Glucose- and phlorrhizin-protected thiol groups in pig intestinal brush-border membranes.
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ABSTRACT:
SUBMITTER: Smith MW
PROVIDER: S-EPMC1165491 | biostudies-other | 1975 Jun
REPOSITORIES: biostudies-other
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Project description:This work highlights the utility of brush border membrane vesicles (BBMV) from the pig small intestine as a reliable model for gathering information about the reaction mechanisms involved in the human digestion of dietary carbohydrates. Concretely, the elucidation of the transgalactosylation mechanism of pig BBMV to synthesize prebiotic galacto-oligosaccharides (GOS) is provided, unravelling the catalytic activity of mammalian small intestinal ?-galactosidase towards the hydrolysis of GOS. This study reveals that pig BBMV preferably synthesizes GOS linked by ?-(1???3) bonds, since major tri- and disaccharide were produced by the transfer of a galactose unit to the C-3 of the non-reducing moiety of lactose and to the C-3 of glucose, respectively. Therefore, these results point out that dietary GOS having ?-(1???3) as predominant glycosidic linkages could be more prone to hydrolysis by mammalian intestinal digestive enzymes as compared to those linked by ?-(1???2), ?-(1???4), ?-(1???1) or ?-(1???6). Given that these data are the first evidence on the transglycosylation activity of mammalian small intestinal glycosidases, findings contained in this work could be crucial for future studies investigating the structure-small intestinal digestibility relationship of a great variety of available prebiotics, as well as for designing tailored fully non-digestible GOS.
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Project description:Aminopeptidase N (EC 3.4.11.2), when isolated from pig intestine in either the proteinase- or detergent-released form, frequently appears to contain three polypeptide chains, here termed alpha, beta and gamma. We have established by an immunological technique that the beta- and gamma-polypeptides are derived from the alpha-chain and that the intact enzyme is a dimer, alpha 2. Each alpha-chain of the detergent form was shown to contain a hydrophobic anchor peptide about 35 amino acid residues in length, which included the N-terminal sequences. A peptide bond in the alpha-chain was very sensitive to proteolysis. Its cleavage generated the commonly observed forms: alpha beta gamma and beta 2 gamma 2. The gamma-fragment, which lacked the anchor peptide, was derived from the C-terminal part of the alpha-chain.
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Project description:An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.
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Project description:D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose.
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Project description:Transporting epithelial cells build apical microvilli to increase membrane surface area and enhance absorptive capacity. The intestinal brush border provides an elaborate example with tightly packed microvilli that function in nutrient absorption and host defense. Although the brush border is essential for physiological homeostasis, its assembly is poorly understood. We found that brush border assembly is driven by the formation of Ca(2+)-dependent adhesion links between adjacent microvilli. Intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips and interact to form a trans-heterophilic complex. The cytoplasmic domains of microvillar protocadherins interact with the scaffolding protein, harmonin, and myosin-7b, which promote localization to microvillar tips. Finally, a mouse model of Usher syndrome lacking harmonin exhibits microvillar protocadherin mislocalization and severe defects in brush border morphology. These data reveal an adhesion-based mechanism for brush border assembly and illuminate the basis of intestinal pathology in patients with Usher syndrome. PAPERFLICK:
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Project description:A dipeptide hydrolase from the brush border of guinea-pig intestinal mucosa was purified. The enzyme resembles another dipeptide hydrolase isolated from the cytosol fraction of intestinal mucosa. Studies on the binding of cytosol peptide hydrolase to brush-border membranes indicate that the enzyme found in the brush border may be a cytoplasmic contaminant.
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Project description:A series of experiments were conducted to examine the possible effects of subcutaneous administration of the synthetic glucocorticoid dexamethasone (100 micrograms/day per 100 g body wt.) on the lipid fluidity and lipid composition of rat proximal-small-intestinal brush-border membranes. After 4 days of treatment, membranes and their liposomes prepared from treated animals possessed a greater fluidity than did their control (diluent, 0.9% NaCl) counterparts, as assessed by steady-state fluorescence-polarization techniques using several different fluorophores. Examination of the effects of temperature on the anisotropy values of 1,6-diphenylhexa-1,3,5-triene, using Arrhenius plots, moreover, revealed that the mean break-point temperatures of the treated preparations were approx. 3-4 degrees C lower than those of their control-preparation counterparts. Changes in the sphingomyelin/phosphatidylcholine (PC) molar ratio as well as in certain of the fatty acids of the PC fraction of treated membranes, secondary to alterations in membrane PC levels and in lysophosphatidylcholine acyltransferase activities respectively, were also noted after dexamethasone administration. These compositional alterations appeared to be responsible, at least in part, for the differences in fluidity noted between treated and control plasma membranes. These results therefore demonstrate that dexamethasone administration can modulate the lipid fluidity and lipid composition of rat proximal-small-intestinal brush-border membranes.
| S-EPMC1148563 | biostudies-other
Project description:1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22mumol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7-0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37 degrees C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.
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Project description:Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.
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Project description:The amounts of lactase (beta-D-galactosidase, EC 3.2.1.23), sucrase (sucrose alpha-D-glucohydrolase, EC 3.2.1.48), maltase (alpha-D-glucosidase, EC 3.2.1.20) microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.-) in tangentially sectioned biopsies from jejunum were studied by quantitative immunoelectrophoresis and enzymic assays. All enzymes had their maximum activities near the mid-region of the villi and their lowest activities at the bases of the crypts. The ratio between enzyme activity and immunoreactive protein was constant along the villus-crypt axis. This result is consistent with a continuous brush-border-enzyme synthesis as the enterocytes migrate up the villi.
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