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PubMed1997

Synthesis by an alpha-glucosidase of glycosyl-trehaloses with an isomaltosyl residue.

Glycosyl-trehaloses with an isomaltosyl residue were synthesized by alpha-glucosidase from Aspergillus niger by using maltotetraose as a glucosyl donor and trehalose as the acceptor. The one trisaccharide and two tetrasaccharides formed were isolated by successive column chromatography. The results of an enzymatic digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were alpha-isomaltosyl alpha-glucoside, alpha-isomaltotriosyl alpha-glucoside and alpha-isomaltoside. These oligosaccharides were not fermented to an acid by Streptococcus mutans, and they effectively inhibited water-insoluble glucan synthesis from sucrose by glucosyltransferase. In an in vitro utilization test with human intestinal bacteria, these oligosaccharides were predominantly utilized by Bifidobacteria.

Bacteroides↗
PubMed1985

Optimization of the urinary acid alpha-glucosidase determination.

The optimization of the method for acid alpha-glucosidase determination (EC 3.2.1.3) in human urines, employing the synthetic substrate 4-nitrophenyl-alpha-D-glucopyranoside, is reported. Storage conditions of the specimens and their pretreatment were particularly investigated. The precision of the whole analytical procedure (including gel filtration) is good (within-run CV = 7.4% for normal samples and 3.7% for elevated ones). The correlation with the method using maltose as substrate is excellent (y = -0.01 + 0.13 x; r = 0.9893).

Chromatography, Gel↗
PubMed1981

Non-competitive inhibition of honeybee haemolymph pNP-alpha-D-glucosidase by chloramphenicol.--A study in vitro.

The haemolymph pNP-alpha-D-glucosidase activity of emerging worker bees shows a tendency towards negative cooperativity (n = 0.92). The relation between initial velocity and enzyme concentration is not exactly linear (bilogarithmic exponent = 0.91). Between 25 degrees and 31 degrees C, the activation energy, EA = 38.2 kJ/mol. Chloramphenicol administered in vitro decreases the maximum velocity and re-establishes pure Michaelian kinetics (n congruent 1.0). The Hill coefficient for the binding of chloramphenicol to the enzyme, ni = 1.13; the values of Ki = 20.7 mM, K'i = 17.3 mM, and I50 = 17.6 mM are not significantly different from one another. These data indicate that inhibition by chloramphenicol is of the pure, non-competitive type.

Animals↗
PubMed1978

Activity of alpha-1, 4-glucosidase in furazolidone-induced glycogenosis.

Furazolidone (FZ) at 700 and 800 p.p.m. was added to feed mixtures fed turkey poults two and three weeks posthatching, respectively, to induce acute experimental cardiomyopathy. Poults in the control pen received the same ration but without FZ. From EKG data obtained at 2, 4, and 5 weeks of age, control unaffected and experimental affected poults were selected for sacrifice. Poults were sacrificed by cervical dislocation and appropriate samples of hepatic tissue were removed for assays of activity of alpha-1, 4-glucosidase. Results indicate that enzyme activity in affected FZ-treated poults is similar to that in unaffected control poults. Lack of significant differences in activity of this lysosomal enzyme suggests that FZ-induced glycogenosis may be related to the adult form of idiopathic generalized glucogenosis, the etiology of which remains unidentified.

Animals↗
PubMed1989

[Purification and properties of beta-glucosidase from Aspergillus phoenicis].

A beta-glucosidase has been purified to electrophoretically homogeneity from the wheat bran culture of Aspergillus phoenicis by PEG 6000-phosphate biphasic separation, column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and SE-Sephadex C-50. The enzyme showed optimal activity at pH 5.0 and 60 degrees C. It was stable in the pH range of 4.0-7.5 and up to 55 degrees C. The enzyme activity was strongly inhibited by Ag+ and Hg2+. The molecular weight of the enzyme was 118000 as determined by SDS-PAGE and 195000 by gradient-PAGE. The isoelectric point was pI 3.95 as determined by PAGIF.

