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:3.2.1.26 (invertase)
4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Specimens of human duodenal mucosa were obtained at duodenotomy. Superficial mucosal scrapings were homogenized in isotonic sucrose solution and fractionated by differential centrifugation. The distribution of organelles among the subcellular fractions was monitored by assay of suitable marker enzymes. 2. Enterokinase was recovered predominantly in the nuclear+brush-border fraction and 80% of the total activity was found to be particulate; approximately 20% of the enzyme was present in the soluble fraction, compared with 1% of the brush-border markers sucrase and alkaline phosphatase. 3. The brush-border-containing fraction was subfractionated by treatment with hypertonic Tris followed by differential and density gradient centrifugation. Enterokinase was distributed among the subfractions in parallel with brush-border markers and was concentrated in a subfraction which was highly enriched in microvillous membranes. 4. It was concluded that enterokinase is localized primarily to the microvillous membrane of the epithelial cell brush border in man, but that in addition a proportion of the enzyme may be present in a soluble or easily released form in the duodenal mucosa.
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PMID:Subcellular localization of enterokinase in human duodenal mucosa. 58 40

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1alpha-hydroxyvitamin D3 treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.
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PMID:Purification and characterization of chick intestine brush border membrane. Effects of 1alpha(OH) vitamin D3 treatment. 67 42

In the course of work concerned with the inhibition of small intestinal carbohydrate digesting enzymes, experiments were performed on rats and two healthy volunteers using tris as a sucrase inhibitor. The following results were obtained: (1) Tris does not lower the blood glucose in fasting rats after oral or subcutaneous doses up to 500 mg/kg, when administered as neutral solution (pH 7.0). (2) Tris reduces reduces the glycemia in rats and human subjects after a sucrose load. In addition, the insulinemia caused by administration of sucrose is reduced in man. This smoothing effect on both curves is dose-dependent. A delay of gastric emptying by tris could be excluded. (3) After a glucose or matose load in rats, tris has no effect on the blood sugar curve. (4) The marked smoothing effect of tris is after sucrose loading is probably caused by its well-known in vitro inhibitory effect on intestinal sucrase activity of pigs and humans.
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PMID:Inhibition of sucrase by tris in rat and man, demonstrated by oral loading tests with sucrose. 111 Jun 27

About 90% of the protein of hamster intestinal brush borders was solubilised in 0.25% (w/v) sodium dodecyl sulphate without total loss of biological activity. Detergent-polyacrylamide gel electrophoresis of the solubilised proteins separated 10-15 bands and partially resolved maltase, lactase, sucrase-maltase, trehalase and alkaline phosphatase activities. The disaccharidases, which were associated with the higher molecular weight proteins, were preferentially solubilised with 0.1%. (w/v) Triton X-100, butanol or papain, whereas Tris and NaI extracted only the lower molecular weight proteins, possible derived from the core filaments. Electrophoresis of brush border proteins metabolically labelled with [14-C] glucosamine suggested that many of the membrane-bound enzymes are glycoproteins. However, chromatography of a papain digest on Sephadex G-200 showed that the sucrase-maltase complex can be separated nearly free of carbohydrate without total loss of activity. The importance of characterizing membrane proteins solubilised by a number of techniques is discussed.
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PMID:Solubilization of brush borders of hamster small intestine and fractionation of some of the components. 113 70

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.
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PMID:Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls. 157 18

