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PubMed2000

Sequence and expression of Thai Rosewood beta-glucosidase/beta-fucosidase, a family 1 glycosyl hydrolase glycoprotein.

Dalcochinin-8'-O-beta-glucoside beta-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both beta-glucosides and beta-fucosides. To determine its structure and evolutionary lineage, the sequence of the enzyme was determined by peptide sequencing followed by PCR cloning. The cDNA included a reading frame coding for 547 amino acids including a 23 amino acid propeptide and a 524 amino acid mature protein. The sequences determined at peptide level were found in the cDNA sequence, indicating the sequence obtained was indeed the dalcochinase enzyme. The mature enzyme is 60% identical to the cyanogenic beta-glucosidase from white clover glycosyl hydrolase family 1, for which an X-ray crystal structure has been solved. Based on this homology, residues which may contribute to the different substrate specificities of the two enzymes were identified. Eight putative glycosylation sites were identified, and one was confirmed to be glycosylated by Edman degradation and mass spectrometry. The protein was expressed as a prepro-alpha-mating factor fusion in Pichia pastoris, and the activity of the secreted enzyme was characterized. The recombinant enzyme and the enzyme purified from seeds showed the same K(m) for pNP-glucoside and pNP-fucoside, had the same ratio of V(max) for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-beta-glucoside.

Amino Acid Sequence↗
PubMed1989

Single active site mechanism of rabbit liver acid alpha-glucosidase.

An acid alpha-glucosidase was purified from rabbit liver by fractionation with ammonium sulfate, and chromatographies on Sephadex G-100, CM-Toyopearl, Toyopearl HW-55F, and Toyopearl HW-65F column. The resulting preparation showed a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be 1.03 X 10(4) by SDS-disc electrophoresis. The optimum pH was found to be 4.7. The alpha-glucosidase showed relatively high activity not only toward maltose but also toward alpha-glucans, such as shellfish glycogen, soluble starch, beta-limit dextrin, amylopectin, and amylose. The Km values for maltose and shellfish glycogen were 2.1 and 16 mM (the concentration of non-reducing glucose units), respectively, and the ratio of maximum velocities of hydrolysis of the two substrates was 100:133. The nature of the active site catalyzing the hydrolyses of maltose and shellfish glycogen was investigated by electrophoresis in the presence of urea and by kinetic methods. The purified enzyme was not separated into two components, maltase and glycogen hydrolase, in the electrophoretic gel containing 3 M urea, contrary to the report by Belenki and Rosenfeld ((1972) Biochem. Biophys. Res. Commun. 46, 443-448). In experiments with mixed substrates of maltose and glycogen, the kinetic features agreed very closely with those theoretically predicted for a single site mechanism. The essential ionizable groups, 1 (on the acidic side) and 2 (on the alkaline side), were identified as -COO- and -COOH for the hydrolysis of both substrates. Cations, Na+, K+, Mg2+, were about equally effective for the stimulation of enzyme action on maltose and glycogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Sulfate↗
PubMed1994

Substrate specificity and subsite affinities of rabbit liver acid alpha-glucosidase.

The substrate specificity of rabbit liver acid alpha-glucosidase was investigated. The enzyme showed a wide specificity for various substrates, and hydrolyzed alpha-glucans such as glycogen and soluble starch. The k0 values (s-1) for maltose, kojibiose, nigerose, isomaltose, phenyl alpha-glucoside, panose, phenyl alpha-maltoside, soluble starch, beta-limit dextrin, amylopectin, shellfish glycogen, and rabbit liver glycogen were estimated to be 94.8, 18.8, 143, 3.6, 11.8, 27.8, 115, 99.2, 155, 83.5, 126, and 108, and the Km values (concentration of non-reducing terminal) for these substrates were 2.1, 1.8, 7.5, 36, 5.4, 1.9, 1.2, 0.90, 9.1, 1.0, 16, and 13 mM, respectively. Isomaltose and phenyl alpha-glucoside were unfavorable as substrates. The acid alpha-glucosidase is characterized by a relatively high activity toward glycogen. The k0 values (s-1) for maltotriose, -tetraose, -pentaose, -hexaose, -heptaose, and -octaose, and maltodextrin (n = 17) were 140, 140, 131, 132, 134, 132, and 74.3, and the Km values, 2.1, 1.8, 1.9, 3.4, 5.0, 4.9, 4.9 and 2.6 mM, respectively. Based on the rate parameters for the series of maltooligosaccharides, the subsite affinities (Ais) in the active site were evaluated as 0.54 (A1), 5.34 (A2), and 0.34 (A3) kcal/mol for subsites 1, 2, and 3, respectively. These three subsites were considered to be predominantly responsible for the binding of substrates to the active site.

