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PubMed2004

A beta-glucosidase from Sclerotinia sclerotiorum: biochemical characterization and use in oligosaccharide synthesis.

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one beta-glucosidase (beta-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that beta-glu x may be a homodimer. For p-nitrophenyl beta-d-glucopyranoside hydrolysis, apparent Km and Vmax values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55-60 degrees C and pH 5.0, respectively. beta-Glu x was strongly inhibited by Fe2+ and activated about 35% by Ca2+. beta-Glu x possesses strong transglucosylation activity in comparison with commercially available beta-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50 degrees C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 3C nuclear magnetic resonance spectroscopy.

Ascomycota↗
PubMed1993

Characterization of the maltase activity of glucosidase II from rat liver. Kinetic model.

Glucosidase II is a key enzyme in the processing of N-glycoproteins since it removes the two glucose residues from the protein-linked oligosaccharide Glc2Man9GlcNAc2-R. We have studied the kinetics of the purified enzyme, using maltose as substrate. Analysis of data fitting to single and double-hyperbolic equations and the Eadie-Hofstee profile indicate that the enzyme has two binding (active) sites for the hydrolysis of maltose. The Km and Vmax values for the high-affinity site were 0.43 mM and 691 mU/mg, respectively, whereas the values for the low-affinity site were 57.7 mM and 2888 mU/mg, respectively. The Vmax/Km ratios were 1607 and 50.1 ml/min per g for the high- and low-affinity sites, respectively. A new kinetic model for this enzyme is proposed from the equilibria corresponding to the partial competitive inhibition produced by maltose on p-nitrophenyl-glucosidase activity. The amino acid composition of the enzyme has been established.

Amino Acids↗
PubMed1993

Segmental distribution of alpha-glucosidase, ornithine decarboxylase and polyamines in the human epididymis.

Regional differences in the total activities of the epididymal secretory parameter, alpha-glucosidase, were demonstrated in the 20,000 g supernatants of human epididymal homogenates. A comparison of the enzymic activities in the supernatants and the washings from 12 one centimetre segments of human epididymides indicated an activity peak in segments 3-4 which appeared to be largely intracellular and which presumably reflects the acidic isoenzyme. A second peak in the caudal region, the segmental localization of which was more variable and differed in post-mortem and operation specimens, appeared to be primarily intraluminal. The activities of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine pathway, also exhibited regional differences with higher activities in relatively short segments in both caput and caudal portions. Corresponding alterations were also found in the tissue concentrations of the enzyme products, spermidine and spermine. The increased intracellular activities of ODC and alpha-glucosidase in the distal caput segments presumably reflect the transition of epithelium from the efferent to the epididymal ducts.

Adult↗
PubMed1993

Islet glucan-1,4-alpha-glucosidase: differential influence on insulin secretion induced by glucose and isobutylmethylxanthine in mice.

In previous in-vivo studies we have presented indirect evidence for the involvement of islet acid glucan-1,4-alpha-glucosidase (acid amyloglucosidase), a lysosomal glycogen-hydrolysing enzyme, in certain insulin secretory processes. In the present combined in-vitro and in-vivo investigation, we studied whether differential changes in islet acid amyloglucosidase activity were related to the insulin secretory response induced by two mechanistically different secretagogues, glucose and isobutylmethylxanthine (IBMX). It was observed that addition of the selective alpha-glucosidehydrolase inhibitor emiglitate (1 mmol/l) to isolated pancreatic islets resulted in a marked reduction of glucose-induced insulin release. This was accompanied by a pronounced suppression of islet activities of acid amyloglucosidase and acid alpha-glucosidase, whereas other lysosomal enzyme activities, such as acid phosphatase and N-acetyl-beta-D-glucosaminidase, were unaffected. Furthermore, islets first incubated with emiglitate in the presence of high (16.7 mmol/l) glucose released less insulin than untreated controls in response to glucose in a second incubation period in the absence of emiglitate. In contrast, IBMX-induced insulin release was not influenced by emiglitate although accompanied by a marked reduction of islet activities of all three alpha-glucosidehydrolases. Basal insulin secretion (1 mmol glucose/l) was unaffected in the presence of emiglitate. In-vivo pretreatment of mice with highly purified fungal amyloglucosidase ('enzyme replacement'), a procedure known to increase islet amyloglucosidase activity, resulted in a greatly enhanced insulin secretory response to an i.v. glucose load. The increase in insulin release was accompanied by a markedly improved glucose tolerance curve in these animals. In contrast, enzyme pretreatment did not influence the insulin response or the blood glucose levels after an i.v. injection of IBMX. The data lend further support to our hypothesis that islet acid amyloglucosidase is involved in the multifactorial insulin secretory processes induced by glucose but not in those involving direct activation of the cyclic AMP system. The results also indicate separate, or at least partially separate, pathways for insulin release induced by glucose and IBMX.

