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At least 1,207 records · Page 67Linked to original sources
PubMed1965

THE ISOLATION AND CHARACTERIZATION OF SUBCELLULAR COMPONENTS OF THE EPITHELIAL CELLS OF RABBIT SMALL INTESTINE.

1. Homogenization of the epithelial cells of rabbit small intestine in 0.3m-sucrose-5mm-EDTA, pH7.4, maintains intact the microvillus sheets that form the lumenal surface of the cells, the nuclei, the mitochondria and the vesicles (microsomes) formed from the endoplasmic reticulum. 2. These particulate components of the cell, and the cell-sap fraction, have been isolated by differential centrifuging of cell homogenates. 3. The nuclei and microvillus sheets sediment together and it has been impossible to separate these subcellular components by centrifugal methods. 4. The isolated subcellular fractions have been identified by a combination of light-microscopic examination, electron-microscopic examination, chemical analysis and assay for selected enzyme activities.

Acid Phosphatase↗
PubMed1963

[GYNECOLOGY].

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Abortion, Threatened↗
PubMed1997

Mechanism of catalysis by retaining beta-glycosyl hydrolases.

Recent structural studies provide a fresh look at the catalytic mechanism of polysaccharide hydrolysis by retaining beta-glycosyl hydrolases. Highlights include insights into saccharide ring distortion, both upon binding and during the course of catalysis, and evidence for the regulation of the pKa of key catalytic residues.

Affinity Labels↗
PubMed1988

Catabolism of Cyanogenic Glycosides by Purified Vicianin Hydrolase from Squirrel's Foot Fern (Davallia Trichomanoides Blume).

Vicianin hydrolase, which catalyzes the hydrolysis of vicianin (K(m), 4.9 millimolar) to (R)-mandelonitrile and vicianose at an optimum pH of 5.5, was extensively purified from the young fronds and fiddleheads of the squirrel's foot fern (Davallia trichomanoides Blume) using DEAE-cellulose and Ultrogel HA chromatography. The native molecular weight of the enzyme was 340,000, and the isoelectric point was 4.6 to 4.7. SDS-PAGE analysis yielded three polypeptides with molecular weights of 56,000, 49,000, and 32,500. The enzyme hydrolyzed only a narrow range of glycosides and, among cyanogenic glycosides, exhibited a strict requirement for (R)-epimers and a preference for disaccharides over monosaccharides. (R)-Amygdalin, (R)-prunasin and p-nitrophenyl-beta-d-glucoside were hydrolyzed at 27, 14, and 3%, respectively, of the rate of vicianin hydrolysis. Mixed substrate studies showed that (R)-vicianin, (R)-prunasin, and p-nitrophenyl-beta-d-glucoside competed for the same active site. The enzyme was significantly inhibited by castanospermine, delta-gluconolactone, and p-chloromercuriphenylsulfonate. Failure to recognize concanavalin A-Sepharose 4B and to stain with periodic acid-Schiff reagent indicated that the enzyme was not a glycoprotein.

Journal Article↗
PubMed1998

Hydrolase-catalyzed beta-mannosylations.

Transmannosylations catalysed by beta-mannosidase from snail viscera or beta-galactosidase from Aspergillus oryzae were accomplished with 4-nitrophenyl beta-D-mannopyranoside as donor substrate. With suitable hydrophobic acceptor molecules preferentially beta1-4-linked disaccharides were obtained. The activities of both glycosidases in buffer cosolvent mixtures were determined, and conditions for their immobilization were elaborated and optimized. A model of the enzymic transfer mechanism is suggested.

Animals↗
PubMed1967

Enzymic hydrolysis of the carbon-fluorine bond of alpha-D-glucosyl fluoride by rat intestinal mucosa. Localization of intestinal maltase.

1. alpha-d-Glucosyl fluoride was hydrolysed by an extract of rat intestinal mucosa. The pH optimum was 6.6 and the K(m) 0.4mm at 20 degrees . Activity was assayed by release of either glucose or fluoride. 2. The alpha-d-glucosyl fluoride-hydrolase activity of the extract was associated with both mutarotase and alpha-d-glucosidase activities. 3. Tris (5mm) inhibited both the alpha-d-glucosidase and alpha-d-glucosyl fluoride-hydrolase activities by 55% but did not inhibit mutarotase. The K(i) of tris for both enzyme activities was 2mm. 4. The extract did not hydrolyse melibiose and lactose. Mutarotase used both alpha-d-glucose and beta-l-arabinose as substrates but the glucosyl fluoride-hydrolase activity did not extend to beta-l-arabinosyl fluoride. 5. The thermal stability of alpha-d-glucosidase and alpha-d-glucosyl fluoride hydrolase was identical. Mutarotase was more thermolabile. 6. A preparation of the brush border of intestinal epithelial cells contained both alpha-d-glucosyl fluoride-hydrolase and alpha-d-glucosidase activities. In each precipitate and washing the ratio of the two activities was the same. All the mutarotase activity was in the first supernatant. 7. Agidex, a fungal amyloglucosidase, cleaved glucosyl fluoride in addition to maltose. Tris inhibited both activities and in each case the K(i) was 3mm. 8. The probable identity of alpha-d-glucosyl fluoride hydrolase with alpha-d-glucosidase is discussed and a possible mechanism for the reaction suggested. 9. Incubation of intestinal slices with alpha-d-glucosyl fluoride led to complete hydrolysis in 30min. The glucose rapidly entered the cell and was metabolized, leaving the fluoride in the incubation medium. This constitutes a further proof that the intestinal alpha-d-glucosidase, although on the brush border, is located outside the site of active transport of sugars.

