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At least 19 recordsLinked to original sources
PubMed1997

Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase II is a thermostable M(r) 540,000 homohexameric alpha-glucosidase with both exo-alpha-1,4-glucosidase and oligo-1,6-glucosidase activities.

alpha-Glucosidase II of the facultative thermophile Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477; growth over 30-66 degrees C) was purified to a homogeneous state. Its M(r) was estimated as 90000 by SDS/PAGE. However, the enzyme behaved as an active Mr 540000 protein on gel filtration with each of two gels of different matrices as well as on gel electrophoresis under native conditions. The enzyme was not glycosylated. Its isoelectric point was estimated as 5.7. The N-terminal sequence of 20 residues was determined asAla1-Ile-Gln-Pro-Glu-Gln-Asp-Asp-Lys-Thr-Gln-Glu-Asp-Gly- Tyr-Ile-Asp-Ile-Gly-Asn20. The sequence did not resemble those of procaryotic and eucaryotic proteins hitherto reported including the monomeric exo-alpha-1,4-glucosidase and the monomeric oligo-1,6-glucosidase from the same microorganism. The alpha-glucosidase II had no antigenic group shared with the latter two enzymes. Analysis of substrate specificity showed that the alpha-glucosidase II has dual activity towards oligo-1,6-glucosidases and exo-alpha-1,4-glucosidases, but its preference is for non-reducing terminal alpha-1,4 glucosidic bonds in substrates. Kinetic studies proved that both activities are attributed to the same catalytic site. The enzyme was most active at 81 degrees C and pH 7.0. Its half-life at pH 6.8 was 10 min at 81 degrees C, and 5 h at 55 degrees C in 6.4 M urea, 26% ethanol or 2.5% SDS. We suggest that the alpha-glucosidase II is a thermostable, homohexameric enzyme of origin distinct from the exo-alpha-1,4-glucosidase and the oligo-1,6-glucosidase present in the same strain.

Amino Acids↗
PubMed1992

Assignment of Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase I to an exo-alpha-1,4-glucosidase, and its striking similarity to bacillary oligo-1,6-glucosidases in N-terminal sequence and in structural parameters calculated from the amino acid composition.

alpha-Glucosidase I of Bacillus thermoamyloliquefaciens KP1071 (FERM P8477, facultative thermophile) was purified to homogeneity. The relative molecular mass was estimated to be 62,000 Da. From its catalytic properties, the enzyme has been assigned to an exo-alpha-1,4-glucosidase. The enzyme shares its antigenic groups in part with Bacillus stearothermophilus ATCC12016 (obligate thermophile) exo-alpha-1,4-glucosidase. These exo-alpha-1,4-glucosidases strikingly resemble oligo-1,6-glucosidases from B. thermoamyloliquefaciens KP1071 and from Bacillus cereus ATCC7064 in the molecular properties tested, including relative molecular mass, N-terminal sequence of 15 residues, amino acid composition and structural parameters calculated from amino acid composition. We have suggested that bacillary exo-alpha-1,4-glucosidases take the same folded conformation, i.e. an (alpha/beta)8-barrel super-secondary structure in its N-terminal domain, as bacillary oligo-1,6-glucosidases.

Amino Acid Sequence↗
PubMed2000

The aglycone specificity-determining sites are different in 2, 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (Maize beta -glucosidase) and dhurrinase (Sorghum beta -glucosidase).

The maize beta-glucosidase isozyme Glu1 hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc (2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-on e), whereas the sorghum beta-glucosidase isozyme Dhr1 hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-d-glucose). To study the mechanism of substrate specificity further, eight chimeric beta-glucosidases were constructed by replacing peptide sequences within the C-terminal region of Glu1 with the homologous peptide sequences of Dhr1 or vice versa, where the two enzymes differ by 4 to 22 amino acid substitutions, depending on the length of the swapped regions. Five Glu1/Dhr1 chimeras hydrolyzed substrates that are hydrolyzed by both parental enzymes, including dhurrin, which is not hydrolyzed by Glu1. In contrast, three Dhr1/Glu1 chimeras hydrolyzed only dhurrin but with lower catalytic efficiency than Dhr1. Additional domain-swapping within the C-terminal domain of Glu1 showed that replacing the peptide (466)FAGFTERY(473) of Glu1 with the homologous peptide (462)SSGYTERF(469) of Dhr1 or replacing the peptide (481)NNNCTRYMKE(490) in Glu1 with the homologous peptide (477)ENGCERTMKR(486) of Dhr1 was sufficient to confer to Glu1 the ability to hydrolyze dhurrin. Data from various reciprocal chimeras, sequence comparisons, and homology modeling suggest that the Dhr1-specific Ser-462-Ser-463 and Phe-469 play a key role in dhurrin hydrolysis. Similar data suggest that DIMBOAGlc hydrolysis determinants are not located within the extreme 47-amino acid-long C-terminal domain of Glu1.

