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PubMed2001

Copy-DNA cloning and characterisation of a potato alpha-glucosidase: expression in Escherichia coli and effects of down-regulation in transgenic potato.

Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristics of an alpha-glucosidase. Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were alpha-glucosidases and alpha-xylosidases of plant origin. The MAL2 cDNA was expressed in Escherichia coli and the recombinant MAL2 protein was affinity-purified. MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nitrophenyl-alpha-D-glucopyranoside with a pH optimum of 5.5-5.7. The substrate with the lowest Km value was maltotetraose (3.7 mM). The MAL2 expression product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p-nitrophenyl-alpha-D-xylopyranoside or gelatinised potato starch. MAL2 was down-regulated in transgenic potato plants using an antisense approach. In several independent transgenic antisense lines, MAL2 expression was severely down-regulated. Despite this, no decrease in total extractable alpha-glucosidase and alpha-xylosidase activity could be detected in tissues from the transgenic plants. In glasshouse trials, no visible phenotype, change in tuber yield or carbohydrate content was associated with MAL2 down-regulation. The implications of these results are discussed.

Cloning, Molecular↗
PubMed1986

6-Epicastanospermine, a novel indolizidine alkaloid that inhibits alpha-glucosidase.

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

Alkaloids↗
PubMed1991

A quantitative assessment of the importance of barley seed alpha-amylase, beta-amylase, debranching enzyme, and alpha-glucosidase in starch degradation.

Extracts of germinated barley (Hordeum vulgare L.) seeds of 41 different genotypes were analyzed for their activities of alpha-amylase, beta-amylase, alpha-glucosidase, and debranching enzyme and for their abilities to hydrolyze boiled soluble starch, nonboiled soluble starch, and starch granules extracted from barley seeds with water. Linear correlation analysis, used to quantitate the interactions between the seven parameters, revealed that boiled soluble starch was not a good substrate for predicting activities of enzymes functioning in in vivo starch hydrolysis as the extracts' abilities to hydrolyze boiled soluble starch was not correlated with their abilities to hydrolyze native starch granules. Activities of alpha-amylase and alpha-glucosidase were positively and significantly correlated with the seed extracts' abilities to hydrolyze all three starches. beta-Amylase was only significantly correlated with hydrolysis of boiled soluble starch. No significant correlations existed between debranching enzyme activity and hydrolysis of any of the three starches. Interactions between the four enzymes as they functioned together to hydrolyze the three types of starch were evaluated by path coefficient analysis. alpha-Amylase contributed to hydrolyses of all three starches primarily by its direct effect (noninteractive component). This direct contribution increased as the substrate progressed from the completely artificial boiled soluble starch, to the most physiologically significant substrate, native starch granules. alpha-Glucosidase contributed to the hydrolysis of boiled soluble starch primarily by its direct effect (noninteractive) yet contributed to starch granule hydrolysis primarily via its interaction with alpha-amylase (indirect effect). The contribution of beta-amylase to hydrolysis of boiled soluble starch was direct and it did not contribute significantly to hydrolysis of native starch granules.

Glucosidases↗
PubMed1976

Specfic irreversible inhibition of sweet-almond beta-glucosidase by some beta-glycopyranosylepoxyalkanes and beta-d-glucopyranosyl isothiocyanate.

beta-D-Glucopyranosyl-(1S and 1R)-epoxyethanes (I and II), 1-(beta-D-glucopyranosyl)-(2R and 2S)-2,3-epoxypropanes (III and IV), beta-D-glucopyranosyl isothiocyanate (V) and beta-D-galactopyranosylepoxyethane (VI) are active-site-directed irreversible inhibitors of sweet-almond beta-glucosidase B (beta-D-Glucoside glucohydrolase, EC 3.2.1.21). Formation of the covalent bond is preceded by the binding of these inhibitors in the active site of the enzyme. This is testitified by the competitive character of inhibition of beta-glucosidase component B by compounds I-VI at the early period and by the protection of the enzyme from inactivation by its competitive inhibitors D-glucose and 1,5-D-gluconolactone. Epoxides I-IV are bound covalently with componet B at a molar ratio 1 : 1 as shown with the aid of 14C-labelled inhibitors. The release of the label from modified enzyme (E-I covalent) by treatment with hydroxylamine suggests the formation of an ester bond between inhibitors I-IV and the carboxyl group of the enzyme active site. The pH dependence curve of the inactivation rate of beta-glucosidase B is of a bell-shaped form for V and of a sigmoid character for I-IV and points to the involvement of the active site groups with pKa 5.6-5.9 and 4.2-4.4.