Animals↗
PubMed2005

Hyperthermophilic alpha-L: -arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein.

A putative alpha-L: -arabinofuranosidase (AFase) gene belonging to family 51 of glycosyl hydrolases of a hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein (Tm-AFase) was purified to apparent homogeneity by heat treatment (80 degrees C, 30 min), followed by hydrophobic interaction, anion-exchange, and gel permeation column chromatography. Tm-AFase had a molecular mass of 55,284 Da on matrix assisted laser desorption ionization time-of-flight mass spectrometry and approximately 332 kDa on gel permeation column chromatography. Therefore, Tm-AFase comprised six identical subunits as in the case of homologous AFase from Geobacillus stearothermophilus. Regarding substrate specificity, Tm-AFase was active with p-nitrophenyl alpha-L: -arabinofuranoside but not with p-nitrophenyl alpha-L: -arabinopyranoside. Regarding polysaccharides, Tm-AFase hydrolyzed arabinan and debranched arabinan but not arabinoxylan, arabinogalactan, and carboxymethyl cellulose. Tm-AFase was extremely thermophilic, displaying an optimal reaction temperature of 90 degrees C in a 10 min assay. When Tm-AFase was heated at 90 degrees C, no loss of activity was observed for at least 24 h. At 100 degrees C, the activity dropped to approximately 50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of approximately 2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.

Amino Acid Sequence↗
PubMed2002

Differentiation of feruloyl esterases on synthetic substrates in alpha-arabinofuranosidase-coupled and ultraviolet-spectrophotometric assays.

4-Nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside, synthesized by our group (M. Mastihubová, J. Szemesová, and P. Biely), were found to be suitable substrates for determination of activity of feruloyl esterases (FeEs) exhibiting affinity for 5-O- and 2-O-feruloylated alpha-L-arabinofuranosyl residues. One assay is based on coupling the FeE-catalyzed formation of 4-nitrophenyl alpha-L-arabinofuranoside with its efficient hydrolysis by alpha-L-arabinofuranosidase to release 4-nitrophenol. An alternative assay explores the difference in the molar absorbances at 340 nm of the substrate (ferulic acid esters) and the reaction products, which are (1) free ferulic acid and 4-nitrophenyl alpha-L-arabinofuranoside in samples free of alpha-L-arabinofuranosidase and (2) ferulic acid, 4-nitrophenyl alpha-L-arabinofuranoside, and/or 4-nitrophenol in samples containing alpha-L-arabinofuranosidase. The new substrates represent convenient tools to differentiate FeEs on the basis of substrate specificity.

Arabinose↗
PubMed2002

Effects of exogenous propylene on softening, glycosidase, and pectinmethylesterase activity during postharvest ripening of apricots.

Apricots (Prunus armeniaca L. cv. Boccuccia spinosa) picked at the commercial ripening stage [soluble solids content (SSC) 12.6%] were left to reach full ripening in continuously humidified air at 20 degrees C. Changes in the rate of ethylene production, firmness, soluble solids concentration, and titratable acidity were measured. The alpha-D- and beta-D-glucosidases, alpha-L-arabinofuranosidase, alpha-D- and beta-D-galactosidases, beta-D-xylosidase, and alpha-D-mannosidase activities were assayed. To evaluate the influence of ethylene on glycosidase activity, propylene (500 microL x L(-1)) was applied to apricots for 24 and 48 h. In apricots ripened in air, ethylene production increased on the first day and exhibited a typical climacteric pattern. Good edible quality was reached in 5 days when SSC was at least 14% and acidity was between 1.1 and 1.2% (% malic acid). During postharvest ripening, alpha-L-arabinofuranosidase activity increased from 1.9 to 11.6 nkat until day 7. alpha-D-Galactosidase, alpha-D-mannosidase, and beta-D-galactosidase activity increased continuously but at a lower rate. beta-D-Xylosidase activity also increased, but the level of activity was lower than the other glycosidases assayed. Pectinmethylesterase (PME) decreased during the postharvest ripening, and propylene enhanced this pattern, by stimulating ethylene production. Even the activities of alpha-L-arabinofuranosidase, beta-D-xylosidase, alpha-D-mannosidase, and beta-D-galactosidase were greatly stimulated by the propylene treatment, which consequently induced rapid softening of the fruits.

Alkenes↗
PubMed1991

Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions.

In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis.

Base Sequence↗