Animals↗
PubMed2006

Ambulatory electrocardiogram analysis in infants treated with recombinant human acid alpha-glucosidase enzyme replacement therapy for Pompe disease.

PURPOSE: Infantile Pompe disease is caused by deficiency of lysosomal acid alpha-glucosidase. Trials with recombinant human acid alpha-glucosidase enzyme replacement therapy (ERT) show a decrease in left ventricular mass and improved function. We evaluated 24-hour ambulatory electrocardiograms (ECGs) at baseline and during ERT in patients with infantile Pompe disease. METHODS: Thirty-two ambulatory ECGs were evaluated for 12 patients with infantile Pompe disease from 2003 to 2005. Patients had a median age of 7.4 months (2.9-37.8 months) at initiation of ERT. Ambulatory ECGs were obtained at determined intervals and analyzed. RESULTS: Significant ectopy was present in 2 of 12 patients. Patient 1 had 211 and 229 premature ventricular contractions (0.2% of heart beats) at baseline and at 11.5 weeks of ERT, respectively. Patient 2 had 10,445 premature ventricular contractions (6.7% of heart beats) at 11 weeks of therapy. CONCLUSION: Infantile Pompe disease may have preexisting ectopy; it may also develop during the course of ERT. Therefore, routinely monitoring patients using 24-hour ambulatory ECGs is useful. Periods of highest risk may be early in the course of ERT when there is a substantial decrease in left ventricular mass and an initial decrease in ejection fraction.

Cardiomegaly↗
PubMed2001

Corynebacterium freneyi sp. nov., alpha-glucosidase-positive strains related to Corynebacterium xerosis.

Three coryneform strains from clinical specimens were studied. They belonged to the genus Corynebacterium, since they had type IV cell walls containing corynemycolic acids. They had phenotypic characteristics that included alpha-glucosidase, pyrazinamidase and alkaline phosphatase activities and fermentation of glucose, ribose, maltose and sucrose. These are the characteristics of Corynebacterium xerosis. Since this species is very rare in human pathology, the strains were studied in more detail by comparing the 16S-23S intergenic spacers, rDNA sequences and levels of DNA similarity of these three strains and those of the reference strains C. xerosis ATCC 373T and Corynebacterium amycolatum CIP 103452T. According to DNA-DNA hybridization data, the three novel strains are members of the same species (level of DNA similarity >72%). Phylogenetic analysis revealed that these strains are closely related to C. xerosis and C. amycolatum, but DNA-relatedness experiments showed clearly that they constitute a distinct new species, with levels of DNA relatedness of less than 23% to C. xerosis ATCC 373T and less than 5% to C. amycolatum CIP 103452T. Two other alpha-glucosidase-positive strains presenting the same biochemical characteristics were included in the study and proved to be C. amycolatum. This new species can be differentiated from C. xerosis and C. amycolatum strains by carbon source utilization, intergenic spacer region length profiles and some biochemical characteristics such as glucose fermentation at 42 degrees C and growth at 20 degrees C. The name Corynebacterium freneyi sp. nov. is proposed with the type strain ISPB 6695110T (= CIP 106767T = DSM 44506T).

Anti-Bacterial Agents↗
PubMed2000

Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point alpha-glucosidase from barley malt.

High-isoelectric-point (pI) alpha-glucosidase was purified 7, 300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k(cat) = 25 s(-1)), with an optimum at pH 4.5, and catalyzed the hydrolysis by a retaining mechanism, as shown by nuclear magnetic resonance. Acarbose was a strong inhibitor (K(i) = 1.5 microM). Molecular recognition revealed that all OH-groups in the non-reducing ring and OH-3 in the reducing ring of maltose formed important hydrogen bonds to the enzyme in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an alpha-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a cDNA fragment from a barley cDNA library. HvAgl97 encodes a putative 96.6-kD protein of 879 amino acids with 93.8% identity to the protein deduced from U22450. The sequence contains two active site motifs of glycoside hydrolase family 31. Three introns of 86 to 4,286 bp interrupt the coding region. The four exons vary from 218 to 1,529 bp. Gene expression analysis showed that transcription reached a maximum 48 h after the start of germination.