1-Deoxynojirimycin↗
PubMedDate not supplied

Seasonal changes of neutral alpha-glucosidase activity in human semen.

Although there are contradictory reports, the biochemical evaluation of the seminal activity of neutral alpha-glucosidase (NAG) has repeatedly been described as an important parameter to test epididymal patency and is recommended by the WHO. Because, for a number of diagnostic parameters, seasonal variations have been described even in the human, it was the aim of this study to investigate possible circannual changes of NAG. This is an important aspect of andrological diagnosis, as seasonal changes of specific diagnostic parameters might have an impact on the accuracy and predictive power of these parameters, which in turn might have an effect on the therapeutic concept for the patients. In a total of 473 patients, sperm concentration, volume of the ejaculate, total motility, progressive motility, pH value, number of peroxidase-positive cells, concentration of fructose, and NAG as functional markers of the seminal vesicles and the epididymis, respectively, were analyzed according to standard procedures. Seminal activity of NAG was significantly correlated with the sperm concentration (P < .0001), ejaculate volume (P < .0001), and the pH (P = .0025). Moreover, significant (P = .0008) seasonal changes in the activity of seminal NAG with the maximum in spring (76.87 mU/ejaculate) and the minimum in autumn (58.55 mU/ejaculate) were found. The incidence of low-ranged activity of the enzyme was 9.2% in spring, while it was 20.3% in autumn. Thus, our data clearly demonstrate circannual changes of the seminal activity of neutral alpha-glucosidase. This in turn has clinical impact as the predictive power of the test system changes throughout the year.

Adolescent↗
PubMed2002

Acid alpha-glucosidase deficiency (glycogenosis type II, Pompe disease).

Glycogenosis type II (GSDII, Pompe disease) is an autosomal recessive lysosomal storage disease caused by a deficiency of acid alpha-glucosidase (acid maltase, GAA). The enzyme degrades alpha -1,4 and alpha -1,6 linkages in glycogen, maltose, and isomaltose. Deficiency of the enzyme results in accumulation of glycogen within lysosomes and in cytoplasm eventually leading to tissue destruction. The discovery of the acid a-glucosidase gene has led to rapid progress in understanding the molecular basis of glycogenosis type II and the biological properties of the GAA protein. The last decade has seen several developments: 1) extensive mutational analysis in patients with different forms of the disease, 2) characterization of the enzyme biosynthesis, processing, and lysosomal targeting, 3) generation of knockout mouse models, 4) development of viral vectors for gene replacement therapy, 5) the production of recombinant human enzyme, and 6) a shift in the enzyme replacement therapy approach from theory to practice. It is anticipated that the enzyme replacement therapy will be widely available for human use in the near future. Several recent reviews (including the most comprehensive one by R. Hirschhorn and A. Reuser [1]), address clinical, biochemical and genetic aspects of the disease, as well as development of new therapies for GSDII [2, 3, 4]. In this article we will review recent findings in the area including rapidly accumulating molecular genetic data (more than 20 mutations need to be added to the list), transcriptional control of gene expression, studies in mouse models, and new approaches to gene therapy. We will also highlight some emerging questions following the introduction of enzyme replacement therapy.

