Human milk and mucosa-associated disaccharides impact on cultured infant fecal microbiota.

Human milk oligosaccharides (HMOs) are a mixture of structurally diverse carbohydrates that contribute to shape a healthy gut microbiota composition. The great diversity of the HMOs structures does not allow the attribution of specific prebiotic characteristics to single milk oligosaccharides. We analyze here the utilization of four disaccharides, lacto-N-biose (LNB), galacto-N-biose (GNB), fucosyl-α1,3-GlcNAc (3FN) and fucosyl-α1,6-GlcNAc (6FN), that form part of HMOs and glycoprotein structures, by the infant fecal microbiota. LNB significantly increased the total levels of bifidobacteria and the species Bifidobacterium breve and Bifidobacterium bifidum. The Lactobacillus genus levels were increased by 3FN fermentation and B. breve by GNB and 3FN. There was a significant reduction of Blautia coccoides group with LNB and 3FN. In addition, 6FN significantly reduced the levels of Enterobacteriaceae family members. Significantly higher concentrations of lactate, formate and acetate were produced in cultures containing either LNB or GNB in comparison with control cultures. Additionally, after fermentation of the oligosaccharides by the fecal microbiota, several Bifidobacterium strains were isolated and identified. The results presented here indicated that each, LNB, GNB and 3FN disaccharide, might have a specific beneficial effect in the infant gut microbiota and they are potential prebiotics for application in infant foods.

Characterization of Mucosal Disaccharidases from Human Intestine.

In this study, we used a brush border membrane (BBM) preparation from human small intestine to analyze the proportion and the activity of major intestinal disaccharidases, including sucrase-isomaltase (SI), maltase-glucoamylase (MGAM) and lactase-phlorizin hydrolase (LPH). SI, MGAM and LPH respectively constituted 8.2%, 2.7% and 1.4% of total BBM protein. The activity of SI and LPH decreased threefold after purification from the brush border membrane, which highlights the effect of membrane microdomains on the functional capacity of these enzymes. All of the disaccharidases showed optimal activity at pH 6, over 50% residual activity between pH 5 to pH 7, and increasing activity with rising temperatures up to 45 °C, along with a stable functional structure. Therefore the enzymes can withstand mild intraluminal pH alterations with adequate function, and are able to increase their activity with elevated core body temperature. Our data provide a functional measure for characterization of intestinal disaccharidases under different physiological and pathological conditions.

Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (Morinda citrifolia) Juice Exudates.

A new fatty acid ester disaccharide, 2-O-(ß-d-glucopyranosyl)-1-O-(2E,4Z,7Z)-deca-2,4,7-trienoyl-ß-d-glucopyranose (1), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (2), and a new iridoid glycoside, 10-dimethoxyfermiloside (3), were isolated along with 13 known compounds (4-16) from fermented noni fruit juice (Morinda citrifolia). The structures of the new compounds, together with 4 and 5, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds 2 and 7 showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds 4 and 6 exhibited considerable quinone reductase-1 (QR1) inducing effects.

Separation properties of saccharides on a hydrophilic stationary phase having hydration layer formed zwitterionic copolymer.

A novel water-holding adsorbent bonded with a zwitterionic polymer, diallylamine-maleic acid copolymer, was developed. With this adsorbent, hydrophilic solutes are partitioned by a hydration layer that forms on the zwitterions, as a main separating force. When the adsorbent was used to separate saccharides by normal-phase partition chromatography, the saccharides eluted in the order, mono-, di- and trisaccharide. The elution profile for mono- and di-saccharides was similar but not identical to that on anion exchange columns. This indicated that the adsorbent exhibited a complex retention behavior by the existence of both anion and cation exchange moieties in the functional polymer. Selecting Na(+) as a counter-ion of the maleate moiety enhanced the retention of saccharide. When used in an high performance liquid chromatography (HPLC) system with gradient elution, the adsorbent enabled the simultaneous analysis of mono-, di- and oligosaccharides.

Evaluation of anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta (Ker Gawl.) Miers.

We have investigated the in vitro anticandidal and antioxidant activities of phenolic compounds from Pyrostegia venusta flower extracts. We used the HPLC technique to purify the flavonoid (quercetin-3-O-α-l-rhamnopyranosyl-(1→6)-ß-d-galactopyranoside) and two phenylpropanoid glycosides (verbascoside and isoverbascoside); we evaluated the antimicrobial activity of the extracts against Candida strains (Candidaalbicans; Candidakrusei ATCC 6258; and the clinical isolate strains of Candida sp. C. albicans, C. krusei, Candidatropicalis, Candidaparapsilosis, and Candidaguilhermondii). The P. venusta flower extracts displayed antimicrobial and antioxidant activities. The semi-purified fraction of the P. venusta flower extract and the phenylpropanoid glycoside verbascoside exhibited activity similar to that of amphotericin B, which denoted that they are potentially applicable as natural antioxidant and anticandidal agents in the pharmaceutical industries.

