Modulation of Starch Digestion for Slow Glucose Release through "Toggling" of Activities of Mucosal α-Glucosidases

Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of "toggling": differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC(50) values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases.     

Localization of α-Glucosidases I,II, and III in Organs of European Honeybees,Apis mellifera L., and the Origin of α-Glucosidase in Honey

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

Action of Rice α-Glucosidase on Maltose and Starch

Rice seeds possess α-glucosidase I and II, and the action of the α-glucosidases on maltose and starch was studied. The activity on starch was increased 2.3~2.6 times in both enzymes at the concentration of 50 mM of potassium chloride. Such activation was also caused by mono and di-valent cations. The activity on maltose was not influenced by the cations. In mixed substrate experiments, liberation of ¹⁴C-glucose from ¹⁴C-maltose was not inhibited in the presence of starch, and this was also the case with that from ¹⁴C-starch in the existence of maltose. From these results, it was suggested that the α-glucosidases possess maltose-hydrolyzing site and starch-hydrolyzing site separately, and also probably regulatory. The α-glucosidases liberated only glucose from starch, and were presumed to complete hydrolysis of starch after longer incubation.     

-Glucosidase isozymes and the labellar sugar receptor of the blowfly

Some properties of four types of the soluble α-glucosidase, especially of P-II α-glucosidase of Amakawa and others, from the labella of the blowfly were examined and compared with those of the sugar receptor of the blowfly. Tris (hydroxymethyl) aminomethane inhibited four types of α-glucosidase and the electrical response of the sugar receptor, all in a competitive manner, but the inhibition constants (K_i) for these glucosidases were more than 150 times smaller than that for the sugar receptor. l-Serine inhibited α-glucosidases competitively, but the inhibition for the sugar receptor was not clear. The effects of cations and amino acids on α-glucosidases were also examined in relation to those on the sugar receptor.     

Evolutionary History of Eukaryotic α-Glucosidases from the α-Amylase Family

Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria.     

Purification and Substrate Specificity of Honeybee,Apis mellifera L., α-Glucosidase III

α-Glucosidase III, which was different in substrate specificity from honeybee α-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of α-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in α-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl α-glucoside, maltose, and maltotriose, and by extremely high K_m for substrates, compared with those of α-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k₀ values for maltose, sucrose, p-nitrophenyl α-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the K_m values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (A_i’s) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the A_i value at subsite 3 was larger than that of subsite 1.     

A novel metabolic pathway for glucose production mediated by α-glucosidase-catalyzed conversion of 1,5-anhydrofructose

α-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form α-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for α-glucosidases. α-Glucosidase-catalyzed hydration produced α-glucose from AF, which plateaued with time. Combined reaction with α-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger α-glucosidase (31AG), which is stable in organic solvent, produced ethyl α-glucoside from AF in 80% ethanol. The findings indicate that α-glucosidases catalyze trans-addition. This is the first report of α-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.     

Novel easily accessible glucosidase inhibitors: 4-hydroxy-5-alkoxy-1,2-cyclohexanedicarboxylic acids

Glycosidases are very important enzymes involved in a variety of biochemical processes with a special importance to biotechnology, food industry, and pharmacology. Novel structurally simple inhibitors derived from cyclohexane-1,2-dicarboxylic acids were synthesized and tested against several fungal glycosidases from Aspergillus oryzae and Penicilliumcanescens. The presence of at least two carboxylic groups and one hydroxy group was essential for efficient inhibition. Significant selective inhibition was observed for α- and β-glucosidases, the magnitude of which depended on the configuration of substituents; inhibition increased for β-glucosidase by lengthening the alkoxy group of the inhibitor.     

Purification and Properties of Three Forms of α-Glucosidase from Germinated Green Gram (Phaseolus vidissimus Ten.)

Three forms of α-glucosidase have been isolated from 5-day-old green gram (Phaseolus vidissimus Ten.) seedlings, by a procedure including fractionation with ammonium sulfate and polyethylene glycol 6000, DEAE-cellulose column chromatography, SP-Sephadex column chromatography, preparative gel electrofocusing and preparative disc gel electrophoresis. The α-glucosidases isolated were designated as α-glucosidase I, α-glucosidase II–1 and α-glucosidase II–2. They were homogeneous on polyacrylamide disc gel electrophoresis. Their molecular weights were 145,000, 105,000 and 65,000, respectively. The three enzymes hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose and soluble starch liberating glucose, but did not act on sucrose. Their enzymes hydrolyzed phenyl α-maltoside into glucose and phenyl α-glucoside. They hydrolyzed amylose liberating α-glucose. Maltotriose was the main α-glucosyltransfer product formed from maltose by the three α-glucosidases.     

