Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “GLUCOSIDASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources
PubMed1993

An in situ study of beta-glucosidase activity in normal and Gaucher fibroblasts with fluorogenic probes.

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) beta-glucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferyl-beta-glucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.

Cells, Cultured↗
PubMed1988

Primary structure and processing of lysosomal alpha-glucosidase; homology with the intestinal sucrase-isomaltase complex.

Lysosomal alpha-glucosidase (acid maltase) is essential for degradation of glycogen in lysosomes. Enzyme deficiency results in glycogenosis type II. The amino acid sequence of the entire enzyme was derived from the nucleotide sequence of cloned cDNA. The cDNA comprises 3636 nt, and hybridizes with a messenger RNA of approximately 3.6 kb, which is absent in fibroblasts of two patients with glycogenosis type II. The encoded protein has a molecular mass of 104.645 kd and starts with a signal peptide. Sites of proteolytic processing are established by identification of N-terminal amino acid sequences of the 110-kd precursor, and the 76-kd and 70-kd mature forms of the enzyme encoded by the cDNA. Interestingly, both amino-terminal and carboxy-terminal processing occurs. Sites of sugar-chain attachment are proposed. A remarkable homology is observed between this soluble lysosomal alpha-glucosidase and the membrane-bound intestinal brush border sucrase-isomaltase enzyme complex. It is proposed that these enzymes are derived from the same ancestral gene. Around the putative active site of sucrase and isomaltase, 10 out of 13 amino acids are identical to the corresponding amino acids of lysosomal alpha-glucosidase. This strongly suggests that the aspartic acid residue at this position is essential for catalytic function of lysosomal alpha-glucosidase.

Amino Acid Sequence↗
PubMed1995

Induced expression of bacterial beta-glucosidase activity in Saccharomyces.

The bglA gene which encodes a beta-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the yeast CYC-GAL promoter. Strains have been constructed which carry the gene in different locations: in a multicopy plasmid, a single integration at the URA3 locus, or multiple integrations at the RDN1 locus. Integrative transformation at RDN1 yielded genetically stable clones with a high level of beta-glucosidase activity. Coordinated overexpression of the GAL4 inducer protein further increased the level of enzyme activity, although eventually caused the lysis of the cultures. Diploid, triploid and tetraploid strains derived from the transformants with multiple integrations were constructed and expression of beta-glucosidase activity in different conditions of growth was assayed. While per-cell activity increased with ploidy, specific activity was about the same in strains of equivalent genotype regardless of ploidy. Genetically stable and regulated expression in Saccharomyces of beta-glucosidase activity is interesting for the development of strains able to ferment beta-glycosidic sugars (i.e. cellobiose and lactose). From another point of view, the bglA product proved to be a convenient reporter enzyme to monitor heterologous gene expression.

Bacillus↗
PubMed2001

Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells.

We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control. DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing. The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting. Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver. In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable. The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform.

Animals↗
PubMed2000

The crystal structure of beta-glucosidase from Bacillus circulans sp. alkalophilus: ability to form long polymeric assemblies.

Family 1 of glycosyl hydrolases is a large and biologically important group of enzymes. A new three-dimensional structure of this family, beta-glucosidase from Bacillus circulans sp. alkalophilus is reported here. This is the first structure of beta-glucosidase from an alkaliphilic organism. The model was determined by the molecular replacement method and refined to a resolution of 2.7 A. The quaternary structure of B. circulans sp. alkalophilus beta-glucosidase is an octamer and subunits of the octamer show a similar (beta/alpha)(8) barrel fold to that previously reported for other family 1 enzymes. The crystal structure suggested that Cys169 in the active site is substituted. The Cys169 is located near the putative acid/base catalyst Glu166 and it may contribute to the high pH optimum of the enzyme. The crystal structure also revealed that the asymmetric unit contains two octamers which have a clear binding interaction with each other. The ability of the octamers to link with each other suggested that beta-glucosidase from Bacillus circulans sp. alkalophilus is able to form long polymeric assemblies, at least in the crystalline state.

Amino Acid Sequence↗
PubMed1994

Avenacosidase from oat: purification, sequence analysis and biochemical characterization of a new member of the BGA family of beta-glucosidases.