Aspergillus↗
PubMed1988

Activity of arylsulfatase A, B-glucosidase and hexosaminidases in chorionic villi at six, eight and ten weeks' gestational age.

Chorionic villus sampling is becoming an accepted method of prenatally diagnosing metabolic diseases. There are few published data on sampling at various gestational ages. Different investigators, using different methods of chorionic biopsy, perform the sampling at various gestational ages. Accurate interpretation of results is dependent on reliable normal values, which could differ on the basis of gestational age. The enzymatic activities of arylsulfatase A, B-glucosidase and hexosaminidases were compared in trophoblast from six-, eight- and ten-week gestations. No significant differences in activity levels were detected. This finding confirms the accuracy of metabolic diagnosis for these enzyme-deficiency syndromes in trophoblastic tissue at gestational ages of six to ten weeks without a requirement for gestational-age-specific normal values.

Cerebroside-Sulfatase↗
PubMed1979

Quantitative study on anomeric forms of glucose produced by alpha-glucosidases.

Anomeric forms of glucose produced from phenyl alpha-maltoside, maltose, or phenyl alpha-glucoside have been determined quantitatively by simultaneous measurements of optical rotation and reducing power, for eight kinds of glucose-producing 1,4-alpha-glucosyl hydrolases, including glucose-forming amylase from human urine, and alpha-glucosidases from pig serum, honey bee, buckwheat seed, rice seed, sugar beet seed, flint corn seed, and brewer's yeast. All the eight enzymes studied were found to produce alpha-glucose exclusively.

Animals↗
PubMedDate not supplied

Multiple forms of intracellular alpha-glucosidase from Thermoascus aurantiacus Miehe: purification and properties.

The intracellular alpha-glucosidase extracted from the mycelium of Thermoascus aurantiacus was resolved by chromatographic procedures on DEAE-cellulose and hydroxylapatite into 4 major forms which account for 96% of the recovered activity. These forms, purified to electrophoretic homogeneity, display the same affinity toward p-nitrophenyl-alpha-glucoside (Km = 0.24 mM at pH 4 and 70 degrees C), the same molecular weight on Sephadex G-200 (140,000-145,000) and isoelectric point (4.2) and are protected by Cl- and H+ ions from heat denaturation at 70 degrees C. They differ in the carbohydrate content which varies between 14 and 31%, w/w.

Ascomycota↗
PubMedDate not supplied

Mechanism of action and structural properties of an intracellular alpha-glucosidase from Thermoascus aurantiacus Miehe.

The intracellular alpha-glucosidase purified from the mycelium of Th. aurantiacus is an exceptionally stable protein which displays its maximum activity at 70 degrees C and pH 4.2 and is inhibited by 4 M urea, 0.5 M mercaptoethanol, 15 mM Cu++ and 0.04% rose bengal only after incubation at high temperature (60-70 degrees C). Carboxylic groups with pKa = 3.25 appear involved in the catalytic process together with a histidine residue (pKa = 5.7). Plots of Log V vs pH also show that the carboxylic groups dissociate in a cooperative way. A simple reaction mechanism is proposed on the basis of competitive inhibition by delta-gluconolactone, which suggests the formation of a carbonium ion.

Ascomycota↗
PubMed1983

Energy of binding of Aspergillus oryzae beta-glucosidase with the substrate, and the mechanism of its enzymic action.

