Glucosidase was purified from lysosomal membranes isolated from rat liver. Binding and uptake of the purified beta-glucosidase were mediated via an apparently single binding site on rat peritoneal macrophages. The number of sites and the Kd were 4.20 X 10(4)/cell and 1.00 X 10(-7) M, respectively. Neither of the processes was inhibited by ligands for mannose/fucose receptors, mannose 6-phosphate receptors, or scavenger receptors, or by other glycoproteins and sugar compounds. A portion of the beta-glucosidase taken up into the macrophages was degraded rapidly. These results suggested that liver lysosomal beta-glucosidase was endocytosed via a receptor not previously described.

Animals↗

PubMed1995

Synthesis of 2-deoxy-glucooligosaccharides through condensation of 2-deoxy-D-glucose by glucoamylase and alpha-glucosidase.

Glucoamylases from Aspergillus niger and Rhizopus niveus catalyzed condensation of 2-deoxy-D-glucose (dGlc) to yield deoxy-glucooligosaccharides with polymerization degrees of 2-5. The enzymes also gave a small amount of products from 3-deoxy-D-glucose, but no products from 6-deoxy-D-glucose. A. niger alpha-glucosidase also catalyzed condensation of dGlc, while Torula and Saccharomyces alpha-glucosidases had low activity. alpha-1,4-, 1,6-, and 1,3-linked deoxy-glucobioses were isolated and identified as the products of A. niger glucoamylase and A. niger alpha-glucosidase. In the reaction of the glucoamylase, 1,4- and 1,3-linked saccharides decreased with an increase of 1,6-linked one. A. niger alpha-glucosidase produced alpha-1,6-linked disaccharide predominantly during the whole course of the reaction.

Aspergillus niger↗

PubMed2004

Degeneration of beta-glucosidase activity in a foam fractionation process.

Foam fractionation is a promising technique for concentrating proteins because of its simplicity and low operating cost. One such protein that can be foamed is the enzyme cellulase. The use of inexpensively purified cellulase may be a key step in the economical production of ethanol from biomass. We conducted foam fractionation experiments at total reflux using the cellulase component beta-glucosidase to study how continuous shear affects beta-glucosidase in a foam such as a fermentation or foam fractionation process. The experiments were conducted at pH 2.4, 5.4, and 11.6 and airflow rates of 3, 6, 15, 20, and 32 cc/min to determine how beta-glucosidase activity changes in time at these different conditions. This is apparently a novel and simple way of testing for changes in enzyme activity within a protein foam. The activity did not degenerate during 5 min of reflux at pH 5.4 at an airflow rate of 10 cc/min. It was established that at 10 min of refluxing, the beta-glucosidase denatured more as the flow rate increased. At pH 2.4 and a flow rate of 10 cc/min, the activity remained constant for at least 15 min.

Biotechnology↗

PubMed1978

On the properties of alpha-glucosidase and the binding of glucose to the enzyme.

An alpha-glucosidase was purified from baker's yeast. The molecular weight was approximately 44 000 daltons. SDS-disc gel electrophoresis suggested that the enzyme consisted of four subunits. The isoelectric point was at pH 5.4. The Km values for p-nitrophenyl alpha-D-glucopyranoside and maltose were 2.9 X 10(-4) and 2.5 X 10(-2) M, respectively. Binding of 2-(p-toluidino)naphthalene-6-sulfonate to the alpha-glucosidase was associated with a strong increase in fluorescence. The dissociation constant of the enzyme-TNS complex was 8 X 10(-5) M. The fluorescent probe did not interfere with the binding of glucose to the enzyme although the alpha-glucosidase was inhibited by high concentrations of TNS. The formation of an enzyme-glucose complex was indicated by an increase of fluorescence and by a shift in the wavelength for maximal emission which suggests that the binding process is associated with a change in conformation. The dissociation constant of the glucose--alpha-glucosidase complex KD = 0.57 X 10(-3) M, was calculated from the increase in fluorescence as a function of glucose concentration.

Glucose↗

PubMed2000

Determination of acid alpha-glucosidase protein: evaluation as a screening marker for Pompe disease and other lysosomal storage disorders.

