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PubMed1988

Reappearance of embryonic neutral alpha-glucosidase isoenzyme in acid maltase-deficient muscle of Japanese quail.

Two neutral alpha-glucosidase isoenzymes were isolated from the muscle of Japanese quails with late-onset acid maltase deficiency. One isoenzyme is predominantly expressed in embryonic muscle and the other in adult muscle. The time of switching from one to the other of these two neutral alpha-glucosidases was the same as in normal birds. The glycogen content in acid maltase-deficient muscle was not inversely proportional to the amount of embryonic neutral alpha-glucosidase. From the results, we conclude that (i) the transition of neutral alpha-glucosidase from the embryonic to the adult type is not influenced by the disease, and (ii) the embryonic neutral alpha-glucosidase seems not to be directly correlated with glycogen storage in skeletal muscle. In acid maltase-deficient muscle, the activity of the embryonic type began to increase again from 14 days after hatching, and attained a level corresponding to 18% of the total neutral alpha-glucosidase activity at 3 months (P less than 0.025). Its biochemical characteristics were the same as those of the normal embryonic neutral alpha-glucosidase. It should be clarified why the reappearance of the normal embryonic type occurs in acid maltase-deficient adult muscle and whether or not the reappearance of the embryonic neutral alpha-glucosidase represents regenerating muscle.

Animals↗

PubMed1964

beta-Glucosidase Activity in Phytopathogenic Bacteria.

Most of 58 isolates of phytopathogenic and related bacteria comprising 24 species in the genera Agrobacterium, Erwinia, Corynebacterium, Pseudomonas, and Xanthomonas exhibited beta-glucosidase activity, especially the gall-nonforming pathogenic pseudomonads and soft rot organisms. The gall-forming pseudomonads and P. fluorescens exhibited no beta-glucosidase activity, with the exception of one isolate of P. savastanoi which showed slight activity on an inorganic nitrogen-arbutin medium. The best medium for demonstrating beta-glucosidase activity contained peptone as the nitrogen source and arbutin. beta-Glucosidase activity in this medium was indicated by either acid production or browning. P. syringae, in contrast to other bacteria tested, produced most beta-glucosidase in a medium containing large amounts of glucose. Chromatographic analyses confirmed that splitting of the glucoside occurred at the glucosidic linkage. Reaction of sonically treated bacterial cells with indican or p-nitrophenyl-beta-D-glucoside proved a rapid method for assaying relative amounts of beta-glucosidase among bacterial species. Harda's paper-strip method of detecting beta-glucosidase also was useful in revealing the distribution and relative amounts of beta-glucosidase in most bacteria, but did not indicate the relatively greater amount of beta-glucosidase in P. syringae.

Journal Article↗

PubMed1994

Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase.

Small (80-amino acid) glycoproteins or saposins are important for the in vivo function of several lysosomal hydrolases. Four saposins, A, B, C, and D, are encoded by a single locus termed prosaposin. Saposins C and A are thought to function in vivo as activators of acid beta-glucosidase. The physiologic role of saposin C has been confirmed, whereas that of saposin A role has not. To investigate the effects of saposins C and A on acid beta-glucosidase activity, the coding sequence for the individual saposins was expressed in Escherichia coli and the recombinant proteins purified to homogeneity. Recombinant and natural saposins A and C activated acid beta-glucosidase similarly only in micromolar amounts. Saposin C had specific activation of acid beta-glucosidase activity at < 200 nM. A second phase of activation was achieved at > 1 microM. In comparison, saposin A consistently activated acid beta-glucosidase only at > 1 microM. Two mutant saposins C (Cys382-->Phe and Cys382--Gly) were created and shown to compete with saposin C for a site on acid beta-glucosidase. The mutant saposins did not activate the enzyme. Recombinant saposin A (< 200 nM) competed with saposin C for a site on the enzyme but without activating effects. These studies show that saposin A is not an in vitro activator of acid beta-glucosidase at physiologic concentrations, although binding occurs without activating acid beta-glucosidase. The studies with mutant saposins C indicate that the binding and activation effects of saposins C are distinct events. These results indicate that the saposin C-induced conformational change in the enzyme occurs via highly specific, probably multivalent, interactions between acid beta-glucosidase and saposin C.

Amino Acid Sequence↗

PubMed1998

Long-term effect of alpha-glucosidase inhibitor on late dumping syndrome.