A protein consisting of 60 kDa subunits (As-P60) was isolated from etiolated oat seedlings (Avena sativa L.) and characterized as avenacosidase, a beta-glucosidase that belongs to a preformed defence system of oat against fungal infection. The enzyme is highly aggregated; it consists of 300-350 kDa aggregates and multimers thereof. Dissociation by freezing/thawing leads to complete loss of enzyme activity. The specificity of the enzyme was investigated with para-nitrophenyl derivatives which serve as substrates, in decreasing order beta-fucoside, beta-glucoside, beta-galactoside, beta-xyloside. The corresponding orthonitrophenyl glycosides are less well accepted. No hydrolysis was found with alpha-glycosides and beta-thioglucoside. An anti-As-P60 antiserum was prepared and used for isolation of a cDNA clone coding for As-P60. A presequence of 55 amino acid residues was deduced from comparison of the cDNA sequence with the N-terminal sequence determined by Edman degradation of the mature protein. The presequence has the characteristics of a stroma-directing signal peptide; localization of As-P60 in plastids of oat seedlings was confirmed by western blotting. The amino acid sequence revealed significant homology (> 39% sequence identity) to beta-glucosidases that are constituents of a defence mechanism in dicotyledonous plants. 34% sequence identity was even found with mammalian and bacterial beta-glucosidases of the BGA family. Avenacosidase extends the occurrence of this family of beta-glucosidases to monocotyledonous plants.

Amino Acid Sequence↗
PubMed1982

Inhibition by nojirimycin and 1-deoxynojirimycin of microsomal glucosidases from calf liver acting on the glycoprotein oligosaccharides Glc1-3Man9GlcNAc2.

Particulate membrane fractions from calf liver catalyze the release of glucose from GlcNAc2-Man9-Glc1-3-oligosaccharides. Maximal oligosaccharide-glucosidase activity was obtained at pH 6.2 and a detergent concentration of 0.5% Triton X-100. This activity could be distinguished from non-specific alpha-glucosidase activity on the basis of different pH-dependence and lack of activation by detergent. The relative rates for the hydrolysis of the Glc3-, Glc2-, and Glc1-oligosaccharide, estimated from the initial velocity, was 1:12:3. There is no significant difference in the enzyme activity towards free, peptide-bound, or lipid-linked oligosaccharide. Nojirimycin and 1-deoxynojirimycin were strong inhibitors of microsomal oligosaccharide-glucosidases. Hydrolysis of Glc3-oligosaccharide was inhibited by 50% at concentrations of 0.16 mM and 2 microM, respectively. Hydrolysis of the Glc2- and Glc1-oligosaccharide was inhibited to a somewhat lower extent, suggesting the presence of at least two glucosidases, one acting on Glc3- and one acting on Glc1- and Glc2-oligosaccharide.

1-Deoxynojirimycin↗
PubMed1992

Tris discriminates between the different alpha-glucosidase activities from extracts of human neutrophils.

In an attempt to detect acid maltase deficiency in neutrophils from patients with type II glycogenosis, without interference from the 'renal' alpha-glucosidase activity present in these cells, we have evaluated the contribution of the renal component in the total activity measured at pH 4.0 in extracts of human neutrophils. The renal contribution is about 13-25% and renal glucosidase appears to be closely related to the enzyme present on the epithelium of small intestine, which is known to be inhibited by Tris. We have used this compound as a selective inhibitor of the renal component of alpha-glucosidase activity measured at pH 4.0 in total extracts of neutrophils. Our results demonstrate that 0.1 mol/L Tris is an inhibitor of the renal alpha-glucosidase present in neutrophils and can be used to reduce the interference from this enzyme in assays of acid maltase.

Glycoside Hydrolase Inhibitors↗
PubMed1990

Maize genotypes classified as null at the glu locus have beta-glucosidase activity and immunoreactive protein.

Maize beta-glucosidase (beta-D-glucoside glucohydrolase; EC 3.2.1.21) was extracted from coleoptiles of 15 maize genotypes (3 normals, 10 nulls, and 2 hybrids) in two fractions, the soluble and the insoluble. The enzyme activity was measured spectrophotometrically in the soluble fraction and also studied on zymograms after native gel electrophoresis and isoelectric focusing. The enzyme was purified from a normal genotype by anion-exchange chromatography and preparative electrophoresis. Antisera were raised in four rabbits, and the soluble and the insoluble extracts of each genotype were analyzed for a cross-reacting material by ELISA and immunoblotting. The results showed that extracts from both the normal and the null genotypes had beta-glucosidase activity, and the activity measured spectrophotometrically was 2- to 10-fold higher in normals than in nulls. Zymograms of the null genotypes were devoid of distinct bands that were present in those of normals and hybrids from crosses between normals and nulls. Zymograms of both the normal and the null genotypes had a diffuse, smeared zone of activity at the cathodic end of native gels. A cross-reacting antigen was present in extracts of both genotypes when assayed by ELISA and a 60-kD polypeptide (beta-glucosidase monomer) was detected by four different monospecific beta-glucosidase antisera on Western blots by immunostaining. Moreover, six of seven null genotypes had a larger amount of their 60-kD polypeptide in the insoluble fraction than in the soluble fraction. These data show that both the null and the normal genotypes have similar amounts of the enzyme protein, but the enzyme occurs mostly as insoluble or poorly soluble polymers in nulls, and the monogenic inheritance reported for the null alleles of the glu locus is likely to be for a factor encoded by another locus which affects directly or indirectly the solubility of the enzyme by increasing its polymerization into large quaternary structures.