Novel imino sugar α-glucosidase inhibitors as antiviral compounds

Deoxynojirimycin (DNJ) based imino sugars display antiviral activity in the tissue culture surrogate model of Hepatitis C (HCV), bovine viral diarrhoea virus (BVDV), mediated by inhibition of ER α-glucosidases. Here, the antiviral activities of neoglycoconjugates derived from deoxynojirimycin, and a novel compound derived from deoxygalactonojirimycin, by click chemistry with functionalised adamantanes are presented. Their antiviral potency, in terms of both viral infectivity and virion secretion, with respect to their effect on α-glucosidase inhibition, are reported. The distinct correlation between the ability of long alkyl chain derivatives to inhibit ER α-glucosidases and their anti-viral effect is demonstrated. Increasing alkyl linker length between DNJ and triazole groups increases α-glucosidase inhibition and reduces the production of viral progeny RNA and the maturation of the envelope polypeptide. Disruption to viral glycoprotein processing, with increased glucosylation on BVDV E2 species, is representative of α-glucosidase inhibition, whilst derivatives with longer alkyl linkers also show a further decrease in infectivity of secreted virions, an effect proposed to be distinct from α-glucosidase inhibition.     

Change in maltose- and soluble starch-hydrolyzing activities of chimeric α-glucosidases of Mucor javanicus and Aspergillus oryzae

The chimeric α-glucosidases of Mucor javanicus and Aspergillus oryzae, which has high activity toward not only maltooligosaccharides but also soluble starch and has high activity toward maltooligosaccharides but faint activity toward soluble starch, respectively, were constructed by shuffling the C-terminal regions where low homology is observed between the two enzymes. The chimera genes were expressed in Pichia pastoris to produce and secrete the enzymes that have predicted molecular masses in the culture medium. The two chimeric M. javanicus α-glucosidases, of which the N- and C-terminal regions are substituted for those of A. oryzae, respectively, decreased in soluble starch-hydrolyzing activity, however, increased in maltose-hydrolyzing activity by 2.1 and 4.9 times higher than that of the native form of M. javanicus α-glucosidase, respectively. The chimeric enzymes changed on the V_max values for maltose significantly, whereas the K_m values were similar to that of the native enzyme.     

Phylogenetic Origin of the MAL and IMA α-Glucosidases of the International Genetic Line of Saccharomyces cerevisiae S288C

Microbiology - Taking into account the accepted concept of the ancient whole genome duplication (WGD) in the yeast genus Saccharomyces, comparative analysis of the multiple α-glucosidases MAL...     

Wall-bound α-glucosidase of suspension-cultured rice cells

Wall-bound α-glucosidase showed similar properties to other α-glucosidases produced by suspension-cultured rice cells except for weak soluble-starch hydrolysing activity. Thw wall-bound enzyme could not be solubilized from wall pellets with high salt concentrations, detergents or a combination of 8 M urea and 0.1 M sodium sulphite. Three carbohydrates susceptible to α-glucosidase digestion were also contained in the wall pellets. They were composed mainly of α-linked glucose residues, but gave a negative iodine test. One of them is maltotetraose, the others are polysaccharides.     

Synergism between enzymes of Sclerotium rolfsii involved in the solubilization of crystalline cellulose

Synergism between (1→4)-β-d-glucan cellobiohydrolase, endo-(1→4)-β-d-glucanases, and β-d-glucosidases of Sclerotium rolfsii for solubilization of native and amorphous celluloses is discussed. Besides synergism between cellobiohydrolase and endo-β-glucanases of S. rolfsii, a synergistic effect between endo-β-glucanases and β-glucosidases [which behaved rather as (1→4)-β-d-glucan glucohydrolases] was observed for solubilization of crystalline and amorphous celluloses. It seems that a cellobiohydrolase initiates the attack on crystalline cellulose and an endo-β-d-glucanase the attack on amorphous cellulose.     

Glycoside hydrolase family 31 Escherichia coli α-xylosidase

Escherichia coli YicI is a retaining α-xylosidase, which strictly recognizes the α-xylosyl moiety at the non-reducing end, belonging to glycoside hydrolase family 31 (GH 31). We have elucidated key residues determining the substrate specificity at both glycone and aglycone sites of Escherichia coli α-xylosidase (YicI). Detection of distinguishing features between α-xylosidases and α-glucosidases of GH 31 in their close evolutionary relationship has been used for the modification of protein function, converting YicI into an α-glucosidase. Aglycone specificity has been characterized by its transxylosylation ability. YicI exhibits a preference for aldopyranosyl sugars having equatorial 4-OH as the acceptor substrate with 1,6 regioselectivity, resulting in transfer products. The disaccharide transfer products of YicI, α-d-Xylp-(1→6)-d-Manp, α-d-Xylp-(1→6)-d-Fruf, and α-d-Xylp-(1→3)-d-Frup, are novel oligosaccharides, which have never been reported. The transxylosylation products are moderately inhibitory towards intestinal α-glucosidases.     