BACKGROUND: In recent years, there have been significant advances in the development of enzyme replacement and other therapies for lysosomal storage disorders (LSDs). Early diagnosis, before the onset of irreversible pathology, has been demonstrated to be critical for maximum efficacy of current and proposed therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be achieved through a newborn screening program. One approach to the development of such a program is the identification of suitable screening markers. In this study, the acid alpha-glucosidase protein was evaluated as a marker protein for Pompe disease and potentially for other LSDs. METHODS: Two sensitive immunoquantification assays for the measurement of total (precursor and mature) and mature forms of acid alpha-glucosidase protein were used to determine the concentrations in plasma and dried blood spots from control and LSD-affected individuals. RESULTS: In the majority of LSDs, no significant increases above control values were observed. However, individuals with Pompe disease showed a marked decrease in acid alpha-glucosidase protein in both plasma and whole blood compared with unaffected controls. For plasma samples, this assay gave a sensitivity of 95% with a specificity of 100%. For blood spot samples, the sensitivity was 82% with a specificity of 100%. CONCLUSIONS: This study demonstrates that it is possible to screen for Pompe disease by screening the concentration of total acid alpha-glucosidase in plasma or dried blood spots.

Biomarkers↗

PubMed1979

[Coordinate regulation of riboflavin permease and alpha-glucosidase synthesis in the yeast Pichia guilliermondii].

Riboflavin permease of the yeast Pichia guilliermondii appear to be inducible transport system. Its synthesis is induced by sucrose, maltose, alpha-methyl-D-glocoside, melizitose and raffinose, but not by D-glucose, trehalose or cellobiose. The synthesis of riboflavin permease in the presence of sucrose of maltose is depressed by cycloheximide, actinomycin D and 8-hydroxyquinoline. These results suggest that the synthesis of riboflavin permease is regulated on the transcription level. The inducers of riboflavin permease are also able to induce the synthesis of alpha-glucosidase. The mutants have been selected in which the synthesis of riboflavin permease occurs constitutively; the synthesis of alpha-glucosidase in the mutants is also constitutive. Growing of the yeast in a medium with high content of glucose results in a parallel decrease of the riboflavin permease and alpha -- glucosidase activities. These data are indicative of corrdinate regulation of riboflavin permease and alpha -- glucosidase in P. guilliermondii. Suboptimal or excessive content of vitamin B2 in the medium does not affect the level of riboflavin permease in this yeast species.

Ascomycota↗

PubMed1989

The dual effects of alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase.

This report demonstrates the effect of primary alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase. Lineweaver-Burk analyses of the kinetic data revealed a biphasic response; at low concentrations the alcohols increased the Vmax 5--7-fold while at higher concentrations they caused a purely competitive type of inhibition. For example, with n-butyl alcohol, increasing the alcohol's concentration in the assay medium from 0 to 0.14 M (0-1% (v/v)) resulted in a progressive increase in Vmax to a value 7-fold above the basal level without affecting the Km. However, between 0.14 and 0.54 M (1 and 4% (v/v)) n-butyl alcohol, the Km for 4-methylumbelliferyl-beta-D-glucopyranoside increased significantly from 0.14 to 0.93 mM. In contrast to n-butyl alcohol or isobutyl alcohol, which are potent activators, structurally related compounds like sec-butyl alcohol, tert-butyl alcohol, butylurea, and butanesulfonic acid did not stimulate the activity of the cytosolic beta-glucosidase. In the concentration range where activation was observed, conventional secondary replots of 1/delta slope versus 1/[alcohol] yielded perfect straight lines, demonstrating that binding of a single molecule of alcohol to the beta-glucosidase was responsible for the initial phase of activation. Furthermore, the glycohydrolase displayed a propensity to bind the longer chain alcohols, as reflected by the KA (binding constant) values of 555, 146, 34.1, and 7.47 mM for ethanol, n-propyl alcohol, n-butyl alcohol, 1-pentanol, respectively. This phenomenon of nonessential activation by alcohols has led us to speculate on the presence of a physiologic activator for the beta-glucosidase in mammalian tissues which contain this enzyme.

Alcohols↗

PubMed1977

[beta-Glucosidases specific for the cyanogenic glucoside triglochinin from Alocasia macrorrhiza Schott: further characterization of properties (author's transl)].