Amino Acid Sequence↗
PubMed1985

Identity of neutral alpha-glucosidase AB and the glycoprotein processing enzyme glucosidase II. Biochemical and genetic studies.

We have previously partially purified, characterized, and chromosomally mapped a human isozyme of alpha-glucosidase which is active at neutral pH. This isozyme appears as a doublet of enzyme activity on native gel electrophoresis and was termed neutral alpha-glucosidase AB. We now report genetic and biochemical evidence that neutral alpha-glucosidase AB is synonymous with the glycoprotein processing enzyme glucosidase II. We have found that a mutant mouse lymphoma line which is deficient in glucosidase II is also deficient in neutral alpha-glucosidase AB, as defined electrophoretically and quantitatively (less than 0.5% of parental). In contrast, both mutant and parental cell lines exhibited several lysosomal hydrolases which are processed by glucosidase II. We have also further purified the human neutral alpha-glucosidase A component of neutral alpha-glucosidase AB 740-fold from placenta in order to compare its biochemical properties with those described for rat liver and pig kidney glucosidase II. Both glucosidase II and neutral alpha-glucosidase AB are high-molecular mass (greater than 200,000 dalton) anionic glycoproteins which bind to concanavalin A, have a broad pH optima (5.5-8.5), and have a similar Km for maltose (4.8 versus 2.1 mM) and the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (35 versus 19 microM). Similar to human neutral alpha-glucosidase AB, purified rat glucosidase II migrates as a doublet of enzyme activity on native gel electrophoresis. Although rat glucosidase II has been reported to have a subunit size of 67 kDa, pig glucosidase II has been found to have a subunit size of 100 kDa, like the 98-kDa major protein in purified human neutral alpha-glucosidase A. Although we have not demonstrated that neutral alpha-glucosidase AB is microsomal nor that it hydrolyzes the natural substrate of glucosidase II, we believe that the genetic evidence is compelling for and the biochemical data consistent with the hypothesis that neutral alpha-glucosidase AB and glucosidase II are synonymous. These and previous results would localize glucosidase II to the long arm of human chromosome II.

Chromatography, Gel↗
PubMedDate not supplied

The effect of chymotrypsin on the determination of total alpha-glucosidase activity in seminal plasma and the correlation between alpha-glucosidase level and semen parameters.

To evaluate the effect of chymotrypsin on the examination of alpha-glucosidase activity in seminal plasma, thirty-nine samples of fresh liquefied semen with or without chymotrypsin and forty-eight samples of fresh un-liquefied semen with chymotrypsin were determined for the total alpha-glucosidase activity in seminal plasma. The total alpha-glucosidase level of each sample was assayed by the method of glucose oxidase. The correlations between alpha-glucosidase level and semen parameters, including semen volume, pH, sperm concentration, grade a and b motility and total motility, were analyzed with SPSS 11.0 software. The results showed that chymotrypsin had no effect on seminal alpha-glucosidase activity determination. Chymotrypsin could improve the liquefaction for un-liquefied semen, and there was no significant difference of alpha-glucosidase activity between liquefied and un-liquefied semen samples. There were significantly positive correlations between seminal alpha-glucosidase activity (U/ml) and sperm concentration (r = 0.338, p = 0.015) and between total alpha-glucosidase activity (U/ejaculate) and semen volume (r = 0.677, p = 0.000). However, there was no significant correlation between alpha-glucosidase level (U/ml) and semen volume, pH, sperm motility or grade a and b motility (r = -0.234 approximately 0.077, p = 0.099 approximately 0.993). The data indicated that chymotrypsin could be added into the un-liquefied semen samples for alpha-glucosidase activity determination, and there were different correlations between seminal alpha-glucosidase level and various semen parameters.