Dumping syndrome commonly occurs after gastrectomy. The late dumping, which is one of the dumping syndromes, is due to postprandial hypoglycaemia caused by an excessive insulin secretion after a sharp rise in plasma glucose. Several treatments, including operation, dietary fibre and somatostatin, have been attempted to relieve dumping symptoms. These treatments take effect through modulation of plasma insulin and glucose levels, but their efficacy is still under consideration. Alpha-glucosidase inhibitor attenuates the postprandial increase of plasma glucose levels and is widely used for treatment of non-insulin-dependent diabetes mellitus (NIDDM). The acute effect of alpha-glucosidase inhibitor on late dumping syndrome has been reported by some studies with test meals. The purpose of this study was to evaluate a long-term effect of alpha-glucosidase inhibitor treatment with ordinary meals in late dumping patients with NIDDM because administration of alpha-glucosidase inhibitor is only ethically allowed for diabetic patients in Japan. Six late dumping patients with NIDDM were orally administered alpha-glucosidase inhibitor, acarbose (50 or 100 mg), three times a day before each meal for 1 month. Diurnal changes of plasma glucose, insulin and pancreatic glucagon levels were compared before and after the alpha-glucosidase inhibitor treatment. All patients had late dumping-related symptoms, such as weakness, palpitation and dizziness before the induction of alpha-glucosidase inhibitor treatment. Patients suffered from a rapid fall in plasma glucose levels from hyperglycaemia at the same time as dumping symptoms. These late dumping-related symptoms disappeared and a rapid change of plasma glucose and insulin levels were attenuated after the alpha-glucosidase inhibitor treatment. These data suggest a long-term therapeutic efficacy of alpha-glucosidase inhibitor for late dumping patients.

Acarbose↗

PubMed1985

Sucrose gradient analysis of phospholipid-activated beta-glucosidase in type 1 and type 2 Gaucher's disease.

Using sucrose density gradients, differences in delipidated lysosomal beta-glucosidase isolated from control spleen and spleen from patients with nonneurologic (type 1) and neurologic (type 2) Gaucher's disease have been examined. The three enzymes differ in sedimentation properties as well as in their responsiveness to activation by phosphatidylserine and heat-stable factor. The control beta-glucosidase sedimented as an apparent 45,000-Da species whose activity was dependent upon the inclusion of exogenous sodium taurodeoxycholate in the assay medium. Preincubation with a mixture of phosphatidylserine and heat-stable factor converted the control enzyme to a faster-sedimenting form which exhibited considerable activity in the absence of exogenous bile salt. Spleen beta-glucosidase from a patient with type 1 Gaucher's disease exhibited an apparent molecular weight of 154,000 on sucrose gradients. Like the control enzyme, the activity of this form was bile salt dependent. Upon preincubation with phosphatidylserine and heat-stable factor, beta-glucosidase from the type 1 case was also converted to a faster-sedimenting form which was more active in the absence of sodium taurodeoxycholate than in the presence of the bile salt. Spleen beta-glucosidase from the patient with type 2 Gaucher's disease sedimented as a broad peak of activity in the most dense regions of the sucrose gradients, appearing to be much larger than the beta-glucosidase from either the control or the type 1 Gaucher's disease patient. The activity of this large species was strongly dependent upon bile salt, and was not affected by preincubation of the enzyme with phosphatidylserine and heat-stable factor. Using the chaotropic salt, sodium thiocyanate (0.15 M), the spleen beta-glucosidase isolated from the type 1 Gaucher's disease case was converted to a slower-sedimenting species. The control enzyme sedimented slightly farther into the sucrose gradients upon treatment with the NaSCN. Thiocyanate treatment had no effect on the spleen beta-glucosidase isolated from the case of type 2 Gaucher's disease.

Aged↗

PubMed1983

Physical and kinetic properties of a plasma-membrane-bound beta-D-glucosidase (cellobiase) from midgut cells of an insect (Rhynchosciara americana larva).

