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At least 685 records · Page 38Linked to original sources
PubMed1990

Kinetic mechanism of beta-glucosidase from Trichoderma reesei QM 9414.

beta-Glucosidase is a key enzyme in the hydrolysis of cellulose to D-glucose. beta-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl beta-glucosides. The double-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl beta-D-glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 +/- 0.2 mM and V = 2.09 +/- 0.52 mumol.min-1.mg-1 for salicin; K = 1.22 +/- 0.3 mM and V = 1.14 +/- 0.21 mumol.min-1.mg-1 for cellobiose; K = 0.19 +/- 0.02 mM and V = 29.67 +/- 3.25 mumol.min-1.mg-1 for p-nitrophenyl beta-D-glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by beta-glucosidase on p-nitrophenyl beta-D-glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl beta-D-glucopyranoside hydrolysis.

Benzyl Alcohols↗
PubMed1983

Comparative study of alpha-glucosidase activities in blood and seminal plasma of rams.

1. This study was undertaken to characterize the alpha-glucosidase present in blood and seminal plasma of two strains of pure-bred rams which are known as seasonal breeders. 2. pH profiles and activity levels were investigated in blood and seminal plasma using a sensitive spectrophotometric assay with para-nitrophenyl-alpha-D-glucoside as substrate. 3. According to their pH optimum, blood plasma and seminal plasma alpha-glucosidases were typically neutral and acid enzymes and significant differences were recorded in their physico-chemical properties, establishing the tissue specificity of the enzyme. 4. Notwithstanding the tissue under study, the nature of the alpha-glucosidase activity was similar in both strains of pure-bred rams.

Animals↗
PubMed1987

Purification of ram seminal plasma acid alpha-glucosidase.

1. Ram seminal plasma alpha-glucosidase has been purified in order to increase our knowledge of this enzyme and of its role in epididymal physiology. 2. Since the enzyme behaved differently from other known acid alpha-glucosidases and was not affinity-adsorbed on dextran gels, another approach had to be used. 3. The final procedure included an ethanol precipitation step, sequential chromatography on hydroxylapatite and DEAE Sepharose CL-6B, isoelectric focusing in polyacrylamide gels and ultrafiltration. 4. The resulting purification factor of alpha-glucosidase was 9822 with an overall yield of 5%. 5. The purified material consisted of several isoforms with a mol. wt of 105,000 and isoelectric points varying between 4 and 5.

Animals↗
PubMed1987

Simultaneous measurement of seminal L-carnitine, alpha,1-4-glucosidase, and glycerylphosphorylcholine in azoospermic and oligozoospermic patients.

L-carnitine, alpha,1-4-glucosidase, and glycerylphosphorylcholine were measured in seminal plasma of a selected group of azoospermic men and in an unselected group of oligozoospermic men. In vasectomized subjects the epididymal indices (mean +/- standard error: L-carnitine, 276.9 +/- 27.5 nmol/ejaculate; alpha-glucosidase, 1.2 +/- 0.1 U/ejaculate; and glycerylphosphorylcholine, 1.5 +/- 0.2 mumol/ejaculate) were always below the normal range of fertile subjects (1757.4 +/- 76.7 nmol/ejaculate; 16.4 +/- 0.9 U/ejaculate; and 17.3 +/- 0.7 mumol/ejaculate, respectively). On the contrary in a large number of patients affected by azoospermia because of seminiferous tubular damage (750.4 +/- 83.6 nmol/ejaculate; 6.8 +/- 0.9 U/ejaculate; and 6.1 +/- 0.6 mumol/ejaculate; respectively) and in a few oligozoospermic subjects (1193.7 +/- 72.3 nmol/ejaculate; 10.3 +/- 0.7 U/ejaculate; and 10.8 +/- 0.7 mumol/ejaculate; respectively) the epididymal indices were found in the range of vasectomized subjects, showing an association between seminiferous tubular lesion and epididymal dysfunction. In conclusion, in spite of the low levels of epididymal indices found in patients with obstructive azoospermia, the presence of a large number of subjects with seminiferous tubular lesions without obstruction with similar low values of L-carnitine, alpha-glucosidase, and glycerylphosphorylcholine reduces the usefulness of these indices in differential diagnosis of azoospermia.