Chymotrypsin↗
PubMed2004

Localization of alpha-glucosidases I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of alpha-glucosidase in honey.

Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an alpha-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey alpha-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.

Amino Acid Sequence↗
PubMed1964

BETA-GLUCOSIDASE SYSTEM OF NEUROSPORA CRASSA. I. BETA-GLUCOSIDASE AND CELLULASE ACTIVITIES OF MUTANT AND WILD-TYPE STRAINS.

Eberhart, Bruce (University of North Carolina, Greensboro), David F. Cross, and Lewis R. Chase. beta-Glucosidase system of Neuspora crassa. I. beta-Glucosidase and cellulose activities of mutant and wild-type strains. J. Bacteriol. 87:761-770. 1964.-A mutant strain, gluc-1, of Neurospora crassa was isolated and characterized by its low level of beta-glucosidase activity. The mutant was selected by testing irradiated colonies for extracellular beta-glucosidase activity. Strains containing the gluc-1 gene were also visibly detected by their reduced ability to destroy esculin in their growth media. The mutant strain grew at wild-type rates with cellobiose or carboxymethylcellulose as carbon sources. This auxotrophic similarity with wild type is explained by the presence of at least two beta-glucosidases (and possibly two cellulases) in Neurospora that act complementarily. The thermolabile beta-glucosidase was destroyed after 1 min of incubation at 60 C. This enzyme was present in mycelia but absent in conidial extracts. A second beta-glucosidase that is comparatively stable at 60 C was present in both mycelia and conidia. A partial separation of these enzymes was achieved with ammonium fractionation of mycelial extracts of gluc-1 and wild-type strains. Thermolabile beta-glucosidase and cellulase activity appear not to be affected by the gluc-1 mutation, whereas the thermostable glucosidase is greatly reduced in gluc-1 strains.

Bacteriological Techniques↗
PubMed1990

Inhibitory effect of pseudo-aminosugars on oligosaccharide glucosidases I and II and on lysosomal alpha-glucosidase from rat liver.

We examined the inhibitory effect of three pseudo-aminosugars (validamine, valienamine, and valiolamine), which were isolated from the broth of Streptomyces hygroscopicus, on the oligosaccharide-processing glucosidases I and II involved in glycoprotein biosynthesis in rat liver. Both glucosidases I and II were inhibited to the same extent by the pseudoaminosugars, and valiolamine had a more potent inhibitory activity than validamine or valienamine. A 50% inhibition of valiolamine was observed at 12 microM for glucosidase I and glucosidase II activities acting respectively on the substrates Glc3Man9GlcNAc2 and p-nitrophenyl alpha-D-glucopyranoside. Further, in order to investigate further the ability of valiolamine to inhibit glucosidase I, reaction products were analyzed by gel filtration on a Bio-Gel P-4 column. We also compared the inhibitory action of these pseudo-aminosugars on the acid alpha-glucosidase of rat liver lysosomes. They competitively inhibited the hydrolysis of both substrates, maltose and glycogen. Valiolamine again had a more potent lysosomal alpha-glucosidase inhibitory activity than the other two. The Ki values of valiolamine for the hydrolysis of maltose and glycogen were 8.1 and 11 microM, respectively. Valiolamine is a particularly effective inhibitor of oligosaccharide glucosidases I and II and of lysosomal alpha-glucosidase. Hence valiolamine might be useful as a research tool in investigations of carbohydrate metabolism.

Amino Sugars↗
PubMed1982

Glycohydrolases in diabetes: characterization of acid alpha-glucosidase from liver and neutral alpha-glucosidase from sera of diabetic patients and controls.