Amino Acid Sequence↗
PubMed2005

Endoplasmic reticulum glucosidase II is required for pathogenicity of Ustilago maydis.

We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic alpha-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant-fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response.

Amino Acid Sequence↗
PubMed2005

Characterization of different crystal forms of the alpha-glucosidase MalA from Sulfolobus solfataricus.

MalA is an alpha-glucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. It belongs to glycoside hydrolase family 31, which includes several medically interesting alpha-glucosidases. MalA and its selenomethionine derivative have been overproduced in Escherichia coli and crystallized in four different crystal forms. Microseeding was essential for the formation of good-quality crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets could be collected. The most suitable crystals for structure determination are the monoclinic form 4 crystals, belonging to space group P2(1), from which data sets extending to 2.5 A resolution have been collected. Self-rotation functions calculated for this form and for the orthorhombic (P2(1)2(1)2(1)) form 2 indicate the presence of six molecules in the asymmetric unit related by 32 symmetry.

Carbohydrates↗
PubMed1992

Rapid and reversible inhibition of tyrosinase activity by glucosidase inhibitors in human melanoma cells.

The dependence of constitutively expressed tyrosinase (dopa oxidase) activity on glycosylation in lightly pigmented human melanoma cells (MM96E) was determined using tunicamycin (TM), which prevents transfer of oligosaccharide chains to nascent protein (core glycosylation), the glucosidase inhibitors castanospermine (CS) and deoxynojirimycin (dNM), and the mannosidase inhibitors deoxymannojirimycin (dMM) and swainsonine (SW). TM caused irreversible inhibition of tyrosinase activity and carbohydrate synthesis as judged by incorporation of 3H-fucose. Tyrosinase in CS- and dNM-treated cells showed 50% loss of activity within 5 h but recovered rapidly when the drugs were removed; dMN and SW had little effect. Expression of the tyrosinase 2B7 epitope and of an 80-kDa melanosomal antigen (B8G3) was inhibited by TM but not by CS, dNM, dMM, or SW. CS and dNM appeared to decrease the half-life of active tyrosinase. Overall, these results indicate that 1) in addition to the requirement for core glycosylation the removal of glucose residues plays a critical role in the formation of active human tyrosinase; 2) glucosidase inhibitors appear to cause an accumulation of inactive tyrosinase and increase the degradation rate of active enzyme; and 3) later stages in oligosaccharide processing are not required for maintaining tyrosinase activity.

1-Deoxynojirimycin↗
PubMed1990

The influence of inflammation of the human male genital tract on secretion of the seminal markers alpha-glucosidase, glycerophosphocholine, carnitine, fructose and citric acid.

Biochemical analysis was made of specific accessory gland products in the ejaculates of 362 men suffering from various acute inflammatory diseases of the reproductive tract and 33 normozoospermic patients acting as controls. The ejaculate content of the epididymal markers alpha-glucosidase and L-carnitine, but not glycerophosphocholine, was significantly reduced in ejaculates from men with epididymitis; citric acid was reduced in men suffering from prostatitis; both citric acid and alpha-glucosidase were reduced in men suffering from adnexitis. The ejaculate content of epididymal and prostatic markers in prostato-urethritis (adnexitis), where the exact localization of the inflammation was unclear, was not as low as in epididymitis or prostatitis. Seminal vesicle function, as judged from semen volumes and seminal fructose, was not different in these groups of patients. The results, although strongly related to the clinical diagnosis, were unrelated to the microbiological flora of the semen and indicate that both the epididymis and the prostate glands are involved in some forms of adnexitis.

Adolescent↗
PubMed1991

Prolactin and alpha-1,4-glucosidase activity in normal and poorly coagulated human semen.