Carnitine↗
PubMed1991

Changes in the activities of jejunal glucosidases in experimental intestinal tumorigenesis induced by 1,2 dimethylhydrazine in rats fed different diets.

The purpose of the present study was to establish the effect of the carcinogen 1,2 dimethylhydrazine on the activities of the jejunal glucosidases and to assess the possible modifying effect of different diets. Two control groups of Wistar albino rats were used - fed standard pellet diet and fed the same diet + 1,2 dimethylhydrazine treatment. Six experimental groups treated with 1,2 dimethylhydrazine were provided. One of them was fed standard diet, containing 30% of wheaten bran and the other 5 groups received high-lipid diets, containing 30% of different fats. The rats were injected subcutaneously once a week for 12 weeks with 20 mg 1,2 dimethylhydrazine/kg b.m. and left for 12 weeks in order to develop a tumor growth. The activities of 5 glucosidases (lactase, maltase, sucrase, palatinase and cellobiase) were determined in homogenates from jejunal mucosa taken near by the tumors and in homogenates from the tumors themselves. An expressed decrease of the jejunal glucosidase activities near the tumors and in the tumors was established. The animals fed 30% wheaten bran diet did not develop tumorigenesis and showed comparatively slight decrease of the enzyme activities. In general, the high-fat regimens did not exert such a preventive effect.

1,2-Dimethylhydrazine↗
PubMed1982

Immunohistochemical demonstration of acid alpha-glucosidase in fetal rat hepatocytes and glycogenosomes of newborn rat hepatocytes.

The electron microscopic distribution of acid alpha-glucosidase in fetal and newborn rat hepatocytes was studied by a peroxidase labeled method. In fetal and newborn rat hepatocytes, acid alpha-glucosidase was found abundantly. The reaction products had a small granular appearance in each hepatic area under the light microscope. By electron microscopy, the enzyme was found to be located in the lysosomes, especially on the lysosome membranes, and sometimes within their contents. Glycogenosomes appearing in the hepatocytes obtained from newborn rats at 6 hours after birth showed a positive immunohistochemical reaction on their membranes and also within their contents. Furthermore, positive reactions were apparent on the Golgi apparatus in newborn rat hepatocytes. These results indicated that the glycogenosomes appearing in the early neonatal rat hepatocytes did not depend on a deficiency of acid alpha-glucosidase.

Animals↗
PubMed1982

Factors determining steric course of enzymic glycosylation reactions: glycosyl transfer products formed from 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol by alpha-glucosidases and an inverting exo-alpha-glucanase.

Glycosyl transfer products were formed from 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) by purified alpha-glucosidases from Candida tropicalis and rice and by an inverting exo-alpha-glucanase (glucodextranase) from Arthrobacter globiformis. The products were structurally defined through 1H and 13C NMR (nuclear magnetic resonance) spectra of their crystalline per-O-acetates in comparison with those of authentic methyl 1-deoxy-alpha- and methyl 1-deoxy-beta-D-gluco-heptuloside. 1-Deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-heptenitol and 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-D-gluco-heptulose were produced by both the Candida alpha-glucosidase and the glucodextranase; 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 5)- and 1-deoxy-alpha-D-gluco-heptulosyl-(2 leads to 7)-D-gluco-heptuloses by the rice alpha-glucosidase. These results, together with our earlier findings of sterospecific hydration of heptenitol catalyzed by the same enzymes [Hehre, E. J., Brewer, C. F., Uchiyama, T., Schlesselmann, P., & Lehmann, J. (1980) Biochemistry 19, 3557-3564], show the inadequacy of the long-accepted notion that carbohydrase-catalyzed reactions always lead to retention (or always lead to inversion) of substrate configuration. In particular, the finding that glucodextranase forms transfer products of alpha configuration and a hydration product of beta configuration from the same substrate provides a clear example of the functioning of acceptors rather than donor substrates in selecting the steric course of reactions catalyzed by a glycosylase. The circumstances under which acceptor cosubstrates might be expected to show this significant effect are discussed. The opportunity presumably would exist whenever carbonium ion mediated reactions are catalyzed by glycosylases that provide oppositely oriented approaches of different acceptors to the catalytic center.