Acid alpha-glucosidase activity measured in the supernatant fraction of liver homogenates obtained from adult-onset diabetic patients is significantly decreased when compared to controls (2.86 +/- 1.18 and 5.79 +/- 1.82 nmoles/min/mg protein +/- S.D., respectively). The biochemical properties (Km values, thermostability, pH optimum, isoelectric focusing profiles) of acid alpha-glucosidase obtained from the livers of diabetic patients were similar to those of acid alpha-glucosidase obtained from the livers of controls. Mixing studies gave additivity of acid alpha-glucosidase activity suggesting that neither inhibitors nor activators are present (in diabetic and control livers, respectively). Neutral alpha-glucosidase activity measured in the sera of diabetic patients was significantly increased when compared to controls (4.35 +/- 1.82 and 2.44 +/- 1.05 nmoles/h/ml +/- S.D., respectively). Neutral alpha-glucosidase in the sera of diabetic and control patients has a similar pH optimum but the enzyme in the serum of diabetics has a slightly lower apparent Km value for the 4-methylumbelliferyl substrate (0.6 vs. 0.9 mmol/L) and slightly increased thermostability. Experiments involving dialysis of patient serum, addition of glucose to patient serum and mixing of control and diabetic patient sera all suggest that glucose exhibits only slight inhibition of serum neutral alpha-glucosidase activity. Isoelectric focusing indicates that neutral alpha-glucosidase activity in the sera of diabetic patients is consistently different from the enzyme in the sera of control patients in that a significantly smaller percentage of activity is found in the acidic region with pI values less than 4.8.

Adolescent↗
PubMed1982

Identity of alpha-glucosidase of human kidney with urine F-1 alpha-glucosidase.

alpha-Glucosidase was extracted from a homogenate of human kidney, initially with 0.02 M Tris-HCl buffer, pH 7.6, and subsequently with a mixture of 0.5% cholate and 0.5% Triton X-100 in the same buffer, pH 7.6. The enzyme in each of these two fractions was purified to the electrophoretically pure state by fractional precipitation with ammonium sulfate, column chromatographies on DEAE-cellulose, hydroxyapatite, Bio Gel A-1.5 m and affinity chromatography on heated glutinous rice. The two purified alpha-glucosidase preparations obtained were the same in enzymatic and proteochemical properties, and the molecular weight and isoelectric point estimated were 3 x 10(5) and 4.2, respectively. No evidence for subunit structure was obtained. The optimum pH for activity was 5.6 and the activity was drastically inhibited by Nojirimycin. The alpha-glucosidase readily hydrolyzed maltose, starch, and glycogen, producing only glucose. It hydrolyzed maltotriitol to split the non-reducing end glucose, but scarcely hydrolyzed maltitol or various other heteroglucosides examined. All these proteochemical and enzymatic properties of kidney alpha-glucosidase were the same as those of urine F-1 alpha-glucosidase. Also, kidney tissue alpha-glucosidase produced a clear precipitin line with antisera against urine F-1 alpha-glucosidase. These facts suggest that F-1 alpha-glucosidase in urine originates from kidney tissue.

Glucosidases↗
PubMed2000

A specific beta-glucosidase-aggregating factor is responsible for the beta-glucosidase null phenotype in maize.

Maize (Zea mays L.) beta-glucosidase was extracted from shoots of a wild-type (K55) and a "null" (H95) maize genotype. Enzyme activity assays and electrophoretic data showed that extracts from the null genotype had about 10% of the activity present in the normal genotype. Zymograms of the null genotype were devoid of any activity bands in the resolving gel, but had a smeared zone of activity in the stacking gel after native polyacrylamide gel electrophoresis. When extracts were made with buffers containing 0.5% to 2% sodium dodecyl sulfate, the smeared activity zone entered the resolving gel as a distinct band. These data indicated that the null genotypes have beta-glucosidase activity, but the enzyme occurs as insoluble or poorly soluble large quaternary complexes mediated by a beta-glucosidase-aggregating factor (BGAF). BGAF is a 35-kD protein and binds specifically to beta-glucosidase and renders it insoluble during extraction. BGAF also precipitates beta-glucosidase that is added exogenously to supernatant fluids of the null tissue extracts. The specific beta-glucosidase-aggregating activity of BGAF is unequivocally demonstrated. These data clearly show that the monogenic inheritance reported for the null alleles at the beta-glucosidase gene is actually for the BGAF protein, and BGAF is solely responsible for beta-glucosidase aggregation and insolubility and, thus, the apparent null phenotype.

Phenotype↗
PubMedDate not supplied

Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe. Purification and characterization of intracellular beta-glucosidase.

