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beta-Glucosidase can be located after nondenaturing polyacrylamide gel electrophoresis by incubating the gel with 0.1% esculin and 0.03% ferric chloride. The esculetin released from esculin by beta-glucosidase action reacts with ferric ion to produce a black band, corresponding to the beta-glucosidase, against the transparent background.
Lactobacillus brevis is found together with the yeast Brettanomyces lambicus during the overattenuation process in spontaneously fermented lambic beer. An isolated L. brevis strain has been shown to produce an alpha-glucosidase with many similarities to the glucosidase earlier found in B. lambicus. The enzyme was purified by ammonium sulfate precipitation, gel (Sephadex G-150 and Ultrogel AcA-44) filtration, and ion-exchange chromatography (DEAE-Sephadex A-50). The molecular weights of the enzyme, as determined by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were about 50,000 and 60,000, respectively. Optimum catalytic activity was obtained at 40 degrees C and pH 6.0. The enzyme showed a decrease of hydrolysis with an increase in the degree of polymerization of the substrate. The K(m) values for p-nitrophenyl-alpha-d-glucopyranoside, maltose, and maltotriose were 0.51, 3.0, and 5.2 mM, respectively. There was lack of inhibition by 0.15 mM acarbose and 0.5 M turanose, but the enzyme was inhibited by Tris (K(i) value of 25 mM). The alpha-glucosidase of L. brevis together with the enzyme of B. lambicus seems to be a key factor in the overattenuation of lambic beer, although the involvement of other lactic acid bacteria (pediococci) cannot be excluded.
BACKGROUND/AIM: The reliability of a quantitative method for the non-invasive assessment of intestinal disaccharide hydrolysis was assessed. METHODS: Differential excretion of intact disaccharide, expressed as ratios of lactulose to appropriate hydrolysable disaccharides in urine collected following combined ingestion, has been investigated in healthy volunteers with drug induced alpha-glucosidase inhibition, in subjects with primary hypolactasia, and patients with coeliac disease. RESULTS: Oral administration of the alpha-glucosidase inhibitor 'Acarbose' (BAY g 5421, 200 mg) together with sucrose and lactulose increased the urinary sucrose/lactulose excretion ratios (% dose/10 h) fivefold. The effect was quantitatively reproducible, a higher dose of 'Acarbose' (500 mg) increasing the excretion ratio to about 1.0 indicating complete inhibition of intestinal sucrase activity. The suitability of the method for measuring differences in dose/response and duration of action was assessed by comparing three different alpha-glucosidase inhibitors (BAY g 5421, BAY m 1099, and BAY o 1248) and found to be satisfactory. Subjects with primary adult hypolactasia had urine lactose/lactulose excretion ratios raised to values indicating reduced rather than complete absence of lactase activity whereas sucrose/lactulose ratios were not significantly affected. 'Whole' intestinal disaccharidase activity assessed by this method demonstrated impairment of lactase, sucrase, and isomaltase in eight, one, and seven, respectively, of 20 patients with coeliac disease. By contrast in vitro assay of jejunal biopsy tissue indicated pan-disaccharidase deficiency in all but five of these patients. This shows the importance of distinguishing between 'local' and 'whole' intestinal performance. CONCLUSIONS: Differential urinary excretion of ingested disaccharides provides a reliable, quantitative, and non-invasive technique for assessing profiles of intestinal disaccharidase activity.
Gaucher disease is an autosomal recessive inborn error of glycosphingolipid metabolism caused by the deficient activity of the lysosomal hydrolase, acid beta-glucosidase. Three phenotypically distinct subtypes result from different acid beta-glucosidase mutations encoding enzymes with absent or low activity. A severe neonatal type 2 variant who presented with collodion skin, ichthyosis, and a rapid neurodegenerative course had two novel acid beta-glucosidase alleles: a complex, maternally derived allele, E326K+L444P, and a paternally inherited nonsense mutation, E233X. Because the only other non-pseudogene-derived complex allele, D140H+E326K, also had the E326K lesion and was reported in a mild type 1 patient with a D140H+E326K/K157Q genotype, these complex alleles and their individual mutations were expressed and characterized. Because the E233X mutation expressed no activity and the K157Q allele had approximately 1% normal specific activity based on cross-reacting immunologic material (CRIM SA) in the baculovirus system, the residual activity in both patients was primarily from their complex alleles. In the type 1 patient, the D140H+E326K allele was neuroprotective, encoding an enzyme with a catalytic efficiency similar to that of the N370S enzyme. In contrast, the E326K+L444P allele did not have sufficient activity to protect against the neurologic manifestations and, in combination with the inactive E233X lesion, resulted in the severe neonatal type 2 variant. Thus, characterization of these novel genotypes with non-pseudogene-derived complex mutations provided the pathogenic basis for their diverse phenotypes.
