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PubMed1990

Developmental regulation of glucosidase I, an enzyme involved in the processing of asparagine-linked glycoproteins in rat mammary gland.

Glucosidase I involved in the processing of N-linked glycoproteins was purified to homogeneity from the lactating rat mammary gland. The purified enzyme exhibited a single band at 85 kDa on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Polyclonal antibodies raised against the enzyme recognized a similar band on Western blots and also inhibited the enzyme activity. The enzyme levels gradually increased until the midlactation stage and thereafter declined sharply during the period of postlactation. A similar profile of the levels of immunoreactive glucosidase I was observed. These findings suggest that the accumulation of glucosidase I is modulated as a function of gland ontogeny. The results on hormonal regulation of glucosidase I indicate that the synthesis of the enzyme is stimulated by a combination of insulin, hydrocortisone, and prolactin; additionally, epidermal growth factor may play a role in this regulation. The above observation was substantiated by immunoprecipitation of [35S]methionine-labeled microsomal extracts with anti-glucosidase I antibodies. The immunoprecipitation of soluble extracts from [35S]methionine-labeled tissue with anti-rat alpha-lactalbumin antibodies indicates that these hormones not only stimulate the synthesis of alpha-lactalbumin but also play an important role in its glycosylation.

Animals↗
PubMed1980

Substrate specificities of rat liver microsomal glucosidases which process glycoproteins.

The substrate specificities of rat liver microsomal glucosidases I and II, which hydrolyze oligosaccharides of the composition Glc3-1Man9GlcNAc, were investigated. Glucosidases I and II were shown to have a specific and necessary interaction with the 6'-pentamannosyl branch of their oligosaccharide substrates; activity was reduced when mannose residues were removed from this branch. Both glucosidases responded to features at the reducing end of the oligosaccharide substrates; activity was lower towards lipid-linked and peptide-linked substrates than towards free oligosaccharide substrates. In addition, the glucosidases appeared to discriminate between oligosaccharides linked to lipid and oligosaccharides linked to peptide. We conclude that rat liver microsomal glucosidases I and II interact with extensive portions of oligosaccharide structure in addition to the glucose residues which they hydrolyze.

Animals↗
PubMed1997

[Neutral alpha-glucosidase, a specific marker for epididymal secretion in seminal pathology].

Semen samples were obtained from 153 patients attending the Centre de recherche en Reproduction Humaine et en Démographie (CRRHD, Bénin) and seminal neutral alpha glucosidase activity was evaluated. Semen contains testicular and accessory gland secretions. Oligozoospernia asthenozoospernia and leucospermia are involved in reducing the fertilizing ability of the spermatozoa. The present work was undertaken to evaluate the variations of seminal alpha glucosidase activity as a specific epididymal enzyme marker in these diseases. A significant positive correlation was demonstrated (P < 0.01) between alpha glucosidase activity and (1) number of spermatozoa in oligozoospermic and normozoospermic men (2) motility of spermatozoa in asthenozoospermic men. No correlation occurred between alpha glucosidase activity and leucospermia. The results of the present study suggest that alpha glucosidase specific activity of the semen was involved in the maturation and acquisition of motility by the germ cells. Otherwise, in our sample, epididymis is scarcely involved in various inflammatory diseases of the reproductive tract.

Biomarkers↗
PubMed1987

Inhibition of bovine alpha-glucosidase by Castanospermum australe and its effect on the biochemical identification of heterozygotes for generalised glycogenosis type II (Pompe's disease) in cattle.

All 18 2-year-old Brahman bulls grazing in a paddock containing Castanospermum australe trees were diagnosed as heterozygotes for Pompe's disease by measurement of mononuclear cell alpha-glucosidase activity. However, removal of the bulls to a paddock free of C. australe and retesting 2 months later indicated that 15 were homozygous normal. An in vitro assay demonstrated that a crude aqueous extract of seeds from these C. australe trees contained a potent inhibitor of mononuclear cell alpha-glucosidase. Two Hereford steers were dosed with 0.6 g C. australe seed/kg bodyweight for 6 days. The alpha-glucosidase activity in blood mononuclear cells declined to 5% of normal within 48 h of commencement of dosing. It was therefore assumed that the bulls had consumed C. australe seeds. A means of differentiating true heterozygotes from animals consuming the toxic seed, using the ratio of plasma alpha-glucosidase activity at pH 5.6 to that at pH 3.7, is proposed.