Effect of Tenuifoliside A isolated from Polygala tenuifolia on the ERK and PI3K pathways in C6 glioma cells.

Tenuifoliside A (TFSA) is a bioactive oligosaccharide ester component of Polygala tenuifolia Wild, a traditional Chinese medicine which was used to manage mental disorders effectively. The neuroprotective and anti-apoptotic effects of TFSA have been demonstrated in our previous studies. The present work was designed to study the molecular mechanism of TFSA on promoting the viability of rat glioma cells C6. We exposed C6 cells to TFSA (or combined with ERK, PI3K and TrkB inhibitors) to examine the effects of TFSA on the cell viability and the expression and phosphorylation of key proteins in the ERK and PI3K signaling pathway. TFSA increased levels of phospho-ERK and phospho-Akt, enhanced release of BDNF, which were blocked by ERK and PI3K inhibitors, respectively (U0126 and LY294002). Moreover, the TFSA caused the enhanced phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 site, the effect was revoked by U0126, LY294002 and K252a. Furthermore, when C6 cells were pretreated with K252a, a TrkB antagonist, known to significantly inhibit the activity of brain-derived neurotrophic factor (BDNF), blocked the levels of phospho-ERK, phospho-Akt and phosphor-CREB. Taking these results together, we suggested the neuroprotection of TFSA might be mediated through BDNF/TrkB-ERK/PI3K-CREB signaling pathway in C6 glioma cells.

The inhibition of JNK MAPK and NF-κB signaling by tenuifoliside A isolated from Polygala tenuifolia in lipopolysaccharide-induced macrophages is associated with its anti-inflammatory effect.

The root of Polygala tenuifolia Willd. (Polygalaceae) is well known for its use in the treatment of neurasthenia, amnesia, and inflammation. In this study, we isolated phenyl propanoid type metabolite tenuifoliside A, one of the phenylpropanoids from P. tenuifolia, and investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 and murine peritoneal macrophages. The results showed that tenuifoliside A inhibited the production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PG E2), and cyclooxygenase (COX)-2. In addition, tenuifoliside A suppressed the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. We also evaluated the effects of tenuifoliside A on the activation of nuclear factor-kappaB (NF-κB). Tenuifoliside A inhibited the translocation of the NF-κB subunit p65 into the nucleus by interrupting the phosphorylation and degradation of inhibitor kappa B (IκB)-α in LPS-stimulated murine peritoneal macrophages. Moreover, we confirmed that the suppression of the inflammatory process by tenuifoliside A was mediated through the mitogen-activated protein kinases (MAPKs) pathway based on the fact that tenuifoliside A significantly decreased p-c-Jun N-terminal kinase (p-JNK) protein expression in LPS-stimulated murine peritoneal macrophages. Taken together, the anti-inflammatory effects of tenuifoliside A were mediated by the inhibition of the NF-κB and MAPK pathways. This study is the first report on the anti-inflammatory effects of tenuifoliside A, and the strong anti-inflammatory effects of tenuifoliside A provide potential compound to be developed as therapeutic for inflammatory diseases.

Tinospora cordifolia, a novel source of extracellular disaccharidases, useful for human disaccharidase deficiency therapy.

Disaccharide intolerance is the inability to digest certain carbohydrates due to a lack of one or more intestinal disaccharidases (e.g., lactase, maltase, isomaltase and sucrase). Symptoms include diarrhea, abdominal distention and flatulence. Management of the disorder by external enzymes supplementation has not yet been attempted. We report that the medicinal plant Tinospora cordifolia contains substantial amounts of all disaccharidases required for intestinal digestion of carbohydrates. The plant is also a rich source of saccharifying amylase. We recovered (units/100 g fresh stem) amylase: 49,000+500, maltase: 400+50, isomaltase: 130+50, sucrase: 4500+500, acid lactase: 350+30, cellobiase: 35+10 and trehalase: 40+10 by buffer extraction of the blended stem. Crude enzymes in the forms of stem powder, lyophilized aqueous extract and ethanol precipitated protein were found to be stable. Disaccharidases were optimally active at 50 (0) C in the pH range of 4-5. Lactase was an acid lactase similar to the type linked with human lactose intolerance. Enzymes were catalytically stable in the pH range of 2-7 and temperature range of up to 40 (0) C. T. cordifolia enzyme was non-toxic up to a dose of 200 mg protein/kg body weight.

Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis.

An unidentified Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) gene was cloned and heterologously expressed in Escherichia coli M15 cells. The over-expressed BlTreA was purified to apparent homogeneity by metal-affinity chromatography and its molecular mass was determined to be approximately 65.9 kDa. The temperature and pH optima for BlTreA were 30 °C and 8.0, respectively. The enzyme hydrolyzed p-nitrophenyl-α-d-glucopyranoside (pNPG) and trehalose-6-phosphate efficiently, but it was inactive toward five other p-nitrophenyl derivatives. Steady-state kinetics with pNPG showed that BlTreA had a K(M) value of 5.2mM and a k(cat) value of 30.2s(-1). Circular dichroism analysis revealed that the secondary structures of BlTreA did not altered by 5-10% acetone and 10-20% ethanol, whereas 5-10% SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.14 M guanidine hydrochloride (GdnHCl) and reached the unfolded intermediates, [GdnHCl](0.5,N-I) and [GdnHCl](0.5,I-U), at 1.02 and 2.24 M, respectively. BlTreA was unfolded completely by 8M urea with [urea](0.5,N-U) of 4.98 M, corresponding to a free energy change of 4.29 kcal/mol for the N→U process. Moreover, the enzyme was unfolded by GdnHCl through a reversible pathway and the refolding reaction exhibited an intermediate state. Taken together, the characterization data provide a foundation for the future structure-function studies of BlTreA, a typical member of glycoside hydrolase family 13.

Isolation of genes involved in biofilm formation of a Klebsiella pneumoniae strain causing pyogenic liver abscess.

BACKGROUND: Community-acquired pyogenic liver abscess (PLA) complicated with meningitis and endophthalmitis caused by Klebsiella pneumoniae is an emerging infectious disease. To investigate the mechanisms and effects of biofilm formation of K. pneumoniae causing PLA, microtiter plate assays were used to determine the levels of biofilm formed by K. pneumoniae clinical isolates and to screen for biofilm-altered mutants from a transposon mutant library of a K. pneumoniae PLA-associated strain. METHODOLOGY/PRINCIPAL FINDINGS: The biofilm formation of K. pneumoniae was examined by microtiter plate assay. Higher levels of biofilm formation were demonstrated by K. pneumoniae strains associated with PLA. A total of 23 biofilm-decreased mutants and 4 biofilm-increased mutants were identified. Among these mutants, a biofilm-decreased treC mutant displayed less mucoviscosity and produced less capsular polysaccharide (CPS), whereas a biofilm-increased sugE mutant displayed higher mucoviscosity and produced more CPS. The biofilm phenotypes of treC and sugE mutants also were confirmed by glass slide culture. Deletion of treC, which encodes trehalose-6-phosphate hydrolase, impaired bacterial trehalose utilization. Addition of glucose to the culture medium restored the capsule production and biofilm formation in the treC mutant. Transcriptional profile analysis suggested that the increase of CPS production in ΔsugE may reflect elevated cps gene expression (upregulated through rmpA) in combination with increased treC expression. In vivo competition assays demonstrated that the treC mutant strain was attenuated in competitiveness during intragastric infection in mice. CONCLUSIONS/SIGNIFICANCE: Genes important for biofilm formation by K. pneumoniae PLA strain were identified using an in vitro assay. Among the identified genes, treC and sugE affect biofilm formation by modulating CPS production. The importance of treC in gastrointestinal tract colonization suggests that biofilm formation contributes to the establishment and persistence of K. pneumoniae infection.

Immobilization of flavonol 3-O-beta-heterodisaccharidase on porous glass and production of rutinose from rutin.

Flavonol 3- O-beta-heterodisaccharidase (FHG 1) was isolated from the dried herb of Fagopyrum esculentum, immobilized on porous glass, and used for the release of rutinose from rutin. The stability of the enzyme in its free or immobilized form was observed continuously at two different temperatures (4 degrees C and 25 degrees C). T(1/2) values were determined to be about 48 h for the free enzyme and about 300 h for the immobilized enzyme. The rutinose released was isolated by fractionated ethanol precipitation.

Disaccharidase activities in camel small intestine: biochemical investigations of maltase-glucoamylase activity.

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.

Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrovirus (family Baculoviridae).