Tris and two of its hydroxylated amine analogs were examined in a metal-free, universal n-butylamine buffer, for their interaction with intestinal brush border sucrase. Our recent three-proton-families model (Vasseur, van Melle, Frangne and Alvarado (1988) Biochem. J., 251, 667-675) has provided the sucrase pK values necessary to interpret the present work. At pH 5.2, 2-amino-2-methyl-l-propanol (PM) causes activation whereas Tris has a concentration-dependent biphasic effect, first causing activation, then fully competitive inhibition. The amine species causing activation is the protonated, cationic form. The difference between the two amines is related to the fact that Tris has a much lower pKa value than PM (respectively, 8.2 and 9.8). Even at pH 5.2, Tris (but not PM) exists as a significant proportion of the free base which, by inhibiting the enzyme fully competitively, overshadows the activating effect of the cationic, protonated amine. Above pH 6.8, both Tris and PM act as fully competitive inhibitors. These inhibitions increase monotonically between pH 6.5 and 8.0 but, above pH 8, inhibition by 2.5 mM Tris tends to diminish whereas inhibition by 40 mM PM increases abruptly to be essentially complete at pH 9.3 and above. As pH increases from 7.6 to 9.0, the apparent affinity of the free amine bases decreases whereas that of the cationic, protonated amines, increases. In this way, the protonated amines replace their corresponding free bases as the most potent inhibitors at high pH. The pH-dependent inhibition by 300 mM Li+ is essentially complete at pH 8, independent of the presence or absence of either 2.5 mM Tris or 40 mM PM. Even at pH 7.6, an excess (300 mM) of Li+ causes significant increases in the apparent Ki value of each Tris, PD (2-amino-2-methyl-1-3-propanediol) and PM, suggesting the possibility of a relation between the effects of Li+ and those of the hydroxylated amines which in fact are mutually exclusive inhibitors. The inhibitory results are interpreted in terms of a mechanistic model in which the free bases bind at two distinct sites in the enzyme's active center. Binding at the glucosyl sub-site occurs through the amine's free hydroxyl groups. This positioning facilitates the interaction between the lone electron pair of the deprotonated amino group with a proton donor in the enzyme's active center, characterized by a pK0 around 8.1. When this same group deprotonates, then the protonated amines acting as proton donors replace the free bases as the species giving fully competitive inhibition of sucrase.
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PMID:pH-dependent inhibitory effects of tris and lithium ion on intestinal brush-border sucrase. 209 67

A detergent solubilised sucrase from monkey small intestine has been purified 388-fold to gel electrophoretic homogeneity with an overall recovery of 36%. The molecular weight of the enzyme was 263 kDa by gel filtration. Electrophoresis in the presence of SDS indicates that the enzyme is a hetero-dimer. Mixed substrate inhibition studies and inhibition by PCMB and Tris suggest the presence of two catalytically active sites in the form of maltase and sucrase with isomaltase activity being common to both sites. Polyclonal antiserum against the purified enzyme showed a single continuous precipitin line with the purified antigen.
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PMID:Some properties of monkey intestinal sucrase. 211 33

Disaccharidases of oral bacteria, especially alpha-glucosidase and beta-fructofuranosidase, are considered to play an important role in the induction of dental caries. Upon the examination of disaccharidases from several strains of saccharolytic oral bacteria, we found all of those bacteria to be capable of hydrolyzing the glycosidic linkage of sucrose. One species of bacteria, Rothia dentocariosa, was found to contain a single disaccharidase, alpha-glucosidase. This enzyme was partially purified by ammonium sulfate precipitation, gel filtration and ion-exchange column chromatography. The optimum pH and temperature for the enzyme activity was found to be 6.8-7.0 and 40 degrees C, respectively. The enzyme activity was strongly inhibited by Ag+, Hg2+, Cu2+, Fe2+ and Tris (Hydroxymethyl) aminomethane.
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PMID:Partial purification and characterization of alpha-glucosidase from Rothia dentocariosa. 263 58