Alleles↗
PubMed1989

Biochemical microanalysis of alpha-glucosidase activity in preneoplastic and neoplastic hepatic lesions induced in rats by N-nitrosomorpholine.

Preneoplastic and neoplastic hepatic lesions were induced in male Sprague-Dawley rats by oral administration of N-nitrosomorpholine (NNM) for 7 weeks at a concentration of 200 mg/l of drinking-water (stop model). Using a laser dissection technique and biochemical microanalysis, the activity of the lysosomal enzyme alpha-glucosidase was measured in glycogen storage foci emerging early, and in mixed or basophilic cell populations (foci and carcinomas) appearing later during hepatocarcinogenesis. In the liver tissue of normal appearance in both untreated controls and NNM-treated animals a slight gradient of alpha-glucosidase activity was observed leading from relatively high activities in zone 1 to lower activities in zone 3 of the liver lobule. In preneoplastic glycogen storage foci a considerable relative reduction in alpha-glucosidase activity was detected, suggesting that a decrease in the hydrolytic glycogen degradation contributes to the disturbance in phosphorylytic glycogen breakdown observed earlier in the majority of the glycogenotic foci. In contrast with glycogen storage foci, mixed and basophilic cell foci and particularly hepatocellular carcinomas showed a marked increase in alpha-glucosidase activity compared with that of normal liver tissue. The gradual enhancement in enzyme activity appeared to be closely related to the reduction in glycogen initially stored in excess during the later stages of hepatocarcinogenesis. The results support the concept that a fundamental shift in carbohydrate metabolism is characteristic of neoplastic transformation of hepatocytes.

Animals↗
PubMed2000

Molecular cloning of a cDNA encoding alpha-glucosidase in the digestive gland of the shrimp, Litopenaeus vannamei.

The complete sequence of the 3-kb cDNA and the 5' genomic structure are reported for the gene encoding the shrimp alpha-glucosidase. Alpha-glucosidase cDNA was isolated from a shrimp digestive gland cDNA library. The 2830-base pair cDNA contains an open reading frame that encodes 919 amino acids. The shrimp alpha-glucosidase cDNA shows a high level of identity with that of the human sucrase-isomaltase, human maltase-glucoamylase, and human acid lysosomal alpha-glucosidase, indicating that the protein shares the same structural domains. The similarities among these proteins are found as clusters and characterize the glycosyl hydrolase family 31. To our knowledge, this is the first report to describe a satellite sequence in the 5' genomic structure before the TATA box in an invertebrate sequence.

5' Untranslated Regions↗
PubMed2003

cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea.

We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity.

Amino Acid Sequence↗
PubMed2002

Molecular characterisation of a xyloglucan oligosaccharide-acting alpha-D-xylosidase from nasturtium (Tropaeolum majus L.) cotyledons that resembles plant 'apoplastic' alpha-D-glucosidases.

We report the isolation, sequencing and analysis of the cDNA corresponding to an alpha-D-xylosidase involved in the mobilisation of xyloglucan from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds. The translated open reading frame (2,808 bp including the stop codon), gave a polypeptide of 935 amino acids. It included the sequences of eleven peptides obtained by endo-proteinase digestion of the protein, and a putative hydrophobic signal sequence characteristic of a protein that is directed through the plasma membrane. The deduced molecular weight of the translated protein was appreciably higher than the molecular weight determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, suggesting post-translational modification. The protein sequence showed high homology (76.0% identity over 896 amino acids) with a putative alpha-xylosidase sequence from Arabidopsis thaliana and there was homology with several alpha-glucosidases, notably those associated with the plant cell apoplast. The enzyme is a member of Family 31 of the glycosyl hydrolases and it fits into Clade 1 of the phylogenic analysis of alpha-glucosidases. Although in vivo the nasturtium enzyme catalyses mobilisation of cell wall xyloglucan, the homology of its primary sequence with alpha-glucosidases prompted study of its action on a range of alpha-glucosides. It was active against several alpha-(1-->4)-and alpha-(1-->6)-linked substrates, the former being hydrolysed faster. The functional and evolutionary relationships between this alpha-D-xylosidase and plant "apoplastic" alpha-D-glucosidases are discussed.

Amino Acid Sequence↗
PubMed2003

Production of beta-glucosidase using immobilised Piromyces sp. KSX1 and Orpinomyces sp. 478P1 in repeat-batch culture.

Two anaerobic fungi, one a monocentric strain ( Piromyces sp. KSX1) and the other a polycentric strain ( Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. beta-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, beta-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more beta-glucosidase than strain 478P1, the maximum specific beta-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor.

Anaerobiosis↗
PubMed2005

Purification and characterization of an intracellular beta-glucosidase from the methylotrophic yeast Pichia pastoris.

