Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.20 (alpha-glucosidase)
4,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.
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PMID:Purification and some properties of an extracellular maltase from Bacillus subtilis. 0 2

Maltase-glucoamylase, a microvillous membrane ectoenzyme, was solubilized from rat intestinal mucosa by digestion with papain and subsequently purified to homogeneity with an overall yield of 10--20%. An antibody to the purified enzyme formed a single precipitin line in immunodiffusion experiments with an intestinal homogenate. The enzyme was shown to be an acidic glycoprotein (20% sugar by weight) which contained low amounts of cysteine and no sialic acid. At pH3--6, maltase activity was slowly lost, but the enzyme was re-activated by re-adjustment of the pH to neutrality. However, in the presence of sodium dodecyl sulphate, acid pH values inactivated maltase irreversibly, and at the same time converted the enzyme (mol.wt. 500000 approx.) into five new species with apparent molecular weights ranging from 134000 to 480000 as judged by polyacrylamide-gel electrophoresis. The same five fragments were also formed by boiling the enzyme for brief periods in the presence of sodium dodecyl sulphate or urea either with or without reducing agents. The dissociated species were stable on re-electrophoresis, and amino acid analysis showed them to be very similar to each other and to the original enzyme. The bands migrated anomalously on polyacrylamide gels of different concentration, thereby preventing the assignment of precise molecular weights. It is possible that the five species may represent stable aggregates of a common monomer of the enzyme.
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PMID:Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH. 2 2

The neutral maltase-glucoamylase complex has been purified to homogeneity from the brush-border membrane of rabbit intestine and kidney. Chemical modification of the amino acid side chains was carried out on the purified enzymes. Studies on the kidney enzyme revealed that tryptophan, histidine and cysteine were essential for both maltase and glucoamylase activities, whereas tryptophan, histidine and lysine were essential for the maltase and glucoamylase activities of the intestinal enzyme. Though there was no difference in the amino acids essential for the hydrolysis of maltose and starch by any one enzyme, starch hydrolysis seems to require two histidine residues instead of the one which is required for maltose hydrolysis. This appears to be true for both the intestinal and kidney enzymes.
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PMID:A comparison of the active site of maltase-glucoamylase from the brush border of rabbit small intestine and kidney by chemical modification studies. 200 4

Human intestinal bacteria were grown in a 3-stage continuous culture system on a medium containing complex polysaccharides and proteins as carbon and nitrogen sources. Selected bacterial populations were enumerated and glycosidase, protease and arylamidase activities measured. Comparison of arylamidase and glycosidase activities in the multichamber system (MCS) and faeces showed that the predominant faecal enzymes were also produced by bacteria growing in the MCS. After 48 d operation, porcine gastric mucin (5.8 g/d) was independently fed to vessel 1. Elevated levels of volatile fatty acid (VFA) formation showed that the glycoprotein was actively fermented. The increase in carbohydrate availability as a result of breakdown of the mucin oligosaccharides stimulated bacterial growth and activities. The enzymological measurements showed that mucin increased production of both cell-bound and extracellular glycosidases, such as beta-galactosidase, alpha-glucosidase and N-acetyl-beta-glucosaminidase. Protease activities were profoundly influenced by mucin. These were largely cell-bound in non-mucin cultures but were predominantly extracellular and collagenolytic when mucin was present. Experiments with protease inhibitors showed that cysteine proteases were the major cell-bound and extracellular enzymes in both mucin and non-mucin cultures, but that serine and metalloproteases were also present. The effect of mucin on arylamidase formation was less marked, although there was increased production of these enzymes in vessels 1 and 2 of the MCS. These results suggest that host-produced substances such as mucin glycoprotein may play a role in modulating the growth and activity of bacteria growing in the human large intestine.
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PMID:Influence of mucin on glycosidase, protease and arylamidase activities of human gut bacteria grown in a 3-stage continuous culture system. 266 79

We determined the complete nucleotide sequence of the yeast MAL6R gene from the Saccharomyces carlsbergensis MAL6 locus. The MAL6R gene encodes a transacting protein required for the inducible, coordinate expression of the two divergently transcribed structural genes, MAL6T (maltose permease), and MAL6S (maltase) at this locus. The transcription initiation sites for MAL6R were determined by primer extension experiments. The MAL6R gene contains an open reading frame of 473 amino acids with a calculated Mr of 54,892. The N-terminus of the deduced protein contains an amino acid sequence isologous to a consensus sequence for cysteine-zinc associated DNA binding fingers found in other fungal DNA binding proteins. The MAL6R gene was mapped to chromosome VIII by using OFAGE (orthagonal field alternating gel electrophoresis) gels and hybridization with specific chromosome and MAL6 probes.
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PMID:Primary structure of the regulatory gene from the MAL6 locus of Saccharomyces carlsbergensis. 285 10

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.
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PMID:Production and properties of alpha-glucosidase from Lactobacillus acidophilus. 641 77

