Antihyperglycemic effect of polyphenols from Acerola (Malpighia emarginata DC.) fruit

A crude acerola polyphenol fraction (C-AP) was prepared by subjecting an acerola extract to a C18 cartridge column, and eluting the adsorbed fraction with ethanol containing 10% of acetic acid. C-AP appeared in a previous study to have an inhibitory effect on alpha-glucosidase and particularly on maltase activities. To elucidate the antihyperglycemic effect of C-AP further, we examined the regulation by C-AP of glucose uptake in Caco-2 cell; this resulted in the inhibition of glucose uptake. We next conducted single administration tests of glucose and maltose to ICR mice to investigate whether C-AP really controlled the intestinal glucose absorption in an animal body. The results showed that C-AP significantly suppressed the plasma glucose level after administering both glucose and maltose, suggesting that C-AP had a preventive effect on hyperglycemia in the postprandial state. The mechanism for this effect is considered to have been both suppression of the intestinal glucose transport and the inhibition of alpha-glucosidase. Despite such a preventive effect, the therapeutic effect of C-AP on hyperglycemia appeared to be low from the experiment with KKAy mice.     

The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle

1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.     

Kinetics of alpha-amylase synthesis from Bacillus amyloliquefaciens

The microbial production of alpha-amylase from Bacillus amyloliquefaciens was investigated. The microorganism was grown using media containing glucose or maltose at 37 degrees C and under aerobic conditions in a 16-L fermentor. The alpha-amylase synthesis from maltose was not found to be inducible but was found to be subject to catabolite repression. The maltose uptake rate was observed to be the rate-limiting step compared to the conversion rate of maltose to glucose by intracellular alpha-glucosidase. The alpha-amylase activity achieved with maltose as a substrate was higher than that achieved with glucose. A slower growth rate and a higher cell density were obtained with maltose. The enzyme production pattern depended upon the nutrient composition of the medium.     

Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena

A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.     

Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP

Escherichia coli shifted from broth at external pH (pH₀) 7·0 to pH₀ 7·0 broth plus glucose rapidly induced marked acid tolerance which also appeared, albeit to a lesser extent, plus maltose, sucrose or lactose. Tolerance appeared without the medium pH becoming acidic. Tolerance was most substantial when glucose was added at pH₀ 7·0 but was also appreciable at pH₀ 7·5, 8·0 and 8·5. Induction of tolerance by glucose was markedly reduced by cyclic AMP and essentially abolished plus NaCl or sucrose ; the induction process was also reduced but not fully inhibited by chloramphenicol, tetracycline and nalidixic acid. Glucose-induced organisms showed less acid damage to DNA and β-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH₀. In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E. coli at pH₀ 7·0 ; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.     

A novel alpha-glucosidase from the moss Scopelophila cataractae

Scopelophila cataractae is a rare moss that grows on copper-containing soils. S. cataractae protonema was grown on basal MS medium containing copper. A starch-degrading activity was detected in homogenates of the protonema, after successive extraction with phosphate buffer and buffer containing 3 M LiCl. Buffer-soluble extract (BS) and LiCl-soluble extract (LS) readily hydrolyzed amylopectin to liberate only glucose, which shows that alpha-glucosidase (EC 3.2.1.20) in BS and LS hydrolyzed amylopectin. The K(m) value of BS for maltose was 0.427. The K(m) value of BS for malto-oligosaccharide decreased with an increase in the molecular mass of the substrate. The value for maltohexaose was 0.106, which is about four-fold lower than that for maltose. BS was divided into two fractions of alpha-glucosidase (BS-1 and BS-2) by isoelectric focusing. The isoelectric points of these two enzymes were determined to be 4.36 (BS-1) and 5.25 (BS-2) by analytical gel electrofocusing. The two enzymes readily hydrolyzed malto-oligosaccharides. The two enzymes also hydrolyzed amylose, amylopectin and soluble starch at a rate similar to that with maltose. The two enzymes readily hydrolyzed panose to liberate glucose and maltose (1 : 1), and the K(m) value of BS for panose was similar to that for maltotriose, whereas the enzymes hydrolyzed isomaltose only weakly. With regard to substrate specificity, the two enzymes in BS are novel alpha-glucosidases. The two enzymes also hydrolyzed beta-limit dextrin, which has many alpha-1,6-glucosidic linkages near the non-reducing ends, more strongly than maltose, which shows that they do not need a debranching enzyme for starch digestion. The starch-degrading activity of BS was not inhibited by p-chloromercuribenzoic acid or alpha-amylase inhibitor. When amylopectin was treated with BS and LS in phosphate buffer, pH 6.0, glucose, but not glucose-1-phosphate, was detected, showing that the extracts did not contain phosphorylase but did contain an alpha-glucosidase. These results show that alpha-glucosidases should be capable of complete starch digestion by themselves in cells of S. cataractae.     