All beta-glucosidases extracted and separated from a plant of Alocasia macrorrhiza are almost entirely specific for triglochinin. The hexameric beta-glucosidase has been shown to dissociate in dimers without any alteration of activity. Reaggregation could only be demonstrated using bifunctional reagents like glutaraldehyde. Treatments of beta-glucosidase with various chemicals (e. g. glutaraldehyde, dodecyl sulfate) decreased the activity for triglochinin more than the activity for 4-nitrophenyl glucoside. On the other hand, specific reagents like bromocondurite or p-chloromercuribenzoate caused identical inactivations measured with various substrates. It seems possible that the different beta-glucosidases splitting triglochinin arose during purification from the hexameric form which occurs in the plant.

Adipates↗

PubMedDate not supplied

Enzyme therapy: II. Effect of covalent attachment of polyethylene glycol on biochemical parameters and immunological determinants of beta-glucosidase and alpha-galactosidase.

The covalent attachment of polyethylene glycol (PEG) to beta-glucosidase from sweet almonds and alpha-galactosidase from green coffee beans results in alterations of their catalytic properties and masking of specific determinant sites on the enzymes. Both enzymes have increased Km and decreased Vmax values against their respective p-nitrophenyl substrate analogs after PEG attachment. When PEG is attached to 30% of alpha-galactosidase epsilon-amino groups, 12% activity remains against ceramide trihexoside, while its ability to convert type B erythrocytes to type H specificity is lost. However, it still is able to cleave terminal galactose residues from human saliva blood group substance B. PEG-beta-glucosidase (38%) did not elicit the production of complement-fixing antibodies, nor did it react with antibodies produced against the native enzyme. Antibody and lectin-specific binding were lost from both modified enzymes (PEG-beta-glucosidase and PEG-alpha-galactosidase). After conjugation with PEG, beta-glucosidase lost its ability to bind to concanavalin A-Sepharose. Antibodies directed against native alpha-galactosidase blocked its enzyme activity, but lost their ability to inhibit activity in progressively higher modified preparations of the enzyme. Antisera against PEG-alpha-galactosidase (53%) did not inhibit enzyme activity in any alpha-galactosidase or PEG-alpha-galactosidase preparation. These results indicate that PEG tends to cover lectin-specific carbohydrate moieties and antigenic determinants and that these sites probably remain cryptic during in vivo processing of PEG-enzymes.

Antigen-Antibody Complex↗

PubMed1983

[Soluble and membrane-bound neutral alpha-glucosidase from the human kidney].

In human kidney cortex neutral alpha-glucosidases 1 and 2 are represented by two forms, soluble (cytosolic) and membrane-bound (brush border) ones. It has been shown that the soluble enzyme preexists in human kidney but does not derive from the membrane-bound form. Similar to the membrane-bound enzyme the soluble form is a glycoprotein. Both enzyme forms possess identical electrophoretic mobility, pH-optimum, heat sensibility and Km values for maltose (0.7 mM) and 4-methylumbelliferyl-alpha-D-glucopyranoside (0.57 mM), but differ by molecular weights as determined by gel filtration chromatography. The molecular weights of the soluble neutral alpha-glucosidases 1 and 2 are lower than those of the comparable brush border enzymes (470 000, 360 000, 520 000 and 440 000, correspondingly). Neutral membrane-bound alpha-glucosidase 1 is a sialylated enzyme with a pI of 4.10 +/- 0.02. The soluble enzyme contains no or only traces of neuraminic acid and has a pI 4.40 +/- 0.03. The soluble and membrane-bound neutral alpha-glucosidases are apparently independent forms of the enzyme, differing by the degree of sialylation and by the presence of an "anchor" in the membrane-bound enzyme. The synthesis of both forms is presumably coded by the same structural gene.

Cell Membrane↗

PubMedDate not supplied

[The use of immunological method for the determination of acid alpha-glucosidase activity in human muscles].

Homogeneous preparation of acid alpha-glucosidase, obtained from human placenta, was used for immunization of rabbits and production of specific antiserum. Immunological procedure was developed for estimation of acid alpha-glucosidase activity from human muscles in presence of the enzyme neutral form. Activity of acid alpha-glucosidase was measured in 20 patients with various types of nervous system impairments. Total activity of alpha-glucosidase in muscles of the patients was 11.4 +/- 0.7 nM/min/g of the tissue and activity of the acid form constituted about 84.8 +/- 1.7% of the total activity.