Adult↗
PubMed1990

Acid beta-glucosidase: enzymology and molecular biology of Gaucher disease.

Human lysosomal beta-glucosidase (D-glucosyl-acylsphingosine glucohydrolase, EC 3.2.1.45) is a membrane-associated enzyme that cleaves the beta-glucosidic linkage of glucosylceramide (glucocerebroside), its natural substrate, as well as synthetic beta-glucosides. Experiments with cultured cells suggest that in vivo this glycoprotein requires interaction with negatively charged lipids and a small acidic protein, SAP-2, for optimal glucosylceramide hydrolytic rates. In vitro, detergents (Triton X-100 or bile acids) or negatively charged ganglioside or phospholipids and one of several "activator proteins" increase hydrolytic rate of lipid and water-soluble substrates. Using such in vitro assay systems and active site-directed covalent inhibitors, kinetic and structural properties of the active site have been elucidated. The defective activity of this enzyme leads to the variants of Gaucher disease, the most prevalent lysosomal storage disease. The nonneuronopathic (type 1) and neuronopathic (types 2 and 3) variants of this inherited (autosomal recessive) disease but panethnic, but type 1 is most prevalent in the Ashkenazi Jewish population. Several missense mutations, identified in the structural gene for lysosomal beta-glucosidase from Gaucher disease patients, are presumably casual to the specifically altered posttranslational oligosaccharide processing or stability of the enzyme as well as the altered in vitro kinetic properties of the residual enzyme from patient tissues.

Amino Acid Sequence↗
PubMed1999

Heavy metal effects on beta-glucosidase activity influenced by pH and buffer systems.

Inhibition of beta-glucosidase activity by Cu(II), Zn(II) and Ni(II) was investigated as a function of pH and buffer type. Both factors were found to exert a strong effect on the activity of the enzyme. All three of the investigated heavy metals inhibited the enzyme activity in acetate buffer. At metal concentrations of 0.6 mM, Zn and Ni reduced the enzyme activity by 25-30% under optimal pH conditions (pH 5-5.2). Under the same conditions, Cu showed an even more pronounced inhibitory effect than Zn and Ni. In presence of 0.6 mM Cu, the enzyme activity was lowered by more than 90% in comparison to metal free systems. In contrast to these results, no enzyme inhibition was observed in citrate buffer, even in the presence of 1 mM Cu. The inhibition of beta-glucosidase activity by Cu increased with increasing pH. Inhibition by Zn and Ni was less pH-dependent in the observed pH range (pH 4-5.5). Copper caused a distinct shift in the pH optimum of enzyme activity, whereas this was not the case for Zn or Ni. The effects of buffer and pH on enzyme inhibition by Cu, Zn and Ni were successfully described using a chemical speciation model, based on the assumption that enzyme activity depends on the protonation of the amino acids at the reactive site and that enzyme activity is inhibited by complexation of the reactive sites by the heavy metal cations. The results show the importance of taking chemical conditions and speciation into account when investigating the effect of heavy metal cations on biological systems.

Buffers↗
PubMed2003

Inhibitory effects of hyssop (Hyssopus officinalis) extracts on intestinal alpha-glucosidase activity and postprandial hyperglycemia.