Binding Sites↗
PubMed1977

Cell-specific differences in membrane beta-glucosidase from normal and Gaucher cells.

Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.

Cell Membrane↗
PubMed1991

Identification of a point mutation in the human lysosomal alpha-glucosidase gene causing infantile glycogenosis type II.

Two patients in a consanguineous Indian family with infantile glycogenosis type II were found to have a G to A transition in exon 11 of the human lysosomal alpha-glucosidase gene. Both patients were homozygous and both parents were heterozygous for the mutant allele. The mutation causes a Glu to Lys substitution at amino acid position 521, just three amino acids downstream from the catalytic site at Asp-518. The mutation was introduced in wild type lysosomal alpha-glucosidase cDNA and the mutant construct was expressed in vitro and in vivo. The Glu to Lys substitution is proven to account for the abnormal physical properties of the patients lysosomal alpha-glucosidase precursor and to prevent the formation of catalytically active enzyme. In homozygous form it leads to the severe infantile phenotype of glycogenosis type II.

Alleles↗
PubMed1991

Cyclophellitol: a naturally occurring mechanism-based inactivator of beta-glucosidases.

The natural product cyclophellitol, isolated from the culture filtrate of a mushroom, Phellinus sp. is found to be a highly specific and effective irreversible inactivator of beta-glucosidases. It inactivates the beta-glucosidases from both almond emulsin and Agrobacter sp. according to pseudo-first order kinetics with inactivation constants of Ki = 0.34 mM, ki = 2.38 min-1, and Ki = 0.055 mM, ki = 1.26 min-1 respectively. No reactivation of the inactivated enzyme is seen upon dialysis, thus providing evidence for the irreversibility of the inactivation. The high specificity of this inactivator is evidenced by the fact that even at very high (12 mM) concentrations of cyclophellitol, no inactivation of yeast alpha-glucosidase was observed, and only extremely slow (t1/2 greater than 5 hours) inactivation of E. coli beta-galactosidase could be detected.

Alcaligenes↗
PubMed1989

Acarbose is a competitive inhibitor of mammalian lysosomal acid alpha-D-glucosidases.

Intraperitoneal injections (approximately 400 mg/kg of body weight) of acarbose, an inhibitor of acid (1----4)-alpha-D-glucosidase, perturb the metabolism of glycogen in the liver, resulting in excess storage of lysosomal glycogen. The metabolism of skeletal muscle glycogen was unaffected, suggesting that acarbose either does not enter the tissue or that the muscle alpha-D-glucosidase is not inhibited. The hydrolysis of maltose and glycogen by the acid alpha-D-glucosidases from rat liver, rat skeletal muscle, and human placenta was inhibited competitively by acarbose. Thus, the lack of effect of acarbose upon the metabolism of muscle glycogen is due to its inability to enter the tissue.

Acarbose↗
PubMed1976

A microfluorometric assay of leukocyte alpha-1,4-glucosidase.

We describe an improved method for detecting deficiency of the acid hydrolase, alpha-1,4-glucosidase in leukocytes, the enzyme defect in glycogen storage disease Type II (Pompe disease). The procedure requires smaller volumes of blood and less time than previous methods. The assay involves the separation of leukocytes by Peter's method for beta-glucosidase and a modification of Salafsky and Nadler's fluorometric method for alpha-glucosidase.

Adult↗
PubMed1976

Two alpha-glucosidases in cultured amniotic fluid cells and their differentiation in the prenatal diagnosis of Pompe's disease.

A sensitive fluorometric assay utilizing 4-methylumbelliferyl-alpha-D-glucopyranoside has been developed for the determination of alpha-glucosidase. The enhanced sensitivity was achieved by increasing the solubility of the substrate with a water miscible organic solvent. With this system, cultured amniotic fluid cells were found to have two major forms of alpha-glucosidase with somewhat overlapping acidic pH optima; one with pH optimum at 4.5 is deficient in Pompe's disease (type II glycogenosis), while one with pH optimum at 6.0 is not affected in this disease. Specificity for the pH 4 form of alpha-glucosidase was achieved by exploiting the greater thermal lability of the pH 6 enzyme. The pH 6 form of the enzyme was also detectable in freshly prepared extracts of cultured fibroblasts. The procedure is direct and simple and has been applied to the prenatal diagnosis in two pregnancies at risk for Pompe's disease.

Amniotic Fluid↗
PubMed1996

Purification and characterization of furostanol glycoside 26-O-beta-glucosidase from Costus speciosus rhizomes.