Baculovirus chitinases and other family 18 glycohydrolases have been shown to possess both exo- and endochitinase activities when assayed against fluorescent chito-oligosaccharides. Homology modelling of the chitinase of Epiphyas postvittana nucleopolyhedrovirus (EppoNPV) against Serratia marcescens chitinase A indicated that the enzyme possesses an N-terminal polycystic kidney 1 (PKD1) domain for chitin-substrate feeding and an alpha/beta TIM barrel catalytic domain characteristic of a family 18 glycohydrolase. EppoNPV chitinase has many features in common with other baculovirus chitinases, including high amino acid identity, an N-terminal secretion signal and a functional C-terminal endoplasmic reticulum-retention sequence. EppoNPV chitinase displayed exo- and endochitinolytic activity against fluorescent chito-oligosaccharides, with K(m) values of 270+/-60 and 240+/-40 microM against 4MU-(GlcNAc)2 and 20+/-6 and 14+/-7 microM against 4MU-(GlcNAc)3 for native and recombinant versions of the enzyme, respectively. In contrast, digestion and thin-layer chromatography analysis of short-chain (GlcNAc)(2-6) chito-oligosaccharides without the fluorescent 4-methylumbelliferone (4MU) moiety produced predominantly (GlcNAc)2, indicating an exochitinase, although low-level endochitinase activity was detected. Digestion of long-chain colloidal beta-chitin and analysis by mass spectrometry identified a single 447 Da peak, representing a singly charged (GlcNAc)2 complexed with a sodium adduct ion, confirming the enzyme as an exochitinase with no detectable endochitinolytic activity. Furthermore, (GlcNAc)(3-6) substrates, but not (GlcNAc)2, acted as inhibitors of EppoNPV chitinase. Short-chain substrates are unlikely to interact with the aromatic residues of the PKD1 substrate-feeding mechanism and hence may not accurately reflect the activity of these enzymes against native substrates. Based upon these results, the chitinase of the baculovirus EppoNPV is an exochitinase.

Expression, isolation, purification, and biochemical properties of trehalose-6-phosphate hydrolase from thermoresistant strain Bacillus sp. GP16.

Here we describe cloning, expression, and purification of the enzyme trehalose-6-phosphate hydrolase from thermoresistant strain Bacillus sp. GP16. Principal biochemical properties of the enzyme at different pH and temperature values were determined. Entropy and enthalpy of activation of the enzyme for substrates trehalose-6-phosphate and p-nitrophenyl glucoside were calculated, and the dependence of the kinetic parameters from ionic strength was established.

Purification and characterization of the phospho-alpha(1,1)glucosidase (TreA) of Bacillus subtilis 168.

The intracellular phospho-alpha(1,1)glucosidase TreA from Bacillus subtilis has been overproduced in Escherichia coli and purified by ion-exchange chromatography and gel filtration. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 64 kDa. Isoelectric focusing indicated homogeneity of the protein, and its pI was determined to be 4.3. Characterization of the enzyme showed a protein which is stable up to 44 degrees C after temperature treatment for 15 min. The temperature optimum was found to be 37 degrees C, and the pH optimum was 4.5. TreA activity is stimulated by high salt concentrations with different efficiencies depending on the kind of salt. When increasing amounts of ammonium sulfate are used, the increase of TreA activity is correlated with a conformational change of the protein or dimerization. The substrate specificity of the purified enzyme was characterized, showing additionally that trehalose is also hydrolyzed, but to a much smaller extent than trehalose-6-phosphate. In vitro, the presence of glucose reduces TreA activity, indicating product inhibition of the enzyme.

Trehalose-6-phosphate hydrolase of Escherichia coli.

The disaccharide trehalose acts as an osmoprotectant as well as a carbon source in Escherichia coli. At high osmolarity of the growth medium, the cells synthesize large amounts of trehalose internally as an osmoprotectant. However, they can also degrade trehalose as the sole source of carbon under both high- and low-osmolarity growth conditions. The modes of trehalose utilization are different under the two conditions and have to be well regulated (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). At low osmolarity, trehalose is transported via a trehalose-specific enzyme II of the phosphotransferase system, encoded by treB. The trehalose-6-phosphate formed internally is hydrolyzed to glucose and glucose 6-phosphate by the key enzyme of the system, trehalose-6-phosphate hydrolase, encoded by treC. We have cloned treC, contained in an operon with treB as the promoter-proximal gene. We have overproduced and purified the treC gene product and identified it as a protein consisting of a single polypeptide with an apparent molecular weight of 62,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes trehalose-6-phosphate with a Km of 6 mM and a Vmax of at least 5.5 mumol of trehalose-6-phosphate hydrolyzed per min per mg of protein. The enzyme also very effectively hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, but it does not recognize trehalose, sucrose, maltose, isomaltose, or maltodextrins. treC was sequenced and found to encode a polypeptide with a calculated molecular weight of 63,781. The amino acid sequence deduced from the DNA sequence shows homology (50% identity) with those of oligo-1,6-glucosidases (sucrase-isomaltases) of Bacillus spp. but not with those of other disaccharide phosphate hydrolases. This report corrects our previous view on the function of the treC gene product as an amylotrehalase, which was based on the analysis of the metabolic products of trehalose metabolism in whole cells.