The midgut caecal cells from Rhynchosciara americana larvae possess a plasma-membrane-bound beta-D-glucosidase (cellobiase, EC 3.2.1.21), which is recovered (75-95%) in soluble form both after treatment with Triton X-100 and after treatment with papain. The Triton X-100-solubilized beta-D-glucosidase displays Mr106000 and pI 5.4, whereas the papain-released beta-D-glucosidase shows Mr65000 and pI 4.7. Thermal inactivations of the detergent-solubilized and the papain-released forms of beta-D-glucosidase both follow apparent first-order kinetics with similar half-lives. The papain-released beta-D-glucosidase, after being purified by density-gradient centrifugation, hydrolyses beta-D-glucosides, beta-D-galactosides and beta-D-fucosides at the same active site, as inferred from experiments of competition between substrates. The beta-D-glucosidase seems to operate in accordance with rapid-equilibrium kinetics, since the Km (0.61 mM) for the enzyme is constant over a wide range of pH. The hydrolysis of the beta-D-glucosidic bond catalysed by the beta-D-glucosidase occurs without inversion of configuration, delta-gluconolactone is a strong (Ki 0.5 microM) inhibitor of the enzyme and substituents in the substrate aglycone affect the catalytic constant of the reaction. These data support the assumption that the mechanism of the reaction catalysed by the beta-D-glucosidase involves the intermediary formation of a carbonium ion, rather than a glucosyl-enzyme intermediate.

Animals↗

PubMed2003

Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp. SAM1606.

The alpha-glucosidase of Bacillus sp. strain SAM1606 is a member of glycosyl hydrolase family 13, and shows an extraordinarily broad substrate specificity and is one of very few alpha-glucosidases that can efficiently hydrolyze the alpha-1,1-glucosidic linkage of alpha,alpha'-trehalose (trehalose). Phylogenetic analysis of family-13 enzymes suggests that SAM1606 alpha-glucosidase may be evolutionally derived from an alpha-1,6-specific ancestor, oligo-1,6-glucosidase (O16G). Indeed, replacement of Pro(273*) and Thr(342*) of B. cereus O16G by glycine and asparagine (the corresponding residues in the SAM1606 enzyme), respectively, was found to cause 192-fold enhancement of the relative catalytic efficiency for trehalose, suggesting that O16G may easily "evolved" into an enzyme with an extended substrate specificity by substitution of a limited number of amino acids, including that at position 273* (an asterisk indicates the amino-acid numbering of the SAM1606 sequence). To probe the role of the amino acid at position 273* of alpha-glucosidase in determination of the substrate specificity, the amino acid at position 273 of SAM1606 alpha-glucosidase was replaced by all other naturally occurring amino acids, and the resultant mutants were kinetically characterized. The results showed that substitution of bulky residues (e.g., isoleucine and methionine) for glycine at this position resulted in large increases in the K(m) values for trehalose and maltose, whereas the affinity to isomaltose was only minimally affected by such an amino-acid substitution at this position. Three-dimensional structural models of the enzyme-substrate complexes of the wild-type and mutant SAM1606 alpha-glucosidases were built to explore the mechanism responsible for these observations. It is proposed that substitution by glycine at position 273* could eliminate steric hindrance around subsite +1 that originally occurred in parental O16G and is, at least in part, responsible for the acquired broad substrate specificity of SAM1606 alpha-glucosidase.

Amino Acid Sequence↗

PubMed1979

Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster.

Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.

Animals↗

PubMed1984

Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal alpha-glucosidase.

The maturation of lysosomal alpha-glucosidase in cultured human skin fibroblasts was studied using a monoclonal antibody that distinguishes between the precursor and mature forms of the enzyme. Monoclonal antibodies against alpha-glucosidase isolated from placenta were produced by the hybridoma technique [Hilkens et al. (1981) Biochim. Biophys. Acta 678, 7-11]. One of these monoclonal antibodies, that synthesized by clone 43G8, reacts with the mature forms, but not with the precursor form of alpha-glucosidase isolated from urine. By means of pulse-labelling studies, it could be shown that monoclonal antibody 43G8 does not react with either the intracellular or the secreted precursor of alpha-glucosidase from cultured human skin fibroblasts. However, the antibody does react with the intermediate and mature forms of alpha-glucosidase. Endocytosis of the precurosor of alpha-glucosidase from urine by fibroblasts is followed by its conversion to a form with lower molecular mass. After endocytosis urinary precursor alpha-glucosidase is converted to a form that binds to monoclonal antibody 43G8. The t 1/2 for this conversion is 2 h. The conversion is inhibited by addition of leupeptin to the culture medium. It is concluded that a thiol proteinase is involved in the maturation of alpha-glucosidase in fibroblasts and the appearance of the antigenic determinant for 43G8.

Antibodies, Monoclonal↗

PubMed1985

Biosynthesis and intracellular transport of alpha-glucosidase and cathepsin D in normal and mutant human fibroblasts.