Animals↗
PubMed1963

GENETIC CONTROL OF BETA-GLUCOSIDASE SYNTHESIS IN SACCHAROMYCES LACTIS.

Herman, Alberta (University of Wisconsin, Madison) and Harlyn Halvorson. Genetic control of beta-glucosidase synthesis in Saccharomyces lactis. J. Bacteriol. 85:901-910. 1963.-Both methyl-beta-d-glucoside (2 x 10(-2)m) and glucose (10(-3)m) induced beta-glucosidase synthesis in selected strains of Saccharomyces lactis. Genetic studies indicated the existence of a single locus specifically affecting beta-methyl glucoside inducibility. Glucose-induced beta-glucosidase synthesis, on the other hand, was nonspecific (other carbohydrases were simultaneously induced) and appeared to be controlled by more than one genetic factor. In both cases, noninducibility was dominant in diploids. The independent expression of these two modes of induction implied that these loci regulated beta-glucosidase induction in a nonsequential manner.

Cellulases↗
PubMed1987

The copurification of beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58.

Two enzyme complexes, each with beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21), beta-xylosidase (beta-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-beta-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75,400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the beta-xylosidase, beta-glucosidase, and 1,3-beta-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl beta-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the beta-glucosidase and 1,3-beta-glucanase of the major complex had inhibition constants which differed by more than 80,000 times. In addition, the inhibition on the 1,3-beta-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.

Chromatography, Ion Exchange↗
PubMed1987

Clinical diversity in glycogenosis type II. Biosynthesis and in situ localization of acid alpha-glucosidase in mutant fibroblasts.

The molecular basis of clinical diversity in glycogenosis type II (Pompe's disease) was investigated by comparing the nature of acid alpha-glucosidase deficiency in cultured fibroblasts from 30 patients. Biosynthetic forms of acid alpha-glucosidase with different molecular mass were separated electrophoretically and identified by immunoblotting. Immuno-electron microscopy was employed to determine the intracellular localization of mutant enzyme. Our studies illustrate that maturation of acid alpha-glucosidase is associated with transport to the lysosomes. Deficiency of catalytically active mature enzyme in lysosomes is common to all clinical phenotypes but, in the majority of cases, is more profound in early onset than in late onset forms of the disease. Thus, the results suggest that the clinical course of glycogenosis type II is primarily determined by the amount of functional acid alpha-glucosidase. The role of secondary factors can, however, not be excluded because three adult patients were identified with very low activity and little enzyme in the lysosomes.

Adult↗
PubMed1985

Defects in synthesis, phosphorylation, and maturation of acid alpha-glucosidase in glycogenosis type II.

Glycogenosis type II is an inherited lysosomal storage disease with acid alpha-glucosidase deficiency as the primary defect. Using cultured skin fibroblasts, we have studied the biosynthesis of acid alpha-glucosidase in clinically different forms of this disease. Three unrelated patients were identified (one with an infantile, one with a juvenile, and one with an adult form of the disease) producing normal quantities of the 110-kDa precursor form of acid alpha-glucosidase. However, post-translational modification to mature 76-kDa enzyme protein was either completely deficient or extremely inefficient. No abnormalities were observed in glycosylation of the mutant precursors, as measured by the incorporation of [3H]mannose, but phosphorylation was only detectable for the precursor synthesized by fibroblasts from the juvenile patient. In three other patients (one with a juvenile and two with adult forms of glycogenosis type II) apparently reduced synthesis of precursor protein was observed, but the processing to mature enzyme seemed to be undisturbed. Finally, neither precursor nor mature forms of acid alpha-glucosidase were detectable in one particular case of infantile glycogenosis type II. The studies reveal an unexpected degree of genetic heterogeneity in this disease and identify various mutants which could be of importance to further elucidate the biosynthetic events during lysosomal enzyme formation.

Adolescent↗
PubMed1998

Translational inefficiency of acid beta-glucosidase mRNA in transgenic mammalian cells.