The effects of administration of an alpha-glucosidase inhibitor and a sulphonylurea compound on lipid profile were investigated in patients with non-insulin-dependent diabetes mellitus, (NIDDM) previously treated with sulphonylurea compounds alone, but in whom metabolic control was inadequate. A group of patients (n = 10) were treated with the alpha-glucosidase inhibitor at a dose of 0.2 mg, three times daily, for 4 weeks. Treatment significantly reduced the post-prandial glucose level and the serum total cholesterol level. In addition, there were non-significant reductions in the triglyceride and very low density lipoprotein levels. These preliminary results suggest that administration of alpha-glucosidase inhibitors might improve the lipid profile of patients with NIDDM.
The effect of adding a very low dose of a sulphonylurea (tolbutamide) to the treatment of 10 patients with noninsulin-dependent diabetes mellitus (NIDDM) was investigated. Patients took 0.1 mg tds of an alpha-glucosidase inhibitor orally for 8 weeks, and 50 mg tds of the sulphonylurea, tolbutamide, for the last 4 weeks of this period. The glycosylated haemoglobin level was significantly reduced during the combined treatment period compared with the level after treatment with alpha-glucosidase inhibitor alone (P = 0.035), although not compared with the pretreatment level. There were no significant changes in post-prandial blood glucose, serum lipid levels or connective peptide immunoreactivities. These preliminary results indicate that the addition of a very low dose of tolbutamide to a recommended diet and treatment with an alpha-glucosidase inhibitor, may improve glucose metabolism without raising insulin secretion or influencing lipid metabolism.
Gaucher disease type 1, the most prevalent lysosomal storage disease, is caused by the defective activity of the lysosomal enzyme, acid beta-glucosidase, or glucocerebrosidase. Infusion of purified acid beta-glucosidase containing alpha-mannosyl-terminated oligosaccharides (alglucerase) is efficacious in reversing hematologic, hepatic, splenic, and bony disease manifestations. The murine tissue distribution and turnover of bolus injections of alglucerase was evaluated by enzymatic activity, quantitative cross-reacting immunologic material analyses, and immunofluorescence studies. Enzyme activity measurements detected distribution to liver, spleen, thymus, kidney, and bone marrow mononuclear cells, but not to lungs and brain. In kidney and thymus, the enzyme was transiently present. In liver and spleen, enzyme activity peaked at about 20 min postinjection followed by a biphasic decrease with t1/2 approximately 40-60 min and approximately 12-14 h. In bone marrow maximal enzyme activity was at 40-60 min with a disappearance t1/2 approximately 60 min. Quantitative cross-reacting immunologic material studies of liver and spleen showed delivery of enzyme with decreased catalytic rate constants whose degradation included denaturation and proteolytic components. By immunofluorescence the human enzyme was distributed primarily to reticuloendothelial cells of the liver and spleen. In autopsy material from a Gaucher disease type 2 patient treated with enzyme, immunohistochemical and activity studies showed distributions similar to those in mice. These studies indicate a complex delivery and intracellular degradation of acid beta-glucosidase with lower intrinsic activity than the administered therapeutic agent.
Glycosidases play a pivotal role in processing of various glycoproteins and glycolipids. It is well known that glycosidases are also involved in a variety of degenerative metabolic disorders such as cancer and AIDS. In order to develop potent alpha-glucosidase inhibitors, we first screened 2,2-diphenyl-1-picrylhydrazyl hydrate as a candidate. 2,2-Diphenyl-1-picrylhydrazyl hydrate was found to inhibit alpha- and beta-glucosidases as well as alpha- and beta-mannosidases. It was also shown to be a non-competitive inhibitor of yeast alpha-glucosidase with a Ki value of 1.1 x 10(-6) M. Taken together, we anticipate that 2,2-diphenyl-1-picrylhydrazyl hydrate may be a potent inhibitor for some incurable metabolic disorders including AIDS.