Electrophoresis, Disc↗

PubMed1998

In vitro cytotoxicity following specific activation of amygdalin by beta-glucosidase conjugated to a bladder cancer-associated monoclonal antibody.

We describe a novel version of antibody-directed enzyme prodrug therapy (ADEPT), with the use of amygdalin as prodrug. Amygdalin is a naturally occurring cyanogenic glycoside, which can be cleaved by sweet almond beta-glucosidase to yield free cyanide. If amygdalin could be activated specifically at the tumour site, then malignant cells would be killed without the systemic toxicity usually associated with chemotherapy. To this end, we have conjugated beta-glucosidase to a tumour-associated monoclonal antibody (MAb) (HMFG1) and the conjugate has been tested in vitro for specificity and cytotoxicity subsequent to activation of amygdalin. Amygdalin was cytotoxic to HT1376 bladder cancer cells only at high concentrations, whereas the combination of amygdalin with HMFG1-beta-glucosidase enhanced the cytotoxic effect of amygdalin by 36-fold. When 2 concentrations of HMFG1-beta-glucosidase were compared, the toxic effect was dose dependent. The cytotoxicity of amygdalin was also enhanced by the MAb-enzyme conjugate even when the unbound conjugate was removed from the medium prior to exposure to amygdalin and the cells were washed. In addition to the cytotoxic effect, we also demonstrated specificity, using a MAb-enzyme conjugate that does not recognise the HT1376 bladder cancer cells. Finally, we studied the cytotoxic effect of the conjugate in co-culture of HMFG1-positive and-negative cell lines (HT 1376 and U87MG cells). We demonstrated that the rate of surviving cells corresponds well to the percentage of U87MG (HMFG1-negative) cells in the flask. Our findings indicate that ADEPT is more effective than non-directed enzyme activation of a prodrug and can result in a non-toxic cancer therapy.

Amygdalin↗

PubMed1997

Consequences of disrupting the gene that encodes alpha-glucosidase II in the N-linked oligosaccharide biosynthesis pathway of Dictyostelium discoideum.

We have identified and disrupted the gene coding for alpha-glucosidase II in Dictyostelium discoideum. This enzyme is responsible for removing two alpha 1,3-linked glucose residues from N-linked oligosaccharides on newly synthesized glycoproteins. Mutagenesis by restriction enzyme-mediated integration (REMI) generated a clone, DG1033, which grows well but forms abnormal fruiting bodies with short, thick stalks. The strain lacks alpha-glucosidase II activity and makes incompletely processed N-linked oligosaccharides that are abnormally large and have fewer sulfate and phosphate esters. The morphological, enzymatic, and oligosaccharide profile phenotypes of the disruption mutant are all recapitulated by a targeted disruption of the normal gene. Furthermore, all of these defects are corrected in cells transformed with a normal, full-length copy of the gene. The phenotypic characteristics of DG1033 as well as chromosomal mapping of the disrupted gene indicate that it is the site of the previously characterized modA mutation. The Dictyostelium gene is highly homologous to alpha-glucosidase II genes in the human and the pig, C. elegans, and yeast. Although various cell lines have been reported to be defective in alpha-glucosidase II activity, disruption of the Dictyostelium gene gives the first example of a clear developmental phenotype associated with loss of this enzyme.

Amino Acid Sequence↗

PubMed2002

Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties.

The thermophilic fungus Chaetomium thermophilum var. coprophilum produced large amounts of extracellular and intracellular beta-glucosidase activity when grown on cellulose or cellobiose as carbon sources. The presence of glucose in the culture medium drastically decreased the level of beta-glucosidase activity, while cycloheximide prevented the induction of the extracellular enzyme activity by cellobiose. An extracellular beta-glucosidase induced by avicel was purified by a procedure involving acetone precipitation and chromatography on two DEAE-cellulose columns. The purified enzyme was a basic protein, with a carbohydrate content of 73%. The deglycosylated enzyme exhibited a molecular mass of 43 kDa, with pH and temperature optima of 5.5 and 65 degrees C respectively. The beta-glucosidase hydrolysed only cellobiose and p-nitrophenyl-beta-D-glucopyranoside, exhibiting apparent Km values of 3.13 mM and 0.76 mM, respectively. The native purified enzyme was stable up to 2 hours at 60 degrees C, and its thermal stability was directly dependent on glycosylation.

Cellobiose↗