The cDNA for acid beta-glucosidase, the Gaucher disease enzyme, was overexpressed in a variety of mammalian cells and in Sf9 insect cells. Whether overexpressed from the MFG-GC retrovirus or the tetracycline transactivator system, there was a large discrepancy between the amounts of mRNA (>750-fold) and acid beta-glucosidase protein (approximately 6- to 14-fold) produced in mammalian cells. This was not observed in Sf9 insect cells. Quantitative evaluation of translation of this mRNA in intact mammalian cells indicated a 55- to 135-fold inefficiency in cell lines compared to normal human skin fibroblasts. In vitro translation efficiency with acid beta-glucosidase mRNAs from overexpressing mammalian or insect cells was similar to that from normal human fibroblasts. A cytoplasmic, heat labile protein was suggested as inhibitory to in vitro translation of these RNAs. North-Western blots and cytoplasmic depletion experiments showed this to be an 80-kDa cytoplasmic mRNA-binding protein that recognized acid beta-glucosidase coding sequences. The cytoplasmic protein was not detected in insect cells. These results implicate acid beta-glucosidase coding sequences and a heat labile cytoplasmic protein in modulating the translation of overexpressed mRNA in transgenic cell lines.

Animals↗
PubMed1996

Use of beta-maltosides (p-nitrophenyl-beta-D-maltoside, 2-chloro-4- nitrophenyl-beta-D-maltoside and 4-methylumbelliferyl-beta-D-maltoside) as substrates for the assay of neutral alpha-glucosidase from human kidney and urine.

The method of assay of neutral alpha-glucosidase from human kidney and urine using beta-maltosides (p-nitrophenyl-beta-D-maltoside [NP-beta-D-maltoside], 2-chloro-4-nitrophenyl-beta-D-maltoside]) [CNP-beta-D-maltoside] and 4-methylumbelliferyl-beta-D-maltosides ([MU-beta-D-maltoside]) as substrates and beta-glucosidase as an auxiliary enzyme is proposed. All three beta-maltosides are suitable substrates for the determination of neutral alpha-glucosidase activity but MU-beta-D-maltoside is the most sensitive due to its methylumbelliferyl moiety. The method is simple, convenient and 10-fold more sensitive than the commonly used alpha-glucosidase assay procedure with the corresponding synthetic alpha-glucosides, p-nitrophenyl- alpha-D-glucoside (NP-alpha-D-glucoside) and 4-methylumbelliferyl-alpha-D-glucoside (MU-alpha-D-glucoside). A modification of the method, with p-nitrophenyl-beta-D-maltoside as substrate, was applied to the semiautomatic assay of urinary alpha-glucosidase in 96-well microtitre plates.

Acetylglucosaminidase↗
PubMed1998

Acid beta-glucosidase: intrinsic fluorescence and conformational changes induced by phospholipids and saposin C.

Acid beta-glucosidase is a lysosomal membrane protein that cleaves the O-beta-D-glucosidic linkage of glucosylceramide and aryl-beta-glucosides. Full activity reconstitution of the pure enzyme requires phospholipids and saposin C, an 80 aa activator protein. The deficiency of the enzyme or activator leads to Gaucher disease. A conformational change of acid beta-glucosidase is shown to accompany activity reconstitution by selected phospholipids or, particularly, phospholipid/saposin C complexes by intrinsic fluorescence spectral shifts, fluorescence quenching, and circular dichroism (CD). Negatively charged phospholipid (NCP) interfaces with unsaturated fatty acid acyl chains (UFAC) induced concordant blue-shifts in tryptophanyl fluorescence spectra and a loss of beta-strand structure by CD. The enzyme required an unsaturated fatty acid acyl chain in proximity (10-11 A) within liposomal membranes for activation, fluorescence blue-shifts, and changes in CD spectra. Activity enhancements were greatest when UFAC and the negatively charged headgroup were present on the same phospholipid. NCPs with UFAC protected the enzyme from fluorescence quenching by aqueous agents (I-, Cs+, acrylamide, TEMPO). Phosphatidylcholine with doxyl spin-labeled fatty acid acyl chains at carbons 7, 10, or 16 quenched enzyme fluorescence only when in NCP/PC liposomes. Saposin C (Trp-free) induced additional activity and fluorescence spectral changes in the enzyme only in the presence of NCP liposomes containing UFA. CD spectral changes indicated saposin C and acid beta-glucosidase interaction only in the presence of NCPs with UFA. These studies show that acid beta-glucosidase requires interfaces composed of NCPs, containing UFAC, for penetration into the outer leaflet of membranes. Furthermore, this interaction induces essential conformational changes for saposin C binding and further enhancement of acid beta-glucosidase catalytic activity.