Pichia pastoris beta-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate, gel filtration, and ion-exchange chromatography with Q-Sepharose and CM-Sepharose. The enzyme is a tetramer (275 kD) made up of four identical subunits (70 kD). The pH optimum is 7.3, and it is fairly stable in the pH range 5.5-9.5. The temperature optimum is 40 degrees C. The purified beta-glucosidase is effectively active on p-/o-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with Km values of 0.12, 0.22, and 0.096 mM and Vmax values of 10.0, 11.7, and 6.2 micromol/min per mg protein, respectively. It also exhibits different levels of activity against p-nitrophenyl-1-thio-beta-D-glucopyranoside, cellobiose, gentiobiose, amygdalin, prunasin, salicin, and linamarin. The enzyme is competitively inhibited by gluconolactone, p-/o-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), and glucose against p-NPG as substrate. o-NPF is the most effective inhibitor of the enzyme activity with Ki value of 0.41 mM. The enzyme is more tolerant to glucose inhibition with Ki value of 7.2 mM for p-NPG. Pichia pastoris has been employed as a host for the functional expression of heterologous beta-glucosidases and the risk of high background beta-glucosidase activity is discussed.

Chromatography, Gel↗
PubMed2006

Phenotypic plasticity of cyanogenesis in lima bean Phaseolus lunatus-activity and activation of beta-glucosidase.

Cyanogenesis, the release of toxic HCN from damaged plant tissues, is generally considered as a constitutive plant defense. We found phenotypic plasticity of cyanogenesis in young leaves of lima bean Phaseolus lunatus based on increased activity of the beta-glucosidase in response to herbivore attack. Two aspects of plant cyanogenesis have to be considered in ecological analyses: (1) the cyanogenic potential (HCNp), which indicates the total amount of cyanide-containing compounds present in a given tissue, and (2) the cyanogenic capacity (HCNc), representing the release of HCN per unit time. This release is catalyzed by specific beta-glucosidases, whose activity is a crucial parameter determining overall toxicity. Enzymatic activity of beta-glucosidase-and, in consequence, the rate of HCN release-was increased significantly after 72 hr of incubation with spider mites as compared to non-infested leaves. Feeding by L1 larvae of Mexican bean beetles also led to enhanced enzymatic activity, whereas mechanical damage of leaf tissue had no effect on beta-glucosidase activity and the release of HCN. The results place plant cyanogenesis in the group of induced resistance traits, whose degree of activity depends on the feeding by a particular herbivore.

Animals↗
PubMed1989

Plant glucosidase II catalyzes a transglucosylation reaction in addition to the hydrolytic reaction.

When the purified plant glucosidase II was incubated with [3H]Glc2Man9GlcNAc in the presence of glycerol and the products were analyzed by gel filtration, a large peak of radioactivity emerged just before the glucose standard. The formation of this peak was dependent on both the presence of Glc2Man9GlcNAc and the presence of glycerol, and the amount of this product increased with time of incubation and amount of glucosidase II in the incubation. When the incubation was performed with [3H]Glc2Man9GlcNAc plus [14C]glycerol, the product contained both 14C and 3H. Strong acid hydrolysis of the purified product gave rise to [14C]glycerol and [3H]glucose. Various other chemical treatments and chromatographic techniques showed that the product was glucosyl----glycerol. Since the glucose was released by alpha-glucosidase, the product must be glucosyl-alpha-glycerol. This study demonstrates that the processing glucosidase II catalyzes a trans-glycosylation reaction in the presence of acceptors like glycerol. Since this transglycosylation reaction may give rise to unexpected products, investigators should be aware of its possible occurrence.

Fabaceae↗
PubMed1990

Purification and properties of an oligo-1,6-D-glucosidase from an alkalophilic Bacillus species.

An isomaltose-hydrolyzing alpha-D-glucosidase from the alkalophilic Bacillus designated strain F5 was purified to an electrophoretically homogeneous state. The molecular weight of the purified glucosidase was 60,000 by SDS-poly(acrylamide) gel electrophoresis, and 63,000 by Sephacryl S-200 gel-filtration chromatography. The enzyme was most active for isomaltose at pH 6.0-6.5 and 45 degrees, and stable up to 50 degrees at pH 7.0 and in the range of pH 6.0-9.0 at 50 degrees by 10-min incubation. The apparent Vmax and Km values for isomaltose were 34.5 mumol.min-1.mg-1 of protein, and 3.33 mM. Panose and isomaltotriose are the best substrates for this enzyme. The restricted substrate specificity indicated the assignment of the enzyme to be an oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase; EC 3.2.1.10), but it was suggested that it could be a new type of oligo-1,6-glucosidase on the basis of its action on a series of (1----4)-alpha-malto-oligosaccharides.

Bacillus↗