Intracellular beta-glucosidase was extracted from the mycelium of Th. aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity. Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM. With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C. beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins. Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).

Amino Acids↗
PubMed1989

Nucleotide sequence of the Clostridium thermocellum bgIB gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases.

The nucleotide sequence of the bglB gene, coding for the thermostable beta-glucosidase B of Clostridium thermocellum was determined. The coding region of 2265 bp was identified by comparison with the N-terminal amino acid sequence of beta-glucosidase B purified from Escherichia coli. The derived amino acid sequence corresponding to a polypeptide of Mr 84,100 was confirmed by sequencing of the C-terminal peptide generated by cleavage with cyanogen bromide. The protein bears no resemblance to other bacterial beta-glucosidase sequences. However, extensive regions of homology were identified between the C. thermocellum enzyme and fungal beta-glucosidases. The N-terminal homologous region contains an amino acid sequence very similar to the active site of beta-glucosidase A3 from Aspergillus wentii. The striking sequence similarities between C. thermocellum beta-glucosidase B and Kluyveromyces fragilis beta-glucosidase suggest the possibility of a genetic exchange between thermophilic anaerobic bacteria and yeasts.

Amino Acid Sequence↗
PubMed1982

Serum beta-N-acetylglucosaminidase, beta-D-glucosidase, alpha-D-glucosidase, beta-D-fucosidase, alpha-L-fucosidase and beta-D-galactosidase levels in acute viral hepatitis, pancreatitis, myocardial infarction and breast cancer.

The specific activities of several glycosidases (beta-N-acetylglucosaminidase, beta-D-glucosidase, alpha-D-glucosidase, beta-D-fucosidase, alpha-L-fucosidase and beta-D-galactosidase) were determined in human sera from a control group to 10 normal subjects and in four groups, each of 10 patients, with acute viral hepatitis, acute pancreatitis, acute myocardial infarction and breast cancer. The results show significantly higher activities in acute viral hepatitis for beta-N-acetylglucosaminidase, beta-D-glucosidase and alpha-D-glucosidase (p less than 0.001); in acute pancreatitis for the first two of these enzymes (p less than 0.001); and in breast cancer for beta-D-glucosidase (p less than 0.001). Further, lower differences were found in the patients with acute viral hepatitis for beta-D-fucosidase and alpha-L-fucosidase (p less than 0.01); in acute myocardial infarction for beta-N-acetylglucosaminidase, beta-D-glucosidase, alpha-D-glucosidase, beta-D-fucosidase and beta-D-galactosidase (p less than 0.01, p less than 0.05, p less than 0.05, p less than 0.01 and p less than 0.01, respectively); and in breast cancer for beta-N-acetylglucosaminidase (p less than 0.01). No significant differences were found for the other glycosidases.

Acetylglucosaminidase↗
PubMed1998

Expression of enzymatically active, recombinant barley alpha-glucosidase in yeast and immunological detection of alpha-glucosidase from seed tissue.

An alpha-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae alpha-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant alpha-glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5-6.3 with an optimum at pH 4.3, classifying the enzyme as an acid alpha-glucosidase. The enzyme had a Km of 1.88 mM and Vmax of 0.054 micromol/min on maltose. The recombinant alpha-glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley alpha-glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa alpha-glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in alpha-glucosidase enzyme activity.

Amino Acid Sequence↗
PubMed2000

A potato alpha-glucosidase gene encodes a glycoprotein-processing alpha-glucosidase II-like activity. Demonstration of enzyme activity and effects of down-regulation in transgenic plants.