In the course of our characterization studies on anti-obese and antidiabetogenic principles in medicinal foodstuffs, we found that the methanolic extract from the stems of Salacia chinensis (Hippocerateaceae) showed potent anti-hyperglycemic effects in oral sucrose or maltose-loaded rats, inhibitory effects on intestinal alpha-glucosidase, rat lens aldose reductase, formation of Amadori compounds and advanced glycation end-products, nitric oxide production from lipopolysaccharide-activated mouse peritoneal macrophage, and radical scavenging activities. Those in vivo and in vitro biological activities were compared with those of S. oblonga and S. reticulata. In addition, we isolated the principal alpha-glucosidase inhibitor, salacinol, from the stems of S. chinensis and examined alpha-glucosidase inhibitory activities of eleven samples of S. chinensis collected in Thailand.
OBJECTIVE: To study the effects of lobster sauce and polysaccharide extracts on alpha-1,4-glucosidase activity by establishing on enzyme-inhibitor model. METHODS: Glucosidase activity was assayed at PH optima by PNPG substrate. Lobster sauce extracts were prepared by soaking, centrifagation. Polysaccharide were prepared by soaking, centrifagation, separation. RESULTS: Lobster sauce and polysaccharide can inhibit alpha-1,4-glucosidase. CONCLUSION: This finding suggested that their hypoglycemic mechanism was similar to Acarbose. Lobster sauce may contain phenyalanine.
Slowly resorbable carbohydrates are preferred in the dietetic therapy of patients with type 2 diabetes. In this study we compared the efficacy of a "müsli" with the alpha-glucosidase inhibitor miglitol in delaying the resorption of carbohydrates. 24 patients with NIDDM took, in randomized order, four different breakfasts with equal amounts of carbohydrate: a standard breakfast with bread and marmelade, a müsli, and both breakfasts with 100 mg miglitol. We calculated the maximal blood glucose concentration, the postprandial difference, the time of the maximum and the area under the curve (AUC). The postprandial blood glucose increase after the müsli breakfast was significantly lower compared with the standard breakfast (maximal blood glucose 12.3 vs. 13.9 mmol/l, postprandial difference 3.6 vs. 5.1 mml/l, AUC 360 vs, 468 mmol/l x min). The blood glucose increase after the standard breakfast with miglitol was even lower (maximum 11.6 mmol/l, postprandial difference 2.9 mmol/l, AUC 241 mmol/l x min). Miglitol also lowered the blood glucose values after the müsli breakfast. This study shows that the alpha-glucosidase inhibitor miglitol in a dose of 100 mg is more effective in lowering the postprandial blood glucose increase than a müsli. In cases of non-acceptance of a modern diet with slowly resorbable carbohydrates, alpha-glucosidase inhibitors may be a therapeutic alternative.
We have characterized a maltodextrin glucosidase, previously described as a maltose-inducible, cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside in Escherichia coli. The gene encoding the enzyme activity, referred to as malZ, is located at 9.3 min on the chromosomal map. We cloned the gene in a high copy number vector and purified the enzyme. It is a monomer, with an apparent molecular weight of 65,000. The enzyme degrades maltodextrins, ranging from maltotriose to maltoheptaose, to shorter oligosaccharides, the final hydrolysis products being maltose and glucose. We measured the kinetic parameters, Km and Vmax, for the hydrolysis to glucose of the five different substrates. The binding of the substrate is enhanced by increasing the number of glucosyl residues in the maltodextrin. In contrast, the maximum rate of hydrolysis (Vmax) is fastest for maltotriose. To study the mode of action of the enzyme, we quantitatively measured the amount of free glucose liberated from the different maltodextrin substrates after a long incubation. More glucose is liberated from the long dextrins, as compared to the shorter ones, showing that the primary hydrolysis product was glucose, not maltose. Furthermore, [14C]maltotriose, specifically labeled at the reducing end, was hydrolyzed to [14C]glucose and unlabeled maltose. These data demonstrate that the malZ gene product is a maltodextrin glucosidase, liberating glucose from the reducing end of malto-oligosaccharides. The nucleotide sequence of malZ and the deduced amino acid sequence showed that malZ encodes a protein with a molecular weight of 68,960. Homology to glucosidases, alpha-amylases, and pullulanases were observed. Conserved regions thought to represent active sites in dextrin hydrolases were found in the MalZ protein.