Use of physiological substrates for zymograms of disaccharidases after separation in immobilized pH gradients.

A new technique is described for in situ visualization of the activity of intestinal disaccharidases after isoelectric focusing in immobilized pH gradients using their physiological substrates. The reaction principle is based on the oxidation of D-glucose, liberated by the disaccharidases, into D-gluconolactone and the production of NADH by glucose dehydrogenase. At the sites of enzymatic activity, tetrazolium salts present in the reaction mixture are reduced to relatively water-insoluble formazans by NADH. The rate of formazan production is increased by the presence of phenazine methosulfate. An additional modification of the technique involves the use of polyvinyl alcohol in the substrate solution. Due to the increase in the viscosity of the substrate solution, leakage of the enzyme from the IPG gels is minimized. Incubation times can thus be prolonged without loss of resolution and band-blurring.

Further characterization of an early expressed glycoprotein of the rabbit small intestinal brush border. Its interaction with some hydrolases.

Mouse 23 B 921 monoclonal antibody recognized a rabbit brush-border antigen with an apparent molecular mass 140 kDa (140-kDa Ag) which, unlike most hydrolases, is expressed in the poorly differentiated crypt cells of the small intestine. Immunoelectron microscopy of brush-border vesicles showed that the 23 B 921 bound to an epitope localized on the outside of the membrane. As is the case with hydrolases the external domain of the 140-kDa Ag constitutes the main part of the molecule, which can be released by papain treatment of brush-border vesicles. The presence of a small hydrophobic domain, anchoring the molecule into the membrane and responsible for its amphipatic character, was shown by its affinity for Triton-X114 micelles. The topological organization in the membrane of 140-kDa Ag seemed to be identical to that of hydrolases. Unlike hydrolases, however, the native structure of the antigen was found from its sensitivity to proteolysis to be very dependent on its integration into the lipid bilayer. Nevertheless, detergent-extracted 140-kDa Ag can be purified by immunoaffinity chromatography although it cannot be stocked for more than 48 h even in the presence of protease inhibitors. The carbohydrate moiety of 140-kDa Ag, bearing the human blood group A determinant, amounts to 20% of the total molecular mass. The existence of some privileged relationship was established between 140-kDa Ag and hydrolases: in the membrane, hydrolases protect the 140-kDa Ag from papain action; after detergent extraction, 140-kDa Ag is strongly associated with several hydrolases particularly disaccharidases.

Phase separation of rat intestinal brush border membrane proteins using Triton X-114.

Rat intestinal microvillus membrane contains at least 24 polypeptides, of which 18 can be solubilized using Triton X-114 at 4 degrees C. Upon phase separation at 32 degrees C, 11 proteins separated nearly completely into the detergent-rich phase, while 9 proteins were found exclusively in the aqueous phase. Enzymes which were uniquely included in the detergent phase were alkaline phosphatase, leucine aminopeptidase, gamma-glutamyl transpeptidase, and Ca2+-Mg2+ ATPase. The proteins which were excluded from the detergent phase and found exclusively in the aqueous phase included the disaccharidases (glucoamylase, sucrase-isomaltase, trehalase, lactase) and the ileal receptor for the intrinsic factor-cobalamin complex. Integral membrane proteins can thus be separated during solubilization into two groups prior to further purification or characterization.

Studies on the intestinal disaccharidases of the pigeon. II. Subcellular localization and solubilization.

In the pigeon, 70-80% of the activities of maltase (alpha-D-glucoside glucohydrolase EC 3.2.1.20), sucrase (alpha-glucohydrolase, EC 3.2.1.48), isomaltase (dextran 6-alpha-D-glucan hydrolase, EC 3.2.1.10) and glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) were found to be localized in the brush-border membrane of intestinal epithelial cells. Of the total glycosidase activities in the mucosal homogenate, nearly 60 to 70% were recovered in the microsomal (105 000 X g) fraction, about 30% in the mitochondrial (22 000 X g) fraction and less than 5% from the cytosol (105 000 X g supernatant) fraction. The hydrolases were solubilized by digestion with papain but not with trypsin, and the phosphate ion had a protective effect in the solubilization. Amongst detergents, Triton X-100 but not sodium deoxycholate, was found to truly solubilize these enzymes.