In order to study the intracellular localization of the proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D in cultured human skin fibroblasts we have used incubation with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap) as described by Jadot et al. [Jadot, M., Colmant, C., Wattiaux-de Coninck, S. & Wattiaux, R. (1984) Biochem. J. 219,965-970] for the specific lysis of lysosomes. When a homogenate of fibroblasts was incubated for 20 min with 0.5 mM Gly-Phe-NH-Nap, a substrate for the lysosomal enzyme cathepsin C, the latency of the lysosomal enzymes alpha-glucosidase and beta-hexosaminidase decreased from 75% to 10% and their sedimentability from 75% to 20-30%. In contrast, treatment with Gly-Phe-NH-Nap had no significant effect on the latency of galactosyltransferase, a marker for the Golgi apparatus, and on the sedimentability of glutamate dehydrogenase and catalase, markers for mitochondria and peroxisomes, respectively. The maturation of alpha-glucosidase and cathepsin D in fibroblasts was studied by pulse-labelling with [35S]methionine, immunoprecipitation, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography. When homogenates of labelled fibroblasts were incubated with Gly-Phe-NH-Nap prior to immunoprecipitation, 70-80% of all proteolytically processed forms of metabolically labelled alpha-glucosidase and cathepsin D was recovered in the supernatant. The earliest proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D appeared to be coupled to their transport to the lysosomes. Although both enzymes are transported via the mannose-6-phosphate-specific transport system, the velocity with which they arrived in the lysosomes was consistently different. Whereas newly synthesized cathepsin D was found in the lysosomes 1 h after synthesis, alpha-glucosidase was detected only after 2-4 h. When a pulse-chase experiment was carried out in the presence of 10 mM NH4Cl there was a complete inhibition of the transport of cathepsin D and a partial inhibition of that of alpha-glucosidase to the lysosomes. Leupeptin, an inhibitor of lysosomal thiol proteinases, had no effect on the transport of labelled alpha-glucosidase to the lysosomes. However, the early processing steps in which the 110-kDa precursor is converted to the 95-kDa intermediate form of the enzyme were delayed, a transient 105-kDa form was observed and the conversion of the 95-kDa intermediate form to the 76-kDa mature form of the enzyme was completely inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonium Chloride↗

PubMed1980

Gaucher's disease II. Studies on the kinetics of beta-glucosidase and the effects of sodium taurocholate in normal and Gaucher tissues.

beta-Glucosidase activity from normal human cultured fibroblasts was not affected by the presence of up to 0.1% (w/v) (1.86 mM) purified sodium taurocholate. At concentrations greater than 0.1%, there was a gradual decrease in activity. Conversely, beta-glucosidase activity from fibroblasts of three patients with juvenile onset Gaucher's disease was preferentially inhibited by the detergent at concentrations as low as 0.025% (0.46 mM). A 40% decrease in activity was observed at 0.1%. Crude sodium taurocholate was more potent in inhibiting beta-glucosidase activity from both the normal and Gaucher fibroblasts. However, very high background fluorescence and inconsistent results were observed when different batches of the crude taurocholate from the same or different sources were used. Similarly, beta-glucosidase activity from Gaucher splenic tissue homogenates, supernatant fluids (40,000 x g) and residue pellets was preferentially inhibited by purified sodium taurocholate. These findings indicate that the reliability and sensitivity of the enzyme assay for Gaucher's disease can be enhanced by determining beta-glucosidase activity in both the absence and presence of purified sodium taurocholate, particularly when variant cases with relatively high residual enzyme activity are encountered. In contrast to the enzyme from fibroblasts and spleens, beta-glucosidase activity from human placenta was markedly activated (greater than 300%) by the presence of 0.08% (1.49 mM) purified sodium taurocholate or 0.1 mM phosphatidyl serine, suggesting the presence of a predominate form of beta-glucosidase, possibly glucocerebroside beta-glucosidase, which is activated by the detergent. The apparent Michaelis constant (Km) for both the soluble and membrane-bound enzyme from normal fibroblasts was 1.6 +/- 0.1 mM. Kms from a patient with severe juvenile Gaucher's disease and two other patients with milder manifestations were 0.8 +/- 0.2 and 3.3 +/- 0.3 mM, respectively.

Adolescent↗

PubMed1985

Acid and neutral alpha-glucosidase in the reproductive organs and seminal plasma of the bull.