The influence of an absorbable glucosidase inhibitor (Bay o 1248) on the endocrine and exocrine rat pancreas was evaluated. Rats fed a standard diet containing Bay o 1248 over 10 days consumed less food, gained about 30% less body weight than controls and showed meteorism. In these animals postprandial plasma insulin and glucose levels were decreased, but the total pancreatic insulin content was not different versus controls. The early insulin secretory response studied by pancreas perfusions was found reduced after a stimulatory glucose load (10 mM). Addition of the glucosidase inhibitor (1 mM) to the incubation medium diminished the glucose-induced insulin release from isolated islets of rats fed a standard diet. The compound added to the perfusion medium (10 microM) induced a slight reduction of half-maximal glucose-induced (10 mM) insulin release from the perfused pancreas. This inhibitory effect disappeared during maximal stimulation (20 mM glucose) of insulin secretion. The compound neither altered basal nor arginine-induced (15 mM) insulin release from the perfused organ. The exocrine pancreas was studied after feeding a Bay o 1248-enriched standard diet over 10 days. Amylase and trypsin concentration and total output into the biliary-pancreatic juice in response to CCK and secretin (20 IU or CU/kg body weight each) were diminished. The pancreatic enzyme content did not differ compared to controls. A significant role of carbohydrate maldigestion, systemic effects of the glucosidase inhibitor, and endocrine-exocrine pancreatic interrelations are discussed to account for the effects of the compound on the rat pancreas.
The development of drugs that target the tumor neovasculature may hold promise in inhibiting tumor growth. Experiments in vivo with castanospermine, an inhibitor of the glucosidases that convert protein N-linked high mannose carbohydrates to complex oligosaccharides, resulted in significant inhibition of tumor growth in nude mice. Angiogenesis to basic fibroblast growth factor in castanospermine-treated C57/BL mice was similarly reduced. Endothelial cell proliferation, invasion of basement membrane, and differentiation are crucial steps during neovascularization. In vitro differentiation models using Matrigel and postconfluent cultures of endothelial cells were used to study the effects of glycosidase inhibitors on endothelial cell behavior. FACS analysis of cell surface oligosaccharides using either Concanavalin A or L-phytohemagglutinin lectins confirmed an increase in high mannose groups and a decrease in tri- and tetra antennary beta-linked galactose-N-acetylglucosamine on mannose residues of Asn-linked oligosaccharides upon drug treatment. Castanospermine and the glucosidase inhibitor N-methyldeoxynojirimycin prevented the morphological differentiation of endothelial cells in vitro. These compounds did not alter the proliferation of cultured endothelial cells or their ability to attach to various extracellular matrix molecules. However, the cells showed a reduced ability to migrate and to invade basement membrane gels in vitro and an increased tendency to form aggregates that was inhibitable by D-mannose. These studies suggest that certain cell surface oligosaccharides are required for angiogenesis and that glucosidase inhibitors that alter these structures on endothelial cells are able to inhibit tumor growth.
The development of the alpha-glucosidase inhibitor acarbose provides a new approach in the management of diabetes. By competitive and reversible inhibition of intestinal alpha-glucosidases, acarbose delays carbohydrate digestion, prolongs the overall carbohydrate digestion time, and thus reduces the rate of glucose absorption. After oral administration of acarbose, the postprandial rise in blood glucose is dose-dependently decreased, and glucose-induced insulin secretion is attenuated. Because of diminished postprandial hyperglycemia and hyper-insulinemia by acarbose, the triglyceride uptake into adipose tissue, hepatic lipogenesis, and triglyceride content are reduced. Therefore, acarbose treatment not only flattens postprandial glycemia, due to the primary and secondary pharmacodynamic effects, but also ameliorates the metabolic state in general. In diabetic animals, acarbose reduced urinary glucose loss, the blood glucose area under the curve, and prevented the decrease in skeletal muscle GLUT4 glucose transporters. As a consequence of the reduced mean blood glucose area under the curve, the amount of protein nonenzymatically glycated was diminished, as was the formation of advanced glycation end-products (AGEs). The prevention of basement membrane glycation and thickening in various tissues indicated that acarbose treatment of diabetic animals produced beneficial effects against the development of nephropathy, neuropathy, and retinopathy. Thus, the alpha-glucosidase inhibitor acarbose may have the potential to delay or possibly prevent the development of diabetic complications.