In plants, spirostanol glycosides (steroid saponins) are known to be formed furostanol glycosides during postharvest treatment and storage. Furostanol glycoside 26-O-beta-glucosidase (F26G) involved in this conversion was purified to apparent homogeneity for the first time from Costus speciosus rhizomes which accumulate these glycosides. The enzyme was highly specific for cleavage of the C-26-boudn glucose moiety of furostanol glycosides showing Km for protogracillin of 50 microM. Glucono-1,5-lactone, a typical beta-glucosidase inhibitor, and diosgenin, an aglycone of spirostanol glycosides, strongly inhibited the enzyme activity. The purified F26G is dimeric with a native apparent molecular weight of 110,000 consisting of subunits of 54,000 and 58,000. The N-terminal sequence of the 54,000 protein has a high similarity to the sequences found in N-terminal regions of known plant beta-glucosidases.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26-O-beta-glucosidase of Costus speciosus.

Furostanol glycoside 26-O-beta-glucosidase (F26G) purified from Costus speciosus rhizomes was digested with endoproteinase, and several internal peptide fragments were obtained. Degenerate oligonucleotide primers based on amino acid sequences of the peptides were used for amplification of F26G cDNA fragments by applying nested polymerase chain reactions to cDNAs from in vitro cultured plantlets of C. speciosus. Using primers based on sequences of the cDNA fragments, the 5'- and 3'-end clones were isolated by rapid amplification of cDNA ends (RACE) methods. Finally, the entire coding portion of F26G cDNA was cloned by using primers designed from sequences of the RACE products. The deduced amino acid sequence of CSF26G1, the protein encoded by the cloned cDNA, consists of 562 amino acids and shows high homology to a widely distributed family of beta-glucosidases (BGA family). Cell-free homogenate of Escherichia coli expressing CSF26G1 cDNA showed beta-glucosidase activity specific for cleavage of the C-26 glucosidic bond of furostanol glycosides.

Amino Acid Sequence↗
PubMed1987

Breakdown of lysosomal glycogen in cultured fibroblasts from glycogenosis type II patients after uptake of acid alpha-glucosidase.

Fibroblast cultures from patients with different clinical subtypes of glycogenosis type II were compared with respect to residual acid alpha-glucosidase activity and lysosomal glycogen content. Lysosomal glycogen storage was most pronounced in fibroblasts from patients with the rapidly progressive infantile form of the disease, and the most severe enzyme deficiency. In fibroblasts from adult patients with more than 10% of the control activity storage did not occur, and 15% of the total cellular glycogen was found in the lysosomes as in control cells. The strict correlation between residual acid alpha-glucosidase activity and lysosomal glycogen accumulation was further illustrated in two adult Pompe patients with an unusually low enzyme activity. The mild clinical course is unexplained in these particular cases. The enzyme deficiency in all the different mutant cell lines was corrected by the uptake of bovine testis acid alpha-glucosidase from the culture medium. As a result of this, the lysosomal glycogen storage disappeared, and the balance between lysosomal and cytoplasmic glycogen was restored to normal. The implications of this study as a model for enzyme replacement therapy are discussed.

Cell Fractionation↗
PubMed1986

In situ radiation-inactivation size of fibroblast membrane-bound acid beta-glucosidase in Gaucher type 1, type 2 and type 3 disease.

The radiation-inactivation size of membrane-bound acid beta-glucosidase in cultured skin fibroblasts of four normal individuals, five Gaucher type 1 (non-neuropathic), four Gaucher type 2 (acute neuropathic) and three Gaucher type 3 (sub-acute neuropathic) patients was determined using the radiation-inactivation method. The radiation-inactivation size of the enzyme in the control, Gaucher type 2 and Gaucher type 3 fibroblasts ranged from 94 000 to 128 800, and no statistical significant difference was found in the enzyme size between the normal and Gaucher cells nor among the Gaucher type 2 and type 3 cells. Contrary to the normal, Gaucher type 2 and Gaucher type 3 enzyme, the radiation-inactivation size of membrane-bound acid beta-glucosidase in all of the Gaucher type 1 fibroblasts tested is significantly higher, ranging from 158 400 to 235 300. The size of the control lysosomal enzyme, sphingomyelinase, also determined by the radiation-inactivation method in fibroblasts of normal individuals and patients with the three Gaucher subtypes, was between 70 000 and 74 500 and indistinguishable from each other. Since the molecular weight of acid beta-glucosidase subunit determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was about 60 000 (Pentchev, P.G., Brady, R.O., Hibbert, S.P., Gal, A.E. and Shapiro, C. (1973) J. Biol. Chem. 248, 5256-5261), the above data suggest that: (i) the normal fibroblast enzyme, as well as the Gaucher type 2 and type 3 mutant enzyme, in the membrane-bound form, exists as a dimer; (ii) the underlying biochemical and genetic defect in non-neuropathic (type 1) and neuropathic (type 2 and type 3) Gaucher disease is very different from each other; and (iii) subunit interaction of the mutant enzyme may be present in Gaucher type 1 fibroblasts, resulting in the formation of a higher-molecular-weight aggregate.