Animals↗
PubMed2005

Different polyphenolic components of soft fruits inhibit alpha-amylase and alpha-glucosidase.

Polyphenol-rich extracts from soft fruits were tested for their ability to inhibit alpha-amylase and alpha-glucosidase. All extracts tested caused some inhibition of alpha-amylase, but there was a 10-fold difference between the least and most effective extracts. Strawberry and raspberry extracts were more effective alpha-amylase inhibitors than blueberry, blackcurrant, or red cabbage. Conversely, alpha-glucosidase was more readily inhibited by blueberry and blackcurrant extracts. The extent of inhibition of alpha-glucosidase was related to their anthocyanin content. For example, blueberry and blackcurrant extracts, which have the highest anthocyanin content, were the most effective inhibitors of alpha-glucosidase. The extracts most effective in inhibiting alpha-amylase (strawberry and raspberry) contain appreciable amounts of soluble tannins. Other tannin-rich extracts (red grape, red wine, and green tea) were also effective inhibitors of alpha-amylase. Indeed, removing tannins from strawberry extracts with gelatin also removed inhibition. Fractionation of raspberry extracts on Sephadex LH-20 produced an unbound fraction enriched in anthocyanins and a bound fraction enriched in tannin-like polyphenols. The unbound anthocyanin-enriched fraction was more effective against alpha-glucosidase than the original extract, whereas the alpha-amylase inhibitors were concentrated in the bound fraction. The LH-20 bound sample was separated by preparative HPLC, and fractions were assayed for inhibition of alpha-amylase. The inhibitory components were identified as ellagitannins using LC-MS-MS. This study suggests that different polyphenolic components of fruits may influence different steps in starch digestion in a synergistic manner.

Anthocyanins↗
PubMed1995

Effects of calcium on phosphatidylserine- and saposin C-stimulated glucosylceramide beta-glucosidase activity.

Glucosylceramide beta-glucosidase is a membrane-bound lysosomal hydrolase that is activated by acidic lipids, the most effective of which is phosphatidylserine (PtdSer), and an activator protein, saposin C. This report documents effects of Ca2+ ions on PtdSer- and saposin C-enhanced beta-glucosidase activity. Ca2+ either increased or decreased enzyme activity, depending on (1) the concentration of phospholipid, and (2) the presence or absence of saposin C. At PtdSer concentrations between 7.6 and 76 microM, in the absence of saposin C, Ca2+ caused an increase in beta-glucosidase activity up to 3 times that measured with PtdSer alone; this was due to both an increase in Vmax, and a decrease in Km. In contrast, at PtdSer concentrations greater than 100 microM, Ca2+ inhibited beta-glucosidase activity by 50%, due to a 2-fold increase in Km. Ca2+ was inhibitory at all PtdSer concentrations tested when both PtdSer and saposin C were present in the assay. Ca2+ ions were also shown to cause changes in the aggregation states of PtdSer. These results suggest that changes in Ca2+ concentration may play a role in regulating beta-glucosidase activity in vivo, thereby modulating sphingolipid metabolism. The implications of these findings are discussed.

Animals↗
PubMed2005

Inhibition of alpha-glucosidase by aqueous extracts of some potent antidiabetic medicinal herbs.

Diabetes mellitus is one of the most prevalant diseases of adults. Agents with alpha-glucosidase inhibitory activity have been useful as oral hypoglycemic drugs for the control of hyperglycemia in patients with type 2; noninsulin-dependent, diabetes mellitus (NIDDM). Investigation of some medicinal herbs: Urtica dioica, Taraxacum officinale, Viscum album, and Myrtus communis with alpha-glucosidase inhibitor activity was conducted to identify a prophylactic effect for diabetes in vitro. All plants showed differing potent alpha-glucosidase inhibitory activity. However, Myrtus communis strongly inhibited the enzyme (IC50 = 38 microg/mL). The inhibitory effect of these plants and some common antidiabetic drugs against the enzyme source (baker's yeast, rabbit liver, and small intestine) were also searched. Approximately all inhibitors used in this study showed quite different inhibitory activities, according to alpha-glucosidase origins. Furthermore, subsequent separation of the active material from Myrtus communis by HPLC showed that only one fraction acted as an a-glucosidase inhibitor.