The onset and progression of long-term complications in diabetes mellitus appear to be related to the degree of hyperglycemia and the overall metabolic control. Therefore, an important goal in the therapy of subjects with diabetes is to avoid wide fluctuations in blood glucose concentrations and increases in lipid levels. The first therapeutic maneuver to achieve glycemic control is to establish a correct diet containing complex carbohydrates and an adequate amount of dietary fibers. Dietary fibers are capable of reducing the intestinal uptake of carbohydrates. An additional strategy to reduce the uptake of carbohydrates across the intestine has recently been proposed by Puls et al. This strategy involves the use of inhibitors of alpha-glucosidase, an intestinal enzyme that participates in the breakdown of polysaccharides into disaccharides and monosaccharides. The inhibition of alpha-glucosidase by these agents is competitive and reversible and results in delayed and reduced uptake of carbohydrates across the intestine. This effect attenuates the post-prandial hyperglycemia and subsequent insulin secretory response particularly in subjects with hyperinsulinemia. The compound acarbose is a member of first generation alpha-glucosidase inhibitors. The administration of high doses of acarbose can be associated with side effects such as flatulence, meteorism, abdominal pain, and diarrhea due to the fermentation of non-absorbed carbohydrates in the intestinal lumen. Usually, these effects subside following a few days of therapy and/or reduction of the initial dose. Acarbose has been effectively used to treat type 2 diabetic patients either as a first choice drug or in association with sulfonylurea agents and in type 1 diabetics in association with insulin therapy.(ABSTRACT TRUNCATED AT 250 WORDS)
We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.
Previous studies have demonstrated that inhibitors of glycoprotein processing glucosidases interfere with the development of oligodendrocyte properties in primary cultures of embryonic rat brain cells (Bhat, J Neurosci Res 20:158-164, 1988). The present study examines the effect of castanospermine, an inhibitor of the processing glucosidases, on the development and differentiation of isolated oligodendrocyte progenitor cells. Treatment of oligodendrocyte progenitors with castanospermine did not affect the developmental progression of the precursors to become committed oligodendrocytes as revealed by comparable increases in the percentages of cells positive for galactocerebroside (a surface marker for terminally differentiated oligodendrocytes) in control and drug-treated cultures. On the other hand, there was an impairment of the expression of differentiated properties of oligodendrocytes [i.e., sulfolipid synthesis, myelin basic protein (MBP)] and 2'3'-cyclic nucleotide 3'-phosphohydrolase in the drug-treated cultures. Immunocytochemical analysis with anti-MBP antibodies revealed a reduced number of MBP-positive cells in inhibitor-treated cultures. Furthermore, a majority of MBP-positive cells in such cultures displayed immunoreactive MBP in their cell body and not the processes, unlike in control cultures where both cell body and the processes of oligodendrocytes stained intensely for MBP. The strong inhibitory effect of castanospermine on the expression of oligodendrocyte-specific activities was contrasted with a relatively smaller effect of swainsonine, a mannosidase inhibitor on oligodendrocyte differentiation. Both castanospermine and swainsonine, however, effectively blocked the formation of complex-type oligosaccharides, suggesting thereby a lack of correlation between the inhibition of the formation of complex-type oligosaccharides and oligodendrocyte differentiation. It is suggested, therefore, that early trimming reactions involving the removal of glucose residues from the high mannose oligosaccharides in the endoplasmic reticulum may be essential for the cell surface localization and function of glycoproteins critically involved in surface interactions of oligodendrocytes with each other and/or with the substratum.