It has been known that Hyssopus officinalis (hyssop) is a herb that grows in the wild and is a source of natural antioxidants. We previously reported that a-glucosidase inhibitors, (2S, 3S)1-O-beta-D-6'-O-cinnamoylglucopyranosyl-3-(3", 5"-dimethoxy-4"-hydroxyphenyl)-1,2,3-propanetriol and (2S, 3S)1-O-beta-D-glucopranosyl-3-(3", 5"-dimethoxy-4"-hydroxyphenyl)-1,2,3-propanetriol, from the dry leaves of hyssop, were isolated. This study examined the alpha-glucosidase inhibitory effects of hyssop extracts on intestinal carbohydrate absorption in rat everted gut sac and carbohydrate-loaded hyperglycemia in mice. In the everted gut sac experiment, 10 mM sucrose- and 5 mM maltose-treated increases in glucose concentration in the serosal compartment were inhibited in the presence of 0.5 and 1.0 mg/ mL hyssop extracts, although a 10 mM glucose-induced increase in serosal glucose was not inhibited by the extracts. Additionally, hyperglycemia in sucrose- and maltose-loaded mice was significantly suppressed at an early stage, within 30 to 60 min by oral pre-administration of 300 and 100 mg/kg hyssop extracts, respectively. These findings suggest that hyssop extracts inhibited the digestion of complex carbohydrates, but not that of absorbable monosaccharide, and might be a useful supplemental food for hyperglycemia.

Animals↗
PubMed1990

Hydrolysis of pyridoxine-5'-beta-D-glucoside by a broad-specificity beta-glucosidase from mammalian tissues.

Research was conducted to evaluate the ability of a broad-specificity beta-glucosidase in mammalian tissues to catalyze the hydrolytic release of free pyridoxine from pyridoxine-5'-beta-D-glucoside, a naturally occurring form of vitamin B6 in plant-derived foods. Activity was detected in liver and intestinal mucosa using tritiated pyridoxine glucoside as a substrate. In the rat and guinea pig, enzyme activity was greater in intestine than in liver or kidney while even greater activity was detected in human intestinal tissue. Reaction rates were, however, low in all tissues. Hydrolysis of the synthetic substrate 4-methylumbelliferyl-beta-D-glucoside was also greatest in intestinal tissue. The characteristics of the enzymatic hydrolysis of pyridoxine glucoside to pyridoxine included: (i) most activity in the soluble tissue fraction, (ii) a pH optimum of approximately 6.0, and (iii) inhibition caused by the addition of sodium taurocholate. These characteristics are very similar to those of the broad-specificity beta-glucosidase in mammalian tissues with respect to the hydrolysis of a variety of naturally occurring and synthetic substrates. The apparent Km was greater than 2 mM for pyridoxine glucoside hydrolysis by intestinal preparations of each species, which is much greater than expected intestinal concentrations derived from dietary sources. In vivo studies have indicated that the intestine is involved in the metabolic utilization of dietary pyridoxine glucoside. The results observed here suggest that an alternate process, possibly involving intestinal microorganisms, may also be involved in the in vivo hydrolysis of pyridoxine glucoside.

Animals↗
PubMed1984

Valiolamine, a new alpha-glucosidase inhibiting aminocyclitol produced by Streptomyces hygroscopicus.

Valiolamine, a new aminocyclitol has been isolated from the fermentation broth of Streptomyces hygroscopicus subsp. limoneus and its structure has been determined to be (1(OH),2,4,5/1,3)-5-amino-1-C-(hydroxymethyl)-1,2,3,4-cyclohexanetetr ol. Valiolamine has more potent alpha-glucosidase inhibitory activity against porcine intestinal sucrase, maltase and isomaltase than valienamine, validamine and hydroxyvalidamine which were reported as building blocks of validamycins and microbial oligosaccharide alpha-glucosidase inhibitors. In addition, valienamine, validamine and hydroxyvalidamine have been isolated from the fermentation broth.

Chemical Phenomena↗
PubMed1999

Serum cytosolic beta-glucosidase elevation and early ischemic injury to guinea pig small intestine.