The beta-D-glucosidase produced by Thermomyces lanuginosus-SSBP was purified to apparent homogeneity. The purified enzyme consisted of two identical subunits with a native molecular mass of 200 kDa. The purified beta-D-glucosidase only hydrolysed the glucoside substrates containing a terminal, non-reducing beta-D-glucose residue and was active on both aryl-beta-glucoside and cellobiose. This enzyme also exhibited less, but significant alpha-D-glucosidase activity and was capable of hydrolysing beta-1,6-linked diglucosides and gentiobiose. The Kapp m, V(max) and k(cat) values for p-nitrophenyl-beta-D-glucopyranoside were calculated to be 0.075 mM, 12.12 units/mg of protein and 44.44 glucose molecules released/s respectively. The beta-D-glucosidase retained its full activity after a 30 min incubation at 50 degrees C but was inactive after the same treatment at 70 degrees C. The enzyme appeared to be stable when the pH of the storage buffer was above 5.0. Maximal beta-D-glucosidase activity occurred at 65 degrees C and pH 6.0. This enzyme was competitively inhibited by glucose, cellobiose and salicin with K(i) values of 0.55, 0.52 and 0.81 mM respectively. The presence of Hg(2+) and N-bromosuccinimide inhibited the enzyme activity completely at 2 mM, while cysteine enhanced beta-D-glucosidase activity.
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PMID:Purification and biochemical characteristics of beta-D-glucosidase from a thermophilic fungus, Thermomyces lanuginosus-SSBP. 1046 23

The de novo design of a molecular adapter for directed association and covalent linkage of two polypeptides is presented. Using peptides containing charged amino acid residues and an additional cysteine residue (AlaCysLys(8) and AlaCysGlu(8)) we demonstrate that the electrostatic interaction promotes the association of two synthetic peptides and, subsequently, disulfide bond formation. The reaction depends on both the redox potential and on the ionic strength of the buffer. Varying the redox potential, the interaction of the peptides was quantified by a Delta G(0') of 6.6 +/- 0.2 kcal/mol. Heterodimerization of the peptides is highly specific, a competition of association by other cysteine containing compounds could not be observed. Two proteins comprising cysteine-containing polyionic fusion peptides, a modified Fab fragment and an alpha-glucosidase fusion, could be specifically conjugated by directed association and subsequent disulfide bond formation. Both proteins retain their functional characteristics within the bifunctional conjugate: enzymatic activity of the alpha-glucosidase and antigen-binding capacity of the Fab fragment are equivalent to the non-conjugated components.
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PMID:Polyionic fusion peptides function as specific dimerization motifs. 1173 96

Glycoside hydrolase family 4 represents an unusual group of glucosidases with a requirement for NAD+, divalent metal cations, and reducing conditions. The family is also unique in its inclusion of both alpha- and beta-specific enzymes. The alpha-glucosidase A, AglA, from Thermotoga maritima is a typical glycoside hydrolase family 4 enzyme, requiring NAD+ and Mn2+ as well as strongly reducing conditions for activity. Here we present the crystal structure of the protein complexed with NAD+ and maltose, refined at a resolution of 1.9 A. The NAD+ is bound to a typical Rossman fold NAD+-binding site, and the nicotinamide moiety is localized close to the maltose substrate. Within the active site the conserved Cys-174 and surrounding histidines are positioned to play a role in the hydrolysis reaction. The electron density maps indicate that Cys-174 is oxidized to a sulfinic acid. Most likely, the strongly reducing conditions are necessary to reduce the oxidized cysteine side chain. Notably, the canonical set of catalytic acidic residues common to other glucosidases is not present in the active site. This, combined with a high structural homology to NAD-dependent dehydrogenases, suggests an unusual and possibly unique mechanism of action for a glycoside-hydrolyzing enzyme.
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PMID:Crystal structure of Thermotoga maritima alpha-glucosidase AglA defines a new clan of NAD+-dependent glycosidases. 1258 67

Disaccharidases (maltase, cellobiase, lactase, and sucrase), alpha-amylase, and glucoamylase in the camel small intestine were investigated to integrate the enzymatic digestion profile in camel. High activities were detected for maltase and glucoamylase, followed by moderate levels of sucrase and alpha-amylase. Very low activity levels were detected for lactase and cellobiase. Camel intestinal maltase-glucoamylase (MG) was purified by DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of camel small intestinal MG4 and MG6 were estimated to be 140,000 and 180,000 using Sephacryl S-200. These values were confirmed by SDS-PAGE, where the two enzymes migrated as single subunits. This study encompassed characterization of MGs from camel intestine. The Km values of MG4 and MG6 were estimated to be 13.3 mM and 20 mM maltose, respectively. Substrate specificity for MG4 and MG6 indicated that the two enzymes are maltase-glucoamylases because they catalysed the hydrolysis of maltose and starch with alpha-1,4 and alpha-1,6 glycosidic bonds, but not sucrose with alpha-1,2 glycosidic bond which was hydrolyzed by sucrase-isomaltase. Camel intestinal MG4 and MG6 had the same optimum pH at 7.0 and temperature optimum at 50 degrees C and 40 degrees C, respectively. The two enzymes were stable up to 50 degrees C and 40 degrees C, followed by strong decrease in activity at 60 degrees C and 50 degrees C, respectively. The effect of divalent cations on the activity of camel intestinal MG4 and MG6 was studied. All the examined divalent cations Ca(2+), Mn(2+), Mg(2+), Co(2+) and Fe(3+) had slight effects on the two enzymes except Hg(2+) which had a strong inhibitory effect. The effect of different inhibitors on MG4 and MG6 indicated that the two enzymes had a cysteine residue.
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PMID:Disaccharidase activities in camel small intestine: biochemical investigations of maltase-glucoamylase activity. 1709 55


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