A synthetic substrate (p-nitrophenyl-alpha-D-glucopyranoside) was used to measure the acid and neutral alpha-glucosidase activity in bull seminal plasma, spermatozoa and in homogenates of bull reproductive organs. Marked differences were observed in the activities of these enzymes in the various tissues studied. Epididymis and particularly its caput region contained the highest specific activity of acid alpha-glucosidase. The activity of neutral alpha-glucosidase was highest in testis and in different parts of the epididymis. Seminal plasma, spermatozoa and seminal vesicle secretion contained only the acid enzyme activity. After fractionation with anion exchange chromatography in HPLC (Mono Q) and chromatofocussing, acid alpha-glucosidase activity of seminal plasma was recovered in two fractions with different pI values. The corresponding activities were found in the secretion of seminal vesicles, which thus form the major secretory source of seminal plasma acid alpha-glucosidase. In the fractionation with gel filtration on Sepharose 6B, the acid alpha-glucosidase had a smaller molecular weight than did the neutral enzyme. In anion exchange chromatography and chromatofocussing the testicular and epididymal homogenates each contained two acid and two neutral isoenzymes. In both fractionations the elution pattern of acid alpha-glucosidase was clearly different from that of the enzymes in seminal plasma. The pH optimum of acid alpha-glucosidase ranged from 3.75 to 4.5 and that of the neutral enzyme from 6.5 to 7.0. The neutral activity was more sensitive to many divalent metal ions and differences were also observed in the response of the enzymes to different concentrations of turanose and KCl.

Animals↗

PubMed2000

Nitric oxide, islet acid glucan-1,4-alpha-glucosidase activity and nutrient-stimulated insulin secretion.

The mechanism of nutrient-evoked insulin release is clearly complex. One part of that mechanism is postulated to be the activation of the glycogenolytic enzyme acid glucan-1,4-alpha-glucosidase. As nitric oxide (NO) has been found to be a potent inhibitor of glucose-stimulated insulin secretion, we have now investigated a possible influence of exogenous NO and inhibition of endogenous NO production on islet acid glucan-1,4-alpha-glucosidase activity in relation to insulin release stimulated by glucose and l-arginine. In isolated islets, NO derived from the intracellular NO donor hydroxylamine inhibited the activation of acid glucan-1, 4-alpha-glucosidase and its isoform acid alpha-glucosidase in parallel with inhibition of glucose-stimulated insulin release. In comparison, other lysosomal enzymes were largely unaffected. Similarly, the spontaneous NO donor sodium nitroprusside, as well as NO gas, when added to islet homogenates, suppressed the activities of these acid alpha-glucosidehydrolases and, to a lesser extent, the activities of other lysosomal enzymes. Finally, in the presence of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester, insulin release from isolated islets stimulated by glucose or l-arginine was markedly potentiated in parallel with an accompanying increase in the activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase. Other lysosomal enzymes and neutral alpha-glucosidase were not influenced. We propose that an important inhibitory effect of NO on the insulin secretory processes stimulated by glucose and l-arginine is exerted via inactivation of islet acid glucan-1,4-alpha-glucosidase, a putative key enzyme in nutrient-stimulated insulin release.

Animals↗

PubMed1983

Genetic heterogeneity in acid alpha-glucosidase deficiency.

Several clinical forms of acid alpha-glucosidase deficiency have been described. Our study was planned to identify differences at the molecular level in acid alpha-glucosidase deficiency. Of nine fibroblast strains derived from patients with the infantile form of the disease, eight were crossreacting material (CRM)-negative and one CRM-positive. This was demonstrated by both agar immunodiffusion and immunotitration. No difference in apparent enzymatic activity was observed between CRM-negative and CRM-positive infantile acid alpha-glucosidase deficiency fibroblasts. In two fibroblast strains with the adult form of acid alpha-glucosidase deficiency, rocket immunoelectrophoresis demonstrated a reduction in the amount of enzyme protein, which was directly proportional to the reduction in enzyme activity. In another fibroblast strain obtained from a patient with the adult form of the disease, the activity was within the range of the infantile form and no CRM could be identified. Fibroblasts with phenotype 2 of acid alpha-glucosidase, considered a normal variant, showed a reduction both in the amount of enzyme protein and in the ability of the enzyme to cleave glycogen. However, the catalytic activity for maltose was normal. The findings demonstrate extensive genetic heterogeneity in acid alpha-glucosidase deficiency. Molecular differences were identified both between the clinical forms of the disease and within the infantile and the adult forms of acid alpha-glucosidase deficiency. It remains unknown whether or not the enzyme deficiency in homozygotes for isozyme 2 of acid alpha-glucosidase will be sufficient to cause glycogen accumulation and lead to the development of muscular dystrophy-like disease later in life.