An inside-alkaline pH gradient (pH 7.7 inside, 5.5 outside) stimulated Cl uptake in brush-border vesicles from rabbit ileum. The addition of HCO3 without changing the pH gradient further stimulated Cl uptake to a level fourfold greater than equilibrated Cl uptake. Although a K diffusion potential stimulated Cl uptake, this was insensitive to inhibition by 4,4-diisothiocyanostilbene-2,2'-disulfonate (DIDS), whereas pH and HCO3 gradient-stimulated Cl uptake was inhibited by DIDS. pH and HCO3 gradient-stimulated Cl uptake was found to be a saturable function of the Cl concentration with a Km of 3.5 mM and a Vmax of 49 nmol X mg prot-1 X min-1. To distinguish between coupling of Na and Cl transport by cotransport or dual exchange (Na-H and Cl-HCO3 exchange), we determined uptake with high (134 mM Tris-HEPES-MES) internal buffer and low (1.34 mM Tris-HEPES-MES) internal buffer concentrations. Inwardly directed gradients of 50 mM Na, 50 mM K, or 50 mM Na and K did not stimulate Cl uptake, and 50 mM Cl, 50 mM K, or 50 mM KCl did not stimulate Na uptake, with high internal buffer, excluding cotransport. In contrast, 50 mM Na stimulated Cl uptake (inhibited by 1 mM DIDS) and 50 mM Cl stimulated Na uptake (inhibited by 1 mM amiloride) in low buffer media. To determine a role for carbonic anhydrase, Na-stimulated Cl uptake was determined in low buffer media, equilibrated with either 100% N2 or 95% N2-5% CO2. Na stimulated Cl uptake 80% (compared with trimethylammonium control) with CO2 but only 30% with N2 (P less than 0.05). Acetazolamide partially inhibited (P less than 0.025) the stimulation of Cl uptake with CO2 but not with N2. Carbonic anhydrase activity was measured in homogenate and brush-border membrane and was enriched 7.9 +/- 0.4-fold, whereas sucrase was enriched 14.0 +/- 1.1-fold. We conclude that coupled Na and Cl transport occurs by dual exchange (Na-H and Cl-HCO3) and carbonic anhydrase, apparently located on the brush-border membrane, facilitates dual exchange by providing HCO3.
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PMID:Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange and mechanism of coupling. 392 45

Sucrase-isomaltase (S-I) and maltase-glucoamylase (M-G) of the brush border have been purified to electrophoretic homogeneity from the pigeon small intestine. Heat-inactivated enzymes of crude homogenates of the pigeon intestinal mucosa, papain-solubilized enzymes and those obtained after chromatographic fractionation behaved in an identical manner. Depending on their sensitivity to heat treatment, the disaccharidases were identified to consist of two maltases; one, the heat-labile maltase, and the other, the heat-stable maltase. Sucrase and isomaltase constituted the thermolabile maltase and could be distinguished from each other. Maltase and glucoamylase formed the thermostable maltase the activities of which however, remained inseparable. Based on these results and in accordance with the nomenclature suggested by Dahlqvist & Telenius (1969), the pigeon intestinal disaccharidases were classified as follows: Maltase Ia = isomaltase, Maltase Ib = sucrase, and Maltase II = glucoamylase. DEAE-Cellulose chromatography did not resolve the two enzyme complexes but gel filtration of the active fractions recovered from the former step, resulted in their separation into two distinct peaks. Sucrase, isomaltase and a part of the maltase activity were recovered in the first peak which eluted close to the void volume. Glucoamylase and the remaining maltase activity were recovered in the second peak which appeared to have been retarded on the column because they were eluted much more slowly. The S-I and M-G complexes have an apparent molecular weight of 195 kd and 209 kd as determined by their gel-filtration pattern on Sepharose 6B. S-I hydrolysed alpha-glucosides such as maltose, sucrose and palatinose with a Km of 3.12 mM, 8 mM and 8.36 mM respectively and did not attack starch or dextran. In contrast, M-G catalysed the hydrolysis of starch, amylose and maltose with a Km of 3.12 mM, 7.59 mM and 3.52 mM respectively, and had no action on sucrose or palatinose. Both S-I and M-G were glycoproteins, and were inhibited by Ag+, Hg2+ and Tris but not by p-hydroxymercuribenzoate, iodoacetamide or imidazole. Na+ on the other hand activated both the enzyme complexes by about 20-25%. It is suggested that the molecular and catalytic properties of intestinal disaccharidases of pigeons do not differ considerably from those of Mammals.
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PMID:Studies on the intestinal disaccharidases of the pigeon. III. Separation, purification and properties of sucrase-isomaltase and maltase-glucoamylase. 620 6


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