Arthrobacter↗
PubMed1985

Glucosidase II, a glycoprotein of the endoplasmic reticulum membrane. Proteolytic cleavage into enzymatically active fragments.

Glucosidase II removes the inner two alpha-linked glucose residues from freshly transferred Asn-linked oligosaccharide chains in the endoplasmic reticulum. This enzyme, whose activity could be measured by the hydrolysis of an artificial substrate (p-nitrophenyl alpha-D-glucopyranoside), was purified 240-fold from a rat liver microsome fraction by DEAE-cellulose, concanavalin A-Sepharose 4B, and hydroxylapatite chromatography. The apparent molecular weight of the active polypeptide was 123 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Glucosidase II has at least one high-mannose oligosaccharide chain that can be cleaved by endoglycosidase H. Trypsin readily cleaved the 123-kilodalton (kDa) form of glucosidase II into a fully active 73-kDa core. The pattern of this cleavage suggests a domain structure for this enzyme. We demonstrate that trypsin first removes a glycosylated 25-kDa domain to yield an apparently unglycosylated 98-kDa product which is further cleaved to yield the active 73-kDa core.

Animals↗
PubMed1986

Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps.

The second-order rate constants (kcat/Km) for the beta-glucosidase-catalyzed hydrolysis of aryl beta-D-glucopyranosides show a bell-shaped dependence of pH. The pKas that characterize this dependence are 4.4 (delta Hion approximately equal to 0) and 6.7 (delta Hion approximately equal to 0). In D2O these pKas are increased by 0.5 (+/- 0.1) unit, but there is no solvent isotope effect on the pH-independent second-order rate constant. Nath and Rydon [Nath, R. L., & Rydon, H. N. (1954) Biochem. J. 57, 1-10] examined the kinetics of the beta-glucosidase-catalyzed hydrolysis of a series of substituted phenyl glucosides. We have extended this study to include glucosides with phenol leaving groups of pKa less than 7. Brønsted plots for this extended series were nonlinear for both kcat/Km and kcat. Brønsted coefficients for those compounds with leaving groups of pKa greater than 7 (for kcat/Km) or pKa greater than 8.5 (for kcat) were nearly equal to -1.0, indicating substantial negative charge buildup on the leaving group in the transition state. The nonlinearity indicates an intermediate in the reaction. This was confirmed by partitioning experiments in the presence of methanol as a competing glucose acceptor. A constant product ratio, [methyl glucoside]/[glucose], was found with aryl glucoside substrates varying over 16,000-fold in reactivity (V/K), indicative of a common intermediate. Viscosity variation (in sucrose-containing buffers) was used to probe the extent to which the beta-glucosidase reactions are diffusion-controlled.(ABSTRACT TRUNCATED AT 250 WORDS)

Deuterium↗
PubMed1977

The separation and characterization of marmoset kidney beta-D-galactosidase and beta-D-glucosidase.

beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.

Animals↗
PubMed1979

The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and beta-glucosidases.

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000-48000 (C1), 30000-35000 (C2), 15000-18000 (C3), 10000-11000 (C4) and 4800-5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2-C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2-C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight beta-glucosidase (component B1, mol.wt. 350000-380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000-47000) predominated in older filtrates. 9. Intermediate molecular species of beta-glucosidase (B2, mol.wt. 170000-180000; B3, mol.wt. 83000-87000) were also found. 10. Cellulases C2-C5 acted in synergism with C1, particularly in the presence of beta-glucosidase.

Cellobiose↗
PubMed1987

The mechanism of action of beta-glucosidase from Botryodiplodia theobromae Pat.

The activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. was affected by added NaCl in such a way that an initial phase of stimulation was followed by a phase of rapid non-linear decrease in velocity and finally by a phase of slow linear decrease in velocity as the concentration of NaCl was increased. In the presence of 0.014 M-sodium acetate/acetic acid buffer (pH 5.0) there was a slight increase in enzymic activity in the presence of low concentrations of dioxan (up to about 10% dioxan) and a rapid decrease in enzymic activity at higher dioxan concentrations, but both effects were mitigated in the presence of 0.1 M buffer. The order of efficiency of added glucosyl acceptors in beta-glucosidase-catalysed reactions was found to be fructose greater than sucrose greater than glycerol greater than methanol. The enzyme was inactivated by the active-site-directed compound conduritol-B-epoxide; but this inactivation was concentration-dependent, was prevented by 10 mM-glucose, and involved an acidic group with pKa 4.3. A rate equation has been derived on the assumption of a mechanism of action involving a solvent-separated and an intimate glucosyl cation-carboxylate ion-pair intermediate and an alpha-glucosyl enzyme intermediate [Umezurike, G. M. (1981) Biochem. J. 199, 203-209]. Calculations based on the application of the derived rate equation and the calculated kinetic parameters show that the rate equation explains the peculiar properties of beta-glucosidase in the presence of added glucosyl acceptors or of NaCl.

Dioxanes↗
PubMed1987

Purification and properties of a stable beta-glucosidase from an extremely thermophilic anaerobic bacterium.

A beta-glucosidase (EC 3.2.1.21) was purified to homogeneity from cell-free extracts of an extremely thermophilic anaerobic bacterium. The enzyme has an Mr of 43,000 as determined by molecular-exclusion chromatography, has a pI of 4.55 and shows optimum activity at pH 6.2. The enzyme is active against a wide range of aryl beta-glycosides and beta-linked disaccharides, with beta-galactosidase activity only slightly less than beta-glucosidase activity, and significant beta-xylosidase activity. Lineweaver-Burk plots for p-nitrophenyl beta-glucoside, o-nitrophenyl beta-glucoside and cellobiose substrates are biphasic concave-downwards. Inhibition of the beta-glucosidase by substrates and glucose is negligible. Thermal inactivation follows first-order kinetics, with t1/2 (65 degrees C) 45 h, t1/2 (75 degrees C) 47 min and t1/2 (85 degrees C) 1.4 min and a deactivation energy of 380 kJ/mol at pH 6.2. At pH 7.0, which is the optimum pH for thermostability, t1/2 (75 degrees C) is 130 min. At 75 degrees C, at pH 6.2, the thermostability is enhanced about 8-fold by 10% (w/v) glycerol, about 6-fold by 0.2 M-cellobiose and about 3-fold by 5 mM-dithiothreitol and 5 mM-2-mercaptoethanol.

Bacteria, Anaerobic↗
PubMed1988

Kinetic studies on the broad-specificity beta-D-glucosidase from pig kidney.

A broad-specificity beta-D-glucosidase from pig kidney cortex was isolated and purified to homogeneity by a rapid purification procedure. The pI (5.14 +/- 0.05), Mr (59,000 +/- 2000) and specific activities with several p-nitrophenyl glycosides (galactopyranoside, glucopyranoside, arabinopyranoside, xylopyranoside) were comparable with those published previously for cytoplasmic beta-D-glucosidase from other sources and organs. Mixed-substrate experiments and inhibition studies with glucono-(1----5)-lactone revealed that a single active centre, containing one catalytic site and one saccharide-binding site, was responsible for the splitting of all four synthetic substrates. Inhibition experiments with substrate analogues demonstrated that (i) the major binding determinant of the glycosides was the aglycone moiety, (ii) an anionic side chain of the enzyme (probably a carboxy group) interacted with the glycosidic linkages and (iii) the properties of the aglycone significantly influenced the binding of the carbohydrate moiety. The inhibition constants of the p-nitrothiophenyl derivatives were in good agreement with the Km values of the corresponding substrates. Therefore the Michaelis constants could be regarded as true equilibrium constants (Ks). The 'three-point-attachment model' of the substrate splitting, proposed by Daniels [(1983) Ph.D. Dissertation, University of Pittsburgh] for the analogous liver enzyme, was applicable for beta-D-glucosidase from pig kidney too. The possible nature of the 'attachments' is discussed.