Prolactin and alpha-1,4-glucosidase levels in seminal plasma were measured in poorly coagulated (I), deficiently coagulated (II) and normally coagulated (III and IV) human ejaculates having 0-20%, 21-50% and 51-100% coagulum respectively 4 min after emission. The prolactin concentration (ng ml-1, mean +/- SEM) in poorly coagulated (5.2 +/- 0.48) and deficiently coagulated (7.6 +/- 0.72) samples was significantly lower than in the normally coagulated groups III (51-75% coagulum, 8.2 +/- 0.43) and IV (76-100% coagulum, 9.9 +/- 0.59) as well as the presumably fertile samples (9.2 +/- 0.74). A highly significant positive correlation was observed between the prolactin level and the percentage coagulum of the ejaculates (r = 0.686, n = 58, P less than 0.001). In contrast, the epididymal marker, alpha-glucosidase showed no relationship to seminal coagulation.

Adult↗
PubMed1994

Relationship between semen characteristics, alpha-glucosidase and the capacity of spermatozoa to bind to the human zona pellucida.

Unfertilized oocytes from an in-vitro fertilization programme were stored in different saline solutions and then utilized in a zona binding assay (ZBA). The four saline solutions tested were identical with regard to the capacity of the zona to bind spermatozoa provided by healthy donors. Spermatozoa from 150 infertile patients were tested in the ZBA. The number of spermatozoa bound to the zona correlated positively with sperm concentration, the percentage sperm motility and the percentage of morphologically normal spermatozoa. The population was then divided into two groups according to the level of alpha-glucosidase activity, an epididymal marker. The average number of spermatozoa bound to the zona was diminished in the group with low alpha-glucosidase activity, even when considering strictly equivalent classes of sperm concentration, motility and morphology, respectively.

Biomarkers↗
PubMed1996

Homozygous deletion of exon 18 leads to degradation of the lysosomal alpha-glucosidase precursor and to the infantile form of glycogen storage disease type II.

We describe two unrelated Dutch patients with typical symptoms of infantile glycogen storage disease type II (GSD II) and virtual absence of acid alpha-glucosidase activity in leukocytes and cultured skin fibroblasts. The patients were identified as homozygotes for a deletion of exon 18 of the acid alpha-glucosidase gene (GAA). The in-frame deletion manifests at the protein level in a characteristic way: the enzyme precursor is smaller than normal and degraded in the endoplasmic reticulum or Golgi complex. These case present an evident example of a genotype-phenotype correlation in glycogen storage disease type II.

Chromosome Deletion↗
PubMed1998

Glycogen storage disease type II: identification of a dinucleotide deletion and a common missense mutation in the lysosomal alpha-glucosidase gene.

In nine Dutch patients with the infantile form of glycogen storage disease type II (GSDII), who were compound heterozygous for either 525delT or exon18del (1), sequence analysis was performed to search for the mutations in the second lysosomal alpha-glucosidase allele. One patient had a novel TG deletion at cDNA position 379 + 380. Surprisingly five of the nine patients had the same two base pair changes: A921 --> T and G925 --> A. The first change is a well-known polymorphism but the second one is a novel mutation and results in the substitution of Gly309 by Arg. By screening 43 other GSDII patients the same mutation was found in two other cases, one from The Netherlands and one from France. To verify its deleterious effect, the mutation was introduced in the wild type lysosomal alpha-glucosidase cDNA and expressed in COS cells.

Alleles↗
PubMed1986

Purification and characterization of Bacillus coagulans oligo-1,6-glucosidase.

A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing oligo-1,6-glucosidase of Bacillus coagulans ATCC 7050 (facultative thermophile) was purified to homogeneity. The relative molecular mass, Stokes radius, sedimentation coefficient at 20 degrees C in water, molecular absorption coefficient at 280 nm and pH 6.8, and isoelectric point were estimated as 60 000, 3.29 nm, 4.8 X 10(-13) s, 1.34 X 10(5) M-1 cm-1, and 4.3, respectively. The amino-terminal amino acid was threonine. There was no common antigenic group between the enzyme and each of its homologous counterparts from Bacillus cereus ATCC 7064 (mesophile) and Bacillus thermoglucosidasius KP 1006 (obligate thermophile). These oligo-1,6-glucosidases strongly resembled one another in their amino acid composition, except that the proline content increased with the elevation of thermostability in the order, mesophile----facultative thermophile----obligate thermophile enzymes.

Amines↗
PubMed1986

Specificity of human glucosylceramide beta-glucosidase towards synthetic glucosylsphingolipids inserted into liposomes. Kinetic studies in a detergent-free assay system.