Several beta-D-glucopyranosides (p-nitrophenyl, phenyl, and ethyl), 1-thio-beta-D-glucopyranosides, and phenyl 2-deoxy, 3-deoxy, 4-deoxy, and 6-deoxy beta-D-glucopyranosides were synthesized and used to study the mechanism of the enzymatic action of Taka-beta-glucosidase [EC 3.2.1.21 Aspergillus oryzae]. Kinetic constants of the enzyme for these glycosides were determined from S/V-S or 1/V-1/S plots, and the hydrolysis rates of these compounds with the enzyme, acid (3 N HCl) and alkali (3 N NaOH) were compared. Inhibition of the enzyme by 1,5-anhydroglucitol, glucal, dihydroglucal, and 1,6-anhydroglucopyranose was also examined. Glucal and 1,5-anhydroglucitol showed strong competitive inhibition. Free energy of binding of each hydroxyl group of glucosidic glucose with the enzyme was estimated from Kms of phenyl beta-glucoside and its deoxy analogues, and also Ki values of some inhibitors. The free energies of binding of 2-OH, 3-OH, 4-OH, and 6-OH were calculated to be 1.1, 2.4, 0.7, and 1.8 kcal/mol, respectively. The free energy of binding of phenoxide at C-1 (0.3 kcal/mol) was calculated from the Km of Ph-beta-Glc and Ki of 1,5-anhydroglucitol. The energy of binding of 5-CH2OH (2.3 kcal/mol) was obtained from the Km of Ph-beta-Glc and that of Ph-beta-Xyl. The sum (6.8 kcal/mol) of each partial binding free energy was close to the value of binding free energy of Ph-beta-Glc (7.0 kcal/mol) calculated by the equation; -delta Gbind = -RT ln Km-T delta Smix, showing that the methods of estimation of each binding energy used in the present study seemed reasonable. Glucal, having a pyranose form distorted slightly, showed strong competitive inhibition and the Ki of this inhibitor was smaller than the Km of Ph-beta-Glc, suggesting that the sugar ring bound to the active site was distored to a half chair form which is labile to acid hydrolysis.

Aspergillus↗
PubMed1983

[A stoichiometric interpretation of the significance of the Hill coefficient of some kinetically cooperative enzymes. Application to alpha-glucosidases of the hemolymph of bees].

In an enzyme complex where active subunits are aggregated in functional groups undergoing steric interactions, the Hill coefficient can reflect the ratio of the partial reaction order of the substrate upon the enzyme one, according to the mass action law. Honeybee haemolymph alpha-glucosidases kinetics support this theory in negative and positive cooperativity cases.

Animals↗
PubMed1984

Leukocyte beta-glucosidase in a child with Gaucher's disease and his kinship.

Beta-glucosidase was determined in leukocyte homogenates of a male child with Gaucher's disease and members of his family. An important difference in enzyme activity was found in the heterozygous gene carriers; in one obligate heterozygote, the mother, a high residual enzyme capacity was detected. Various methods of enzyme determination using various substrates are recommended for the detection of the gene carrier condition.

Child, Preschool↗
PubMed1980

Temperature-dependent change in the rate-limiting step of beta-glucosidase catalysis.

When beta-glucosidase from sweet almonds reacts with p-nitrophenyl-beta-D-glucoside at subzero temperatures, a rapid release of p-nitrophenol, stoichiometric with enzyme concentration, occurs prior to turnover. This observation is interpreted as reflecting rapid formation of a glucose-enzyme intermediate, followed by rate-limiting breakdown. The energies of activation for the formation and breakdown of the intermediate were 6.0 +/- 2 and 19.0 +/- 2 kcal mol-1, respectively. Extrapolation of the reaction rates to higher temperatures suggests an isokinetic temperature circa 24 degrees C. Measurement of kcat in aqueous solution as a function of temperature yields a breakpoint in the Arrhenius plot at 20 degrees C and activation energies above and below this point which correspond to the values at subzero temperatures (6.7 and 17.0 kcal mol-1, respectively). This breakpoint thus reflects a change in the rate-determining step from formation of the intermediate at higher temperatures to breakdown at lower values, and explains why a "burst" of p-nitrophenol is observed at low, but not high, temperatures with this substrate. The significance of this finding to other glycohydrolases, and enzyme catalysis in general, is considered.

Glucosidases↗
PubMed1980

Human neutral alpha-glucosidase C: genetic polymorphism including a "null" allele.