Animals↗
PubMed1986

Affinity chromatography of beta-glucosidase and endo-beta-glucanase from Aspergillus niger on concanavalin A-Sepharose: implications for cellulase component purification and immobilization.

Affinity chromatography of a commercial preparation of beta-glucosidase from Aspergillus niger using concanavalin A-Sepharose (CAS) was employed as a means of purifying this glycoprotein. However, mannose (up to 1.08 M) was ineffective as an eluent of this enzyme from CAS, as were several other sugars and their derivatives, including 0.5 M glucose. Also, washing the CAS: beta-glucosidase complex with buffer at pH 3.5 in the absence of MnCl2 and CaCl2 (required to preserve the binding activity of concanavalin A below pH 5.0) did not result in elution of this enzyme. On the contrary, endoglucanase activity present in a crude cellulase complex (A. niger) which bound to CAS could be eluted by mannose (0.5-0.7 M) and was fractionated into at least two components. The CAS: beta-glucosidase complex hydrolyzed cellobiose to glucose and possessed an activity of 2,158 units/g dry CAS. It could be used, therefore, for continuous cellobiose hydrolysis without leakage of enzyme from the support.

Adsorption↗
PubMed1985

Detection of human acid alpha-glucosidase in fibroblasts using monoclonal antibodies in a biotin-avidin amplified ELISA.

A sensitive and specific immunological assay for detection of human lysosomal alpha-glucosidase was developed using a mouse monoclonal antibody incorporated into a biotin-avidin amplified ELISA. The immunoassay was more than 60 times more sensitive than the currently used enzymatic assay for alpha-glucosidase activity using a fluorimetric substrate. This methodology provides an alternative approach with increased sensitivity for screening individuals for alpha-glucosidase deficiency.

Antibodies, Monoclonal↗
PubMed1999

Human acid alpha-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II.

Pompe's disease or glycogen storage disease type II (GSDII) belongs to the family of inherited lysosomal storage diseases. The underlying deficiency of acid alpha-glucosidase leads in different degrees of severity to glycogen storage in heart, skeletal and smooth muscle. There is currently no treatment for this fatal disease, but the applicability of enzyme replacement therapy is under investigation. For this purpose, recombinant human acid alpha-glucosidase has been produced on an industrial scale in the milk of transgenic rabbits. In this paper we demonstrate the therapeutic effect of this enzyme in our knockout mouse model of GSDII. Full correction of acid alpha-glucosidase deficiency was obtained in all tissues except brain after a single dose of i.v. enzyme administration. Weekly enzyme infusions over a period of 6 months resulted in degradation of lysosomal glycogen in heart, skeletal and smooth muscle. The tissue morphology improved substantially despite the advanced state of disease at the start of treatment. The results have led to the start of a Phase II clinical trial of enzyme replacement therapy in patients.

Animals↗
PubMed1985

Nucleotide sequence of Candida pelliculosa beta-glucosidase gene.

The nucleotide sequence of the DNA fragment containing the beta-glucosidase gene of Candida pelliculosa was determined. Analysis of the sequence revealed three open reading frames which could encode 65,825, and 412 amino acid residues. The presence of the second frame was found to be sufficient for the expression of the beta-glucosidase gene in a heterologous host Saccharomyces cerevisiae. Putative protein encoded by this gene had hydrophobic amino acids, resembling a signal peptide, at its N-terminal region and 19 potential glycosylation sites. Codon usage of Candida genes had the similar pattern shown in S.cerevisiae. Codon bias of the beta-glucosidase gene of Candida was relatively low, compared with that of the highly expressed genes of S. cerevisiae.

Amino Acid Sequence↗