Enzyme Activation↗
PubMed1981

Subcellular Localization of 2-(beta-d-Glucosyloxy)-Cinnamic Acids and the Related beta-glucosidase in Leaves of Melilotus alba Desr.

The distribution of the glucosides of trans- and cis-2-hydroxy cinnamic acid and of the beta-glucosidase which hydrolyzes the latter glucoside was examined in preparations of epidermal and mesophyll tissue obtained from leaves of sweet clover (Melilotus alba Desr.). The concentrations of glucosides in the two tissues were about equal when compared on the basis of fresh or dry weight. Inasmuch as the epidermal layers account for no more than 10% of the leaf volume, the mesophyll tissue contains 90% or more of the glucosides. Vacuoles isolated from mesophyll protoplasts contained all of the glucosides present initially in the protoplasts.The specific activities of the beta-glucosidase in the two tissues were also similar; thus, most of the enzyme is contained in mesophyll tissue. However, the amount of enzyme in mesophyll protoplast extracts amounts to only 1 to 2% of the activity present in leaf homogenates when chlorophyll was the basis for comparison. (This small amount of coumarin-beta-glucosidase present in protoplasts is not associated with chlorophyll-containing fractions.) In contrast, 90% of the uridine diphosphate glucose-o-coumaric acid glucosyl transferase activity present in leaf homogenate was recoverable in protoplasts prepared from intact leaves. Such results indicate that most of the coumarin-beta-glucosidase in M. alba leaves is located in the extracytoplasmic space. Only a small fraction (7%) of this extra cytoplasmic beta-glucosidase was associated with individual cells or cell clusters isolated from clover leaves.

Journal Article↗
PubMed1981

High Production of beta-Glucosidase in Schizophyllum commune: Isolation of the Enzyme and Effect of the Culture Filtrate on Cellulose Hydrolysis.

Optimization experiments with response surface statistical analysis were performed with Schizophyllum commune to obtain high beta-glucosidase yields. The factors in the optimization experiment were the concentrations of cellulose, peptone, and KH(2)PO(4). Their optimal values were 3.2, 3.0, and 0.2 g/100 ml, respectively. Enzyme assays revealed very high beta-glucosidase (22.2 U/ml) and cellobiase (68.9 U/ml) yields. The avicelase yield was low as compared with that from Trichoderma reesei. Mixtures of S. commune and T. reesei culture filtrates caused faster and more extensive saccharification of Avicel than could be achieved by either filtrate alone. A beta-glucosidase was isolated and purified from the optimized culture filtrate of S. commune. The electrophoretic mobility of the purified beta-glucosidase indicated a molecular weight of 97,000. The amino acid composition was similar to that of beta-glucosidase from T. reesei. The acidic (aspartate and glutamate) residues or their amides or both made up approximately 20% of the protein. The NH(2)-terminal amino acid of the enzyme was histidine.

Journal Article↗
PubMed1982

Purification and Properties of beta-Glucosidase from Aspergillus terreus.

A beta-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50 degrees C. The beta-glucosidase had K(m) values of 0.78 and 0.40 mM for p-nitrophenyl-beta-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K(i) of 3.5 mM when p-nitrophenyl-beta-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-beta-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The beta-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta-glucosidases from other organisms being studied for use in industrial cellulose saccharification.

Journal Article↗
PubMed1984

Production and Characterization of Cellulase and beta-Glucosidase from a Mutant of Alternaria alternata.

A mutant of Alternaria alternata excreted enhanced levels of carboxymethylcellulase and particularly beta-glucosidase when grown in cellulose liquid media. Both enzymes were purified two- to four-fold by ammonium sulfate precipitation and gel filtration, and the kinetic data showed K(m) values of 16.64 mg/ml of culture fluid for carboxymethylcellulase and 0.14 mM p-nitrophenyl-beta-d-glucoside and 0.81 mM cellobiose for beta-glucosidase at pH 5. Carboxymethylcellulase and extracellular beta-glucosidase functioned optimally at pH 5 to 6 and 4.5 to 5 and at temperatures of 55 to 60 and 70 to 75 degrees C, respectively. Both temperature optima and thermostability of beta-glucosidase were among the highest ever reported for the same enzyme excreted from cellulase and beta-glucosidase hyperproducing microorganisms.

Journal Article↗