BACKGROUND: The lack of an early, sensitive marker for intestinal ischemia has led to delay in diagnosis and worsended outcome for patients with acute onset of this condition. Our preliminary studies revealed that guinea pig cytosolic beta-glucosidase (CBG) is expressed predominantly in the small intestine, with lower levels in the liver and pancreas and undetectable levels in other organs. Cytosolic beta-glucosidase was investigated as a serum marker of small intestinal ischemia in a guinea pig model. METHODS: Guinea pigs underwent anesthesia, sham laparotomy, 30 minutes of mesenteric ischemia followed by 6 hours of reperfusion 6 hours of sustained mesenteric ischemia, or closed-loop small bowel obstruction. Serum samples were assayed for CBG activity. At the conclusion of the ischemia/reperfusion experiments, small bowel samples were assayed for residual enzyme activity, and paraffin sections were graded for the severity of histologic injury. RESULTS: Serum CBG activity rose rapidly after intestinal ischemia with and without reperfusion. Peak enzyme activities were elevated 23-fold for reperfused animals (P < .001) by 4 hours. For nonreperfused animals, peak serum CBG activities reached 29-fold above baseline and were significantly higher than the CBG activities of reperfused animals at 4 hours (P < .01) and at 6 hours (P < .05). Mucosal injury ranged from undetectable to moderate and corresponded in severity with both peak serum enzyme activity and decreased residual activity in the small bowel. In animals subjected to closed-loop obstruction, there was a mean increase of serum CBG of 9.2-fold from 4 to 6 hours after establishment of obstruction (P < .05). CONCLUSIONS: In the guinea pig model, CBG is a sensitive marker of ischemic injury caused by arterial occlusion or closed-loop obstruction of the small bowel.

Animals↗
PubMed1999

Type 1 Gaucher disease presenting with extensive mandibular lytic lesions: identification and expression of a novel acid beta-glucosidase mutation.

The finding of extensive lytic lesions in the mandible of a 19-year-old Ashkenazi Jewish woman led to the diagnosis of Type 1 Gaucher disease. She had extensive skeletal involvement, marked hepatosplenomegaly, and deficient acid beta-glucosidase activity. Mutation analysis identified heteroallelism for acid beta-glucosidase mutations N370S and P401L, the latter being a novel missense mutation in exon 9. Expression of the P401L allele resulted in an enzyme with a reduced catalytic activity (specific activity based on cross-reacting immunological material approximately 0.21), which was similar to that of the mild N370S mutant enzyme. The expression studies predicted a mild phenotype for the proposita's N370S/P401L genotype which was inconsistent with her severe diffuse skeletal disease and organ involvement. Since lytic mandibular lesions may be complicated by osteomyelitis, pathologic fracture, and tooth loss, regular dental assessments in Type 1 Gaucher patients should be performed.

Child↗
PubMed2000

Routine alpha-amylase assay using protected 4-nitrophenyl-1, 4-alpha-D-maltoheptaoside and a novel alpha-glucosidase.

BACKGROUND: In contrast to numerous methods for measuring alpha-amylase activity, the approved IFCC reference method offers an invariable time-independent constant product pattern, thus avoiding possibly changing stoichiometric calculations. However, reference methods do not lend themselves to routine use, so that such methods need to be modified. METHODS: Ethylidene-blocked 4-nitrophenylmaltoheptaoside (EPS-G7) is degraded to glucose and 4-nitrophenol in a coupled assay with a bacterial alpha-glucosidase under the following measurement conditions: 3.5 mmol/L EPS-G7, 7.1 kU/L alpha-glucosidase, 70 mmol/L sodium chloride, 1 mmol/L calcium chloride, 50 mmol/L HEPES, pH 7.15, at 37 degrees C. The increase of absorbance is continuously monitored for 3 min at 405 nm after a lag phase of 2 min. RESULTS: Catalytic concentrations up to 15-fold higher than the upper reference limit can be determined without dilution. Precision studies in manual performance show CVs of 1.4-2. 6% (within-run) and 1.9-2.8% (day-to-day). There was no interference from 100 mmol/L glucose, 30 mmol/L triacylglycerols, 610 micromol/L bilirubin, and 2.95 g/L hemoglobin. The method closely correlates with other chromogenic assays. The preliminary 0.95 reference interval for adults, not dependent on age and sex, is 33.6-96.2 U/L. CONCLUSION: The procedure is a robust adaptation of the reference method to routine use at 37 degrees C with increased sensitivity, fewer interferences, and reduced cost.