In order to elucidate more fully the function of a potato gene (MAL1) encoding alpha-glucosidase activity, transgenic plants in which MAL1 expression was down-regulated were generated using antisense technology. In transgenic lines severely down-regulated in the expression of MAL1, total alpha-glucosidase activity was not decreased in leaves and tubers, and the contents of starch, glucose, fructose and sucrose remained unchanged in tubers. Phylogenetic analysis indicated that the MAL1 gene product was more similar to the glycoprotein-processing alpha-glucosidase II of mammalian and yeast origin than to other plant alpha-glucosidases. Using [14C-Glc]-labelled Glc2Man9GlcNAc2 as a substrate, it was demonstrated that glucosidase II activity was markedly down-regulated in microsomes isolated from tubers of four independent antisense lines studied in detail, strongly suggesting that MAL1 encodes glucosidase II activity. In field trials (but not in the glasshouse), MAL1 down-regulation produced an extremely stunted phenotype - the leaves were curled and tuber yield was decreased by 90% compared to control values. Microscopic analysis of leaves revealed significant differences between the antisense and control samples. Plants with down-regulated glucosidase II activity showed a greater degree of plasmolysis, and an increase in the size of mesophyll intracellular spaces. Analysis of cell walls also indicated changes in structure as a result of MAL1 down-regulation. In leaves from four antisense lines, the steady-state transcript level corresponding to the endoplasmic reticulum chaperone, BiP, was enhanced. This is diagnostic of stress in the endoplasmic reticulum.

Carbohydrate Metabolism↗
PubMed1986

Isolation of a cDNA for human acid alpha-glucosidase and detection of genetic heterogeneity for mRNA in three alpha-glucosidase-deficient patients.

Lysosomal acid alpha-glucosidase (EC 3.2.1.3) hydrolyzes 1,4-linked alpha-D-glucose polymers present in glycogen. Genetic deficiency of acid alpha-glucosidase results in glycogen-storage disease type II, encompassing a spectrum of disorders of varying severity. To study the molecular basis for this heterogeneity, we sought to clone the coding sequence for human acid alpha-glucosidase. We screened 10(6) recombinant phage from a human liver cDNA expression library with an affinity-purified polyclonal antibody to human acid alpha-glucosidase. When we retested positive phage for reactivity to monoclonal antibodies, we identified a single phage, containing a 2-kilobase (kb) cDNA insert, that reacted with both polyclonal and monoclonal antibodies. The 2-kb cDNA hybridized to a 20-kb EcoRI fragment of human genomic DNA. This 20-kb EcoRI fragment was present only in DNA from somatic cell hybrids that retained the human chromosome 17 segment q21-q23, which contains the gene for human acid alpha-glucosidase. The cDNA also hybridized to a 3.4-kb mRNA, consistent with the size (approximately 105 kDa) of the acid alpha-glucosidase protein. Finally, in one of two infantile-onset acid alpha-glucosidase-deficient cell lines tested, the 3.4-kb mRNA was not detectable, whereas in an adult-onset cell line, an mRNA of reduced size and amount was found. Examination of DNA digested with restriction enzymes did not reveal any major deletions in the genomic DNA of these patients.

Chromosomes, Human, Pair 17↗
PubMed2000

Identification of beta-glucosidase aggregating factor (BGAF) and mapping of BGAF binding regions on Maize beta -glucosidase.

In certain maize genotypes (nulls), beta-glucosidase does not enter the gel and therefore cannot be detected on zymograms. Such genotypes were initially thought to be homozygous for a null allele at the glu1 gene. We have shown that a beta-glucosidase aggregating factor (BGAF) is responsible for the null phenotype, and it specifically interacts with maize beta-glucosidases and forms large insoluble aggregates. To understand the mechanism of the beta-glucosidase-BGAF interaction, we constructed chimeric enzymes by domain swapping between the maize beta-glucosidase isozymes Glu1 and Gu2, to which BGAF binds, and the sorghum beta-glucosidase (dhurrinase) isozyme Dhr1, to which BGAF does not bind. The results of binding assays with 12 different chimeric enzymes showed that an N-terminal region (Glu(50)-Val(145)) and an extreme C-terminal region (Phe(466)-Ala(512)) together form the BGAF binding site on the enzyme surface. In addition, we purified BGAF, determined its N-terminal sequence, amplified the BGAF cDNA by reverse transcriptase-polymerase chain reaction, expressed it in Escherichia coli, and showed that it encodes a protein whose binding and immunological properties are identical to the native BGAF isolated from maize tissues. A data base search revealed that BGAF is a member of the jasmonite-induced protein family. Interestingly, the deduced BGAF sequence contained an octapeptide sequence (G(P/R)WGGSGG) repeated twice. Each of these repeat units is postulated to be involved in forming a site for binding to maize beta-glucosidases and thus provides a plausible explanation for the divalent function of BGAF predicted from binding assays.

Amino Acid Sequence↗