Unexpected high digestion rate of cooked starch by the Ct-maltase-glucoamylase small intestine mucosal α-glucosidase subunit

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies.     

Multiple Forms of α-Glucosidase from Pig Duodenum

Multiple forms of neutral α-glucosidase (pH optima, 6.0~6.5) were purified from pig duodenal mucosa by a procedure including Triton X-100 treatment, fractionation with ammonium sulfate, fractionation with ethyl alcohol, DEAE-cellulose column chromatography and preparative polyacrylamide disc gel electrophoresis. All of the α-glucosidases, I_a, II_a, I_b and II_b, were found to be homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights, isoelectric points and optimum temperatures of α-glueosidases I_a and II_a were 145,000~150,000, pH 3.5~3.7 and 55°C, respectively, and both enzymes were stable up to 55°C on treatment at pH 6.0 for 15 min; whereas those of the other two α-glucosidases, I_b and II_b, were 80,000, pH 4.0~4.1 and 65°C, respectively, and both enzymes were stable up to 70°C on the same treatment. The Km values of enzyme II_a for maltose, maltotriose and amylose were 1.72mm, 0.37 mm and 1.67mg/ml, while those of enzyme II_b were 3.33 mm, 2.61 mm and 11.8 mg/ml, respectively. All enzyme hydrolyzed α-1,4-, α-1,3- and α-1,2-glucosidic linkages in substrates, but showed no activity on sucrose or isomaltose. Enzymes II_a and II_b hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside, and maltotriose was formed as the main α-glucosyltransfer product from maltose. It was revealed that two types of neutral α-glucosidases having no activity toward sucrose or isomaltose existed in pig duodenal mucosa, and that one type comprised α-glucosidase having both maltose- and amylaceous α-glucan-hydrolyzing activities and the other type heat-stable maltooligosaccharidases which hydrolyzed amylaceous α-glucan weakly.     

Mécanismes régulateurs des α-glucosidases épididymaires chez le rat

Epithelial cells of mammalian epididymis produced acid and neutral α-glucosidases (α-G) which are either modulated by hormonal systemic influences or by local factors arising from the rete testis fluid. In this study, ligation of ductuli efferentes in the rat was characterized by a modest elevation of both α-G 15 days after surgery. In adrenalectomized rats, both α-G are lower than in intact animals. Because of the continuance of spermatophagia 2 weeks after ductuli ligation, the observed enzymatic variations cannot be ascribed to paracrine influences; however, the effects of adrenalectomy on α-G levels are obvious and may be attributed to a multifactorial endocrine control (androgens and corticoids).     

Digestive glucosidases in the midgut of Apodiphus amygdali Germar (Hemiptera: Pentatomidae)

Apodiphus amygdali or stink bug of fruit trees is one of the polyphagous species from pentatomid bugs that attack many of fruit trees and ornamental trees. In the current study, activities of α- and β-glucosidases were measured in the midgut of A. amygdali adults. It was found the higher activity of β-glucosidase than α-glucosidase in addition to different enzymatic properties of the enzymes. Optimal pHs for enzymatic activities were found to be 5 and 7 for α- and β-glucosidases, respectively. Values regarding optimal temperatures were obtained at 30?°C for both α- and β-glucosidases. Among ions used on α-glucosidase activity, K⁺ and Ca²⁺ significantly increased enzymatic activity, Na⁺ had no effect, and Cu²⁺, Fe²⁺ and Mg²⁺ had the significant negative effects on the enzyme activity. Ca²⁺ and Fe²⁺ increased β-glucosidase activity in the midgut of A. amygdali, Na⁺ had no effect, and other ions significantly decreased the enzyme activity. Ethylene glycol-bis (β-aminoethylether) N,N,N?,N-tetraacetic acid (EGTA), citric acid, ethylenediamide tetraacetic acid (EDTA) and sodium dodecylsulfate (SDS) significantly decreased α-glucosidase activity but EGTA, triethylenetetramine hexaacetic acid (TTHA), EDTA and SDS decreased β-glucosidase activity in the midgut of A. amygdali. Characterisation of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful for better understanding of enzyme roles in nutritional physiology of insects in addition to reach safe and useful controls of insect pests.