We describe a genetic polymorphism of human neutral alpha-glucosidase C, detected in lymphoid cells by a combination of starch gel electrophoresis and isoelectric focusing. The seven phenotypes observed appear to result from the expression of four different alleles. The distribution of the observed phenotypes fits the expected distribution predicted from calculated gene frequencies in Hardy-Weinberg equilibrium. Family studies are consistent with autosomal inheritance of the gene. The product of one of the alleles is unusual in that it is "silent," with an estimated gene frequency of .174 in an outbred white population. Approximately one-third of the population is heterozygous "null." Homozygosity for the allele has not been associated with any obvious disease state. This is the third example of a "null" allele which has a substantial gene frequency in an outbred population but does not appear to result in disease in the homozygous state.

Alleles↗
PubMed1982

A lectin-resistant mouse lymphoma cell line is deficient in glucosidase II, a glycoprotein-processing enzyme.

Glycosylation of asparagine residues of glycoproteins occurs by the transfer of a glucose3mannose9N-acetylglucosamine2 (Glc3Man9GlcNAc2) oligosaccharide from a lipid carrier to the nascent protein. Normally, this transfer is quickly followed by the stepwise removal of the glucose residues which are arranged in the sequence: Glc1 leads to 2Glc1 leads to 3Glc1 leads to 3Man. We now report studies which demonstrate that a lectin-resistant mutant of the BW5147 mouse lymphoma cell line is deficient in the enzyme which removes the two inner glucose residues. This cell line (PHAR2.7) was selected for resistance to the cytotoxic effects of Phaseolus vulgaris leukoagglutinating lectin (Trowbridge, I. S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Glycopeptides prepared from cells equilibrium-labeled with either [2-3H]mannose or [6-3H]galactose were characterized using lectin affinity chromatography, treatment with specific endo- and exoglycosidases, sizing by paper chromatography, and methylation analysis. Approximately 50% of the radioactivity in [3H]mannose-labeled glycopeptides from the mutant cells is present as glucosylated high mannose-type oligosaccharides whereas parent cell glycopeptides labeled under similar conditions lack detectable amounts of these species. Using [3H]galactose labeling, the major glucosylated oligosaccharides were identified as Glc2Man9GlcNAc2 and Glc2Man8GlcNAc2. In vitro enzyme assays demonstrated that the mutant cells cannot remove either of the two inner 1 leads to 3-linked glucose residues. Removal of the outer 1 leads to 3-linked glucose is normal. We conclude from these data that the PHAR2.7 cell line is deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the oligosaccharides of newly glycosylated proteins.

Animals↗
PubMed1998

Circular permutants in beta-glucosidases (family 3) within a predicted double-domain topology that includes a (beta/alpha)8-barrel.

By predicting the general secondary structure for beta-glucosidases (family 3), in conjunction with existing knowledge of the circular permutants present in B. fibrisolvens and R. albus, we were able to find the canonical elements of the secondary structure. The way these elements are linked suggests that there is a double-domain topology made up of a (beta/alpha)8-barrel domain and a "mainly all-beta" domain. A number of already known conserved motifs are located within (or near) the C-terminal part of the putative parallel beta-strands of the (bet/alpha)8-barrel, which is consistent with what is known about the location of catalytical sites for enzymes that have this domain topology. Within the circular permutants, two beta/alpha units are located at the N-terminal part of the molecule, whereas the other six beta/alpha units are located at the C-terminal end. In this way, the circular permutants can be seen to have a putative discontinuous double-domain topology.

Amino Acid Sequence↗
PubMed1998

Structural basis of increased resistance to thermal denaturation induced by single amino acid substitution in the sequence of beta-glucosidase A from Bacillus polymyxa.

The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions. beta-glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering. This is a well-characterized enzyme, belonging to glycosyl hydrolase family 1. Its natural substrate is cellobiose, but is also active against various artificial substrates. In its native state has an octameric structure. Its subunit conserves the general (alpha/beta)8 barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel. Using random-mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes. The crystal structure of this mutant has been determined by X-ray diffraction and compared with the native structure. The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N-terminus of helix alpha2, to Asp28, located in one of the loops surrounding the active-site cavity. The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure. Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed. However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other. These results show that far-reaching effects can be achieved by a single amino acid replacement within the protein structure. Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions.

Amino Acid Sequence↗