Some properties of human liver acid alpha-glucosidase.

1. Albumin activates human liver acid alpha-glucosidase (alpha-D-glucoside hydrolase, EC 3.2.1.20). From the Arrhenius plot, pH-dependence and Lineweaver-Burk plots it can be concluded that this activation is not only due to stabilisation of the enzyme, but also influences the enzymatic activity. It is proposed that for optimal functioning human liver acid alpha-glucosidase needs a protein environment. 2. Glycogen has a competitive inhibitory effect on the hydrolysis of 4-methylumbelliferyl-alpha-D-glucopyranoside, in contrast to maltose which exhibits a non-competitive type of inhibition. It is concluded that two catalytic sites exist, one for glycogen and one for maltose, while both sites influence each other. With glycogen as substrate a break in the Arrhenius plot is found. This is not the case when maltose is used as substrate. 3. The effect of antibody raised against human liver acid alpha-glucosidase on the activity of human liver acid alpha-glucosidase is studied. No corss-reacting material could be demonstrated in the liver of a patient with glycogen storage disease Type II (M. Pompe, acid alpha-glucosidase deficiency).     

Some properties of two forms of alpha-glucosidase from Saccharomyces cerevisiae-II

The amino acid composition of two forms of alpha-glucosidase from the yeast Saccharomyces cerevisiae-II was established and the values of Km, V, kcat and kcat/Km for maltose, maltotriose and p-nitrophenyl-alpha-D-glucopyranoside (PNPG) were determined. PNPG possessed a much higher affinity for the enzyme as compared to sucrose, maltose and maltotriose. The value of V decreased in the following order: PNPG greater than sucrose greater than maltose greater than greater than maltotriose. No differences between the kinetic parameters of individual forms of alpha-glucosidase were observed. Glucose, fructose and methyl-alpha-glucoside act as competitive inhibitors. The two forms of alpha-glucosidase under study have an identical pH optimum and thermal stability.     

Caffeoylsophorose, a new natural alpha-glucosidase inhibitor, from red vinegar by fermented purple-fleshed sweet potato

The suppressive effect on the postprandial blood glucose rise through alpha-glucosidase (AGH) inhibition was investigated in this study in order to clarify an antihyperglycemic function of 6-O-caffeoylsophorose (CS) from diacylated anthocyanin. The administration of CS (100 mg/kg) following maltose (2 g/kg) to Sprague-Dawley rats resulted in the maximal blood glucose level after 30 min being significantly decreased by 11.1% compared to the control. A reduction in the serum insulin secretion was also observed in parallel to the decrease in blood glucose level. No blood glucose change was apparent when sucrose or glucose was ingested, suggesting that the antihyperglycemic effect of CS was achieved by maltase inhibition, rather than by sucrase or glucose transport inhibition. An AGH inhibitory assay demonstrated that the non-competitive maltase inhibition of CS was partly due to acylation by phenolic acid with sugar, the presence of hydroxyl groups in the aromatic ring, and the presence of an unsaturated alkyl chain in the acylated moiety.     

Postnatal formation of the mechanisms of carbohydrate hydrolysis and transport in the rat small intestine

Using accumulating preparations of the mucose, studies have been made on the rate of accumulation of the glucose from 11 mM solutions of glucose, maltose and starch in proximal, intermediate and distal parts of the small intestine of 2--13-week rats. It was demonstrated that in 2-week animals, rather intensive transmembrane transport of "free" glucose takes place in the proximal and medial parts of the small intestine, the transport of glucose in the form of maltose or starch being absent. At later stages of postnatal life, especially to the onset of definitive nutrition, together with the induction of alpha-glucosidase systems, gamma-amylase and maltase transporting mechanisms are formed which provide for the adaptation of the organism to qualitatively new feeding conditions.     