Adult↗
PubMedDate not supplied

Production, characterization and properties of beta-glucosidase and beta-xylosidase from a strain of Aureobasidium sp.

beta-glucosidase and beta-xylosidase production by a yeast-like Aureobasidium sp. was carried out during solid-state and submerged fermentation using different carbon sources and crude enzymes were characterized. beta-glucosidase and beta-xylosidase gave optimum activity at pH 2.0-2.5 and 3.0, respectively. These enzymes presented maximal activity at 65 degrees, and were stable in a wide pH range and high temperatures.

Carbohydrate Metabolism↗
PubMedDate not supplied

Low frequency of isozyme 2 acid a-glucosidase in Greece.

The isozyme type 2 of acid a-glucosidase, identified by "affinity" starch gel electrophoresis, was found to have a gene frequency of 0.001 in an unselected Greek population from Southwestern Greece. The estimated frequency of the heterozygote in the population is 1 in 500 and of type 2 homozygote 10(-6). This frequency is by far lower than that reported in Europeans and other populations. This may be a characteristic of the Greek gene pool at the acid a-glucosidase locus or it may be present in populations of neighbouring countries as well.

Alleles↗
PubMed1991

Enzymic production of sweet stevioside derivatives: transglucosylation by glucosidases.

For the purpose of improving sweetness and a further study on the structure-sweetness relationship of steviol glycosides, transglycosylation of stevioside by a variety of commercial glucosidases was investigated. It was revealed that two alpha-glucosidases gave glucosylated products. Transglucosylation of stevioside by Pullulanase and pullulan exclusively afforded three products, 13-O-[beta-maltotriosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D-g luc osyl- steviol (1), 13-O-[beta-maltosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D-glucosyl- steviol (2) and 13-O-beta-sophorosyl-19-O-beta-maltotriosyl-steviol (3). All of these products have already been obtained by trans-alpha-1,4-glucosylation of stevioside by the cyclodextrin glucanotransferase starch system, and 1 and 2 have been proven to be tasty and potent sweeteners. Transglucosylation of stevioside by Biozyme L and maltose afforded three new products, 4, 5 and 6, the structures of these compounds being elucidated as 13-O-beta-sophorosyl-19-O-beta-isomaltosyl-steviol (4), 13-O-[beta-isomaltosyl(1----2)-beta-D-glucosyl]-19-O-beta-D-glucosyl- steviol (5) and 13-O-[beta-nigerosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D- glucosyl-steviol (6). A significantly high quality of taste was evaluated for 4.

Carbohydrate Sequence↗
PubMed1992

[Purification and characterization of alpha-glucosidase from an extreme thermophile, Thermus thermophilus HB 8].

A highly thermostable alpha-glucosidase (E C.3.2.1.20) from an extreme thermophile, Thermus thermophilus HB 8, was purified to homogeneous by ammonium sulfate fractionation, DEAE-cellulose chromatography and preparative slab gel electrophoresis. The enzyme was purified 17 fold with 21% recovery of activity. The enzyme had a molecular weight of 67000 by SDS-PAGE. The isoelectric point was pH4.5 by IEF on PAG. The enzyme hydrolized p-nitrophenyl-alpha-glucoside (PN-PG), sucrose and maltose, but not cellobiose, melibiose and soluble starch. The km value for PNPG was 0.4mmol/L, the Vmax was 0.29 mumol.min-1.mg-1. The enzyme exhibited high thermostability. After incubation at 90 degrees C for 10 h in 50 mmol/L acetate buffer pH 5.8, the enzyme retained 90% of its original activity. The half-live (t1/2) at 95 degrees C was 108 min. The enzyme was activated by Mg2+, Mn2+, Ca2+, Ba2+ and strongly inhibited by Hg2+, Cu2+. Modification of the enzyme by EDC or DEPC led to complete loss of activity, which suggests that carboxyl group(s) and histidine residue(s) are essential for activity of alpha-glucosidase.

Thermus thermophilus↗