Adult↗

PubMed1993

Biosynthesis of glucosidase II in suspension-cultured soybean cells.

Since antibody against homogeneous mung bean glucosidase II cross-reacted with a 110-kDa protein from cultured soybean cells and also precipitated this activity from extracts of soybean cells, we used this antibody to examine the biosynthesis, turnover, and cellular localization of glucosidase II in soybean cells. Time course studies of [35S]methionine incorporation into glucosidase II (as well as pulse-chase studies) showed that this enzyme is synthesized as a 110-kDa protein that does not change in size from very early labeling times to those as late as 60 h, indicating the absence of a cleavable signal sequence or extensive modification of the carbohydrate. Furthermore, glucosidase II remained susceptible to digestion by endo-beta-N-acetylglucosaminidase H throughout this time period, and the major oligosaccharide structure was a Man9(GlcNAc)2 with small amounts of Glc1Man9(GlcNAc)2. The half-life of the biosynthesized glucosidase II was about 36 h, and no secretion of this protein occurred. Membranes of gently disrupted cells were separated by sucrose-density gradient centrifugation, and fractions were tested for glucosidase II activity as well as for marker enzymes. The bulk of the glucosidase II activity fractionated with endoplasmic reticulum membranes. Detergent solubility studies with Triton X-114 suggested that glucosidase II did not have a hydrophobic domain and is probably a luminal endoplasmic reticulum protein.

Autoradiography↗

PubMed1987

Cell-free translation of human lysosomal alpha-glucosidase: evidence for reduced precursor synthesis in an adult patient with glycogenosis type II.

Early events in the biosynthesis of alpha-glucosidase (EC 3.2.1.20) were studied in a wheat-germ cell-free translation system, using control and mutant RNA. In vitro, the primary translation product of the alpha-glucosidase mRNA is a 100 kDa protein. When canine microsomal membranes are added to the translation system, the nascent alpha-glucosidase precursor is cotranslationally transported across the microsomal membranes, yielding a 110 kDa glycosylated form. This protein has the same electrophoretic characteristics as the alpha-glucosidase precursor observed after in vivo labeling of control fibroblasts. Inhibition of glycosylation in vivo by tunicamycin or deglycosylation of the in vivo synthesized alpha-glucosidase precursor by glycopeptidase F reveals a core protein similar in molecular mass to the primary translation product. Total RNA from a patient with the adult form of glycogenosis type II is not able to direct the synthesis of normal amounts of alpha-glucosidase in vitro. Northern blot analysis of the RNA, using cloned alpha-glucosidase cDNA sequences as a probe, demonstrates that in this patient the amount of the 3.4 kb alpha-glucosidase mRNA is highly reduced. The results indicate that the synthesis or stability of the mRNA is affected.

Cell-Free System↗

PubMed2004

Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson.

The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity. However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase. Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition. However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity. Other compounds displayed mild activity on both the tests. Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test. Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors. However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity. Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase. This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds. Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R. emodi for the first time. Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus.

Animals↗

PubMed2002

Purification and characterization of beta-glucosidase from honey bees (Apis mellifera).

beta-glucosidase has been purified from the ventriculus and honey sac of Apis mellifera using a combination of anion- and cation-exchange, hydroxyapatite and gel-permeation chromatography. In addition, beta-glucosidase from the hypopharyngeal glands has been partially purified using anion-exchange and gel-permeation chromatography. The purified beta-glucosidase gave a positive result by glycoprotein staining. This beta-glucosidase consists of only one subunit and has M(r) of 72 kDa as determined by SDS-PAGE. IEF-PAGE showed several bands with pIs ranging from 4.5 to 4.8. These multiform proteins have been proposed as having different degrees of glycosylation. The pH optimum of the purified beta-glucosidase from the ventriculus and honey sac are 5.0. These enzymes were stable at temperatures up to 50 degrees C and have a relatively wide pH stability range of 4.0 to 9.0. MALDI-TOF-MS peptide mass maps of purified beta-glucosidase from the ventriculus, honey sac and hypopharyngeal glands showed six matching masses. These results indicate that the beta-glucosidase isolated from the hypopharyngeal glands, honey sac and ventriculus is the same. It is proposed that beta-glucosidase is produced in the hypopharyngeal glands, secreted into the mouth during feeding and then passes to the honey sac. From the honey sac, this enzyme is transferred into honeycomb cells and the ventriculus.

Animals↗