Novel alpha-glucosidase from Aspergillus nidulans with strong transglycosylation activity

Aspergillus nidulans possessed an alpha-glucosidase with strong transglycosylation activity. The enzyme, designated alpha-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to alpha-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other alpha-glucosidases. We propose here that AgdB is a novel alpha-glucosidase with unusually strong transglycosylation activity.     

Temperature-sensitive binding of alpha-glucans by Bacillus stearothermophilus.

Bacillus stearothermophilus was found to bind strongly to starch and related alpha-glucans at 25 degrees C but not at 55 degrees C. The binding at the lower temperature could be assayed either by binding of fluorescein-labeled amylopectin to washed cell suspensions or through the reversible retention of bacteria by affinity chromatography in matrices containing immobilized starch. The bacteria exhibited amylopectin-dependent agglutination. The binding and agglutination were highest in bacteria grown on substrates containing alpha-1,4-glucosidic linkages such as maltose or dextrins. The binding affinity of cells was highest for maltohexaose, lower for maltose, and low or undetectable for glucose, isomaltose, cellobiose, or lactose. The reduced binding at the higher temperature was due to the rapid breakdown of the alpha-glucosides. The bacteria exhibited an extracellular alpha-amylase activity as well as a cell-associated alpha-glucosidase with high activity at 55 degrees C but undetectable activity at 25 degrees C. The inducibility, specificity, and protease sensitivity of the thermophilic alpha-glucosidase in whole cells were similar to those of the binding activity assayed at the lower temperature. Further evidence linking the binding and alpha-glucosidase activities came from a mutant, selected through affinity chromatography, which was reduced in starch binding at room temperature and also reduced in membrane-associated alpha-glucosidase activity at 55 degrees C. These results suggest a novel survival mechanism whereby a bacterium attaches to a macromolecular substrate under nonoptimal growth conditions for possible utilization upon a shift to more favorable conditions.     

Purification and characterization of cyclic maltosyl-(1-->6)-maltose hydrolase and alpha-glucosidase from an Arthrobacter globiformis strain

Cyclic maltosyl-maltose [CMM, cyclo-[-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]], a novel cyclic tetrasaccharide, has a unique structure. Its four glucose residues are joined by alternate alpha-1,4 and alpha-1,6 linkages. CMM is synthesized from starch by the action of 6-alpha-maltosyltransferase from Arthrobacter globiformis M6. Recently, we determined the mechanism of extracellular synthesis of CMM, but the degrading pathway of the saccharide remains unknown. Hence we tried to identify the enzymes involved in the degradation of CMM to glucose from the cell-free extract of the strain, and identified CMM hydrolase (CMMase) and alpha-glucosidase as the responsible enzymes. The molecular mass of CMMase was determined to be 48.6 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and 136 kDa by gel filtration column chromatography. The optimal pH and temperature for CMMase activity were 6.5 and 30 degrees C. The enzyme remained stable from pH 5.5 to 8.0 and up to 25 degrees C. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates, but it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes the alpha-1,6-linkage of CMM. The molecular mass of alpha-glucosidase was determined to be 60.1 kDa by SDS-PAGE and 69.5 kDa by gel filtration column chromatography. The optimal pH and temperature for alpha-glucosidase activity were 7.0 and 35 degrees C. The enzyme remained stable from pH 7.0 to 9.5 and up to 35 degrees C. alpha-Glucosidase degraded maltosyl-maltose to glucose via panose and maltose as intermediates, but it did not degrade CMM. Furthermore, when CMMase and alpha-glucosidase existed simultaneously in a reaction mixture containing CMM, glucose was detected as the final product. It was found that CMM was degraded to glucose by the synergistic action of CMMase and alpha-glucosidase.     

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.     

Acid alpha-glucosidase from human heart

An alpha-glucosidase maximally active at acid pH has been purified from human heart some 2,600-fold and its properties compared to a purified alpha-glucosidase from human liver. Molecular weight was evaluated using three different analytical procedures. The effect of various cations was determined. Thermal lability was evaluated using three different substrates. Affinity and hydrolysis velocity constants for maltose, glycogen and 4-methylumbelliferyl-alpha-D-glucose were determined for both preparations at optimal hydrogen ion concentration. Inhibition studies were carried out using the disaccharide turanose. From this study, we conclude there are no significant differences in molecular weight or kinetic properties between the cardiac and hepatic alpha-glucosidase enzymes.     