Adult↗
PubMed1994

Barley beta-glucosidase: expression during seed germination and maturation and partial amino acid sequences.

Unlike most of the hydrolytic enzymes that participate in endosperm mobilization, beta-glucosidase of barley (Hordeum vulgare) seeds does not increase during germination, even in the presence of exogenously added gibberellic acid. However, the germination process affects the physical properties of beta-glucosidase in terms of charge and apparent molecular weight. Analysis of developing barley grains shows that the enzyme is synthesized two weeks before maturation and is stored in the endosperm of the dry dormant seed. Partial amino acid sequencing of the purified beta-glucosidase demonstrates significant similarity between the barley enzyme and beta-glycosidases that belong to family 1 of glycosyl hydrolases.

Amino Acid Sequence↗
PubMed1991

Species-specific antibodies of Tetrahymena acid alpha-glucosidase.

1. Tetrahymena acid alpha-glucosidases A and B were purified from T. pyriformis W and T. thermophila 399, respectively. The acid alpha-glucosidases A and B were different in immunological properties and thermostability. 2. The acid alpha-glucosidases A and B reflected specific distribution between T. pyriformis and T. thermophila. 3. Type A and B of taurolipid showed a species-specific distribution pattern between T. pyriformis and T. thermophila.

Animals↗
PubMed1996

The enzymatic hydrolysis of 6-acylamino-4-methylumbelliferyl-beta-D-glucosides: identification of a novel human acid beta-glucosidase.

Fluorogenic 6-acylamino-4-methylumbelliferyl-beta-D-glucosides were found to be poor substrates for the three known human beta-glucosidases, i.e., lysosomal and non-lysosomal glucocerebrosidases and cytosolic broad-specificity beta-glucosidase. However, homogenates of human tissues and human cell types showed significant enzymatic hydrolysis of 6-ethanoylamino-4-methylumbelliferyl-beta-D-glucoside (EMGlc) due to the activity of a hitherto undescribed beta-glucosidase, called here EMGlc-ase. It was shown that the isozyme is hardly active towards 4-methylumbelliferyl-beta-D-glucoside or glucosylceramide. EMGlc-ase exhibits maximal activity at pH 4.5 and 5.0 in the absence and presence of sodium taurocholate respectively. It is a soluble lysosomal enzyme with a discrete isoelectric point of about 5.0. EMGlc-ase is not inhibited by conduritol B-epoxide, is activated by sodium taurocholate and binds strongly to Concanavalin A. This enzyme is not deficient in relation to Gaucher disease.

Animals↗
PubMed2005

Trp-262 is a key residue for the hydrolytic and transglucosidic reactivity of the Aspergillus niger family 3 beta-glucosidase: substitution results in enzymes with mainly transglucosidic activity.

Trp-262 of the Aspergillus niger family 3 beta-glucosidase is shown in this report to be a key residue for determining the ratio of this enzyme's hydrolytic and transglucosidic activities. TLC showed that when cellobiose was both the substrate and the acceptor, beta-glucosidases with substitutions (Phe, Ala, Leu, and Cys) for Trp-262 formed very high amounts of transglucosidic adducts. When pNPGlc was the substrate and the acceptor of the substituted beta-glucosidases, only transglucosidic adducts and pNP were produced. Little or no Glc could be detected, indicating that the reactions occurring were mainly transglucosidic. GLC studies with cellobiose quantitatively showed that one Glc was transferred for each free Glc produced. Since this is the maximum level of transglucosidation possible, this again showed that the reaction is predominantly transglucosidic. Analyses of the K(m) and K(i) values of cello-oligosaccharides of increasing length, of the K(i) values of Glc and of the transglucosidic activity at low acceptor concentration, showed that substitution for Trp-262 causes poor binding at the binding site for the non-reducing Glc of the substrate while the affinity for other Glc units is only minimally affected. The acceptor sites become saturated with substrate (acceptor) at the concentrations needed for glucosidic bond cleavage and thus only transglucosidic reactions occur. In addition, the data indicate that substitution for Trp-262 causes the rate of the hydrolysis step (k(3)) to be small.

Amino Acid Substitution↗