The behaviour of highly purified glucosylceramide beta-glucosidase (glucosylceramidase, EC 3.2.1.45) from human placenta [Furbish, F. S., Blair, H. E., Shiloach, J., Pentchev, P. G. & Brady, R. B. (1977) Proc. Natl Acad. Sci. USA 74, 3560-3563] was investigated in the absence of detergents with structurally modified glucosylceramides inserted into unilamellar liposomes. The reaction between the water-soluble enzyme and the liposomal substrates was significantly dependent on the structure of the lipophilic aglycon moiety of glycolipids: glucosyl-N-acetyl-sphingosines (D-erythro and L-threo) were better substrates than the corresponding glucosylceramides. The L-threo derivatives were poorer substrates with higher apparent Km values than the corresponding D-erythro derivatives. For glucosyl-3-keto-ceramide and glucosyl-dihydro-ceramide (D-erythro), higher Km values were found than for glucosylceramide. Sphingosine, glucosylsphingosine and glucosyl-N-acetyl-sphingosine were the most effective inhibitors of the hydrolysis of glucosylceramide. D-erythro-Ceramide and D-galactosyl-N-acetyl-D-erythro-sphingosine inhibited the hydrolysis of amphiphilic glucosylceramide but not that of water-soluble 4-methyl-umbelliferyl-beta-glucoside, suggesting a hydrophobic binding site of the enzyme for the aglycon moiety of its membrane-bound substrate. Dilution experiments suggested that at least a fraction of the enzyme associates with the liposomes and degrades the lipid substrate even in the absence of activator proteins. Acidic phospholipids incorporated into liposomes caused a powerful stimulation (30-40-fold) of the glucosylceramide beta-glucosidase, whereas acidic sphingolipids (sulphatide, gangliosides GM1 and GD1a) incorporated into liposomes stimulated this enzyme only moderately (3-10-fold).

Cerebrosides↗
PubMed1994

Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase. Irrefragable proof supporting the proline rule.

Nine residues of Bacillus cereus ATCC7064 oligo-1,6-glucosidase were replaced stepwise with proline residues. Of the nine residues, Lys121, Glu208 and Glu290 were at second sites of beta turns; Asn109, Glu175 and Thr261 were at N-caps of alpha helices; Glu216, Glu270 and Glu378 were in coils within loops. The replacements were carried out in the order, Lys121-->Pro, Glu175-->Pro, Glu290-->Pro, Glu208-->Pro, Glu270-->Pro, Glu378-->Pro, Thr261-->Pro, Glu216-->Pro and Asn109-->Pro. The resultant nine active mutant enzymes contained 1-9 more proline residues than B. cereus oligo-1,6-glucosidase. The thermostability of these mutants was additively enhanced with the increase in the number of proline residues introduced. The increase in the thermostability was most remarkable when proline residues were introduced at second sites of beta turns or at N-caps of alpha helices. The above results afforded irrefragable proof for the proline rule as an effective principle for increasing protein thermostability [Suzuki, Y., Oishi, K., Nakano, H. & Nagayama, T. (1987) Appl. Microbiol. Biotechnol. 26, 546-551].

Amino Acid Sequence↗
PubMedDate not supplied

The presence of alpha-glucosidase, glycerophosphocholine and carnitine in the epididymis and ejaculate of the vervet monkey (Cercopithecus aethiops) and the Chacma baboon (Papio ursinus).

The epididymis is the site of posttesticular sperm maturation in the male genital tract. Studies on human epididymides are hampered by the practical inaccessibility of epididymides of healthy men in their reproductive years. The limited use of laboratory animals therefore seems unavoidable. The objective was to establish baseline values of the epididymal markers alpha-glucosidase, glycerophosphocholine (GPC) and carnitine in the lumen of the caput, corpus and cauda epididymidis and in the ejaculate of adult male Chacma baboons and vervet monkeys. In both primates, alpha-glucosidase was found throughout the epididymis and in the ejaculate; values did not vary significantly. In monkeys, the highest concentration of GPC was found in the cauda epididymidis, but smaller amounts were found in the other regions and the ejaculate. In baboons, GPC was absent from the caput, but present in the other regions, including the ejaculate. Carnitine concentrations increased significantly from the caput to the cauda in monkeys and from the caput to the corpus in baboons. With this study, the relative concentration ranges in which these markers are present in the epididymides of these primates have been established. In future studies, changes in concentrations of these substances would probably indicate changes in epididymal function.

Animals↗