Human liver lysosomal alpha-glucosidase modified by chemical galactosylation and isolation of specific antibodies

Human liver acid alpha-glucosidase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was modified with water soluble carbodiimide in the presence of p-aminophenyl-beta-D-galactopyranoside. The incorporation of the aminophenyl derivative of galactose into alpha-glucosidase caused some changes in the physiocochemical properties of the enzyme: a blue shift in the absorption maximum, an alteration of the total electric charge affecting electrophoretic mobility upon polyacrylamide gel electrophoresis, and acquisition of the ability to interact specifically with Ricinus communis agglutinin. At the same time, the 'galactosylated' enzyme possessed high stability and exhibited catalytic activity towards maltose. The Km values of the native and modified enzymes with maltose were 6 and 5 mM, respectively. p-Aminophenyl-beta-D-galactopyranoside residues incorporated in alpha-glucosidase and in other proteins were found to be antigenic determinants to which the pure antibodies were obtained.     

Purification and characterization of a maltase from the extremely thermophilic crenarchaeote Sulfolobus solfataricus.

A soluble maltase (alpha-glucosidase) with an apparent subunit mass of 80 kDa was purified to homogeneity from Sulfolobus solfataricus. The enzyme liberates glucose from maltose and malto-oligomers. Maximal activity was observed at 105 degrees C, with half-lives of 11 h (85 degrees C), 3.0 h (95 degrees C), and 2.75 h (100 degrees C). The enzyme was generally resistant to proteolysis and denaturants including aliphatic alcohols. n-Propanol treatment at 85 degrees C increased both Km and Vmax for maltose hydrolysis.     

Inhibition by 2-deoxy-D-glucose of synthesis of glycoprotein enzymes by protoplasts of Saccharomyces: relation to inhibition of sugar uptake and metabolism

The synthesis of the glycoprotein enzymes, invertase and acid phosphatase, by protoplasts of Saccharomyces mutant 1016, is inhibited by 2-deoxy-d-glucose (2-dG) after a 20- to 30-min lag period under conditions (external sugar to 2-dG ratio of 40:1) which cause only a slight decrease in total protein synthesis. Formation of one intracellular enzyme, alpha-glucosidase, is also sensitive, but production of another, alkaline phosphatase, is unaffected. A nonmetabolized glucose analogue, 6-deoxy-d-glucose, had no inhibitory effect. The total uptake of external fructose and maltose was decreased by 2-dG after a lag period of about the same duration as that before the inhibition of synthesis of enzymes or of mannan and glucan; during this time 2-dG was taken up by the protoplasts and accumulated primarily as 2-dG-6-phosphate (2-dG-6-P). Studies in vitro showed that 2-dG-6-P inhibits both yeast phosphoglucose isomerase and phosphomannose isomerase. The intracellular levels of the 6-phosphates of glucose, fructose, and mannose did not increase in the presence of 2-dG. We suggest that the high internal level of 2-dG-6-P blocks synthesis of the cell wall polysaccharides and glycoproteins in two ways. It directly inhibits the conversion of fructose-6-P to glucose-6-P and to mannose-6-P. At the same time, it restricts the transport of fructose and maltose into the cell; however, the continuing limited uptake of the sugars still provides sufficient energy for protein synthesis. The cessation of alpha-glucosidase synthesis is probably a result of depletion of the internal pool of maltose (the inducer). Our findings support the suggestion that restriction of synthesis of the carbohydrate moiety of glycoproteins reduces formation of the active enzyme.     

Diauxic growth in Azotobacter vinelandii.

Azotobacter vinelandii exhibited diauxie when grown in a medium containing both acetate and glucose as carbon sources. Acetate was used as the primary carbon source during the acetate-glucose diauxie. Uptake of acetate was constitutively expressed during both diauxic phases of growth. Induction of the glucose uptake system was inhibited in the presence of acetate. Acetate was also the preferred growth substrate for A. vinelandii grown in a medium containing either fructose, maltose, xylitol, or mannitol. The tricarboxylic acid cycle intermediates citrate, isocitrate, and 2-oxoglutarate inhibited glucose utilization in cells grown in glucose medium containing these substrates, and diauxic growth was observed under these growth conditions. Temporal expression of isocitrate-lyase, ATPase, and nitrogenase was exhibited during acetate-glucose diauxie.