Rat Intestinal Membrane Research Articles

The partial characterization of the binding of avidin-biotin complex to rat liver plasma membrane.

Rat liver plasma membrane and rat intestinal mucosa plasma membrane were examined for their ability to bind avidin-[3H]biotin complex. Avidin-[3H]biotin complex bound specifically to liver plasma membrane with a Kd of approx. 35 nM and Bmax. of 136 pmol/mg of membrane protein. The receptor on the liver plasma membrane was exposed by Pronase, sensitive to tryptic hydrolysis and insensitive to neuraminidase. The optimum pH of specific binding was less than 6.0. Simple carbohydrates, mannan and ovalbumin did not inhibit specific binding. Periodate-treated or alpha-mannosidase-treated avidin was able to inhibit binding of either avidin-[3H]biotin complex or untreated avidin to plasma membrane. [3H]Avidin containing methylated lysine residues did not bind to rat liver plasma membrane. There was no specific binding of the avidin-biotin complex to rat intestinal mucosa plasma membrane.

Effect of prostaglandin A 2 on Na +-K +-ATPase in basolateral plasma membrane of rat intestine In vitro: Inhibition of activation of K +-dependent p-nitrophenylphosphatase by Na + and ATP

1. 1. The activation of K +-dependent p-nitrophenylphosphatase (EC 3.6.1.7) by both Na + and ATP in rat intestinal basolateral plasma membrane is inhibited by prostaglandin A 2. 2. 2. The drug's inhibition of the activation of the enzyme by both Na + and ATP is due to a decrease in the affinity of the enzyme for Na + and in the V max of the enzyme. 3. 3. The K i values for this drug for Na + and ATP in the activation of the enzyme were 45 and 70 μm. respectively.

The role of cholesteryl ester hydrolysis and synthesis in cholesterol transport across rat intestinal mucosal membrane a new concept

The rate of accumulation of cholesteryl ester in rat intestinal sacs is enhanced up to 3-fold by the addition of porcine cholesterol esterase to the uptake medium. For this the enzyme must be catalytically active and does not exert its effect via its activity in the medium. A mechanism for this rate enhancement is proposed in which the action of cholesterol esterases on both sides of a bilayer membrane can catalyze the net transmembrane movement of cholesterol.

Angiotensin I converting enzyme of rat intestinal and vascular surface membrane

High levels of angiotensin I converting enzyme are present in rat intestinal mucosa and in intestinal arteries. Homogenates of both tissues were subfractionated and fractions enriched in vascular plasma membrane or intestinal brush border were prepared. The preparations were identified and their purities established by marker enzyme enrichment and/or electron microscopy. Converting enzyme activity was highly enriched on both the vascular plasma membrane and the intestinal brush border. Subsequently the properties of these membrane-bound enzymes were compared. Both surface membrane-bound enzymes were highly sensitive to inhibition by captopril (SQ 14225) and teprotide (SQ 20881). Similar to converting enzyme isolated from other sources, they were also inhibited by bradykinin, angiotensin I, EDTA and o-phenanthroline. Finally, both membrane-bound enzymes were relatively resistant to activation by sonication, freezing and thawing or detergent. These results demonstrate significant similarities between surface membrane-bound converting enzyme from vascular and non-vascular sites. In addition, in view of the possible relationship of kinins and angiotensins to gastrointestinal function and blood flow, inhibition of gastrointestinal converting enzyme by captopril may affect some aspects of intestinal physiology.

Alterations in labeling of cell-surface glycoproteins from normal and diabetic rat intestinal microvillous membranes

The effect of chronic streptozotocin-induced diabetes was studied on intestinal microvillous membrane surface carbohydrate groups. After 7 weeks of diabetes, purified microvillous membranes were prepared from rat small intestine and surface galactoproteins identified by labeling with galactose oxidase/sodium boro[ 3H]hydride. Membrane surface sialic acid residues were labeled using the sodium metaperiodate/sodium boro[ 3H]hydride technique. Membranes were solubilized in SDS and protein labeling analyzed by acrylamide electrophoresis. Membranes from diabetic rats showed an 81% increase in galactoprotein labeling ( P< 0.02) while labeling of sialic acid residues was unchanged. The greatest increase in galactoprotein labeling occurred in protein monomers of M r 116 000–200 000, where there was a 155% increase in labeling ( P< 0.005). These results indicate that intestinal microvillous membrane protein glycosylation is altered in chronic diabetes. This increase in surface membrane carbohydrates could explain the decreased rates of proteolytic degradation previously described for at least one microvillous protein. An increase in membrane galactose groups has also been noted in hepatocyte and kidney glomerular basement membranes, which suggests the presence of a systematic change in membrane protein glycosylation occurring as a result of the diabetic state.

Rat intestinal microvillus membrane sucrase-isomaltase is a single high molecular weight protein and fully active enzyme in the absence of luminal factors

Sucrase-isomaltase immunoprecipitated from brush border of an intestinal transplant lacking pancreatic proteases was found to be a single, high molecular weight protein. Elastase digestion converted this protein into two subunits which co-migrated on electrophoresis with those normally found on the microvillus membrane. The high molecular weight from had full sucrase and isomaltase activities.

Partial characterization of the carbohydrate units of rat intestinal sucrase--isomaltase.

Sucrase--isomaltase was purified from rat intestinal microvillus membranes after injection of D-[2-3H]mannose and L-[6-3H]fucose, using a column of monoclonal antibody-protein A-Sepharose. After Pronase digestion and gel filtration of the glycopeptides labelled from both precursors, a major part of the radioactivity was recovered in asparagine-linked complex oligosaccharides, and a smaller amount in partially alkali-labile high-molecular-weight glycopeptides. Only a small amount of [3H]mannose was found in endo-beta-N-acetylglucosaminidase H-sensitive high-mannose oligosaccharides.

Inhibition of Na +-stimulated glucose transport function and perturbation of intestinal microvillus membrane vesicles by ethanol and acetaldehyde

The effects of ethanol and acetaldehyde on rat intestinal microvillus membrane integrity and glucose transport function were examined in vitro with purified membrane vesicles. Ethanol could influence glucose transport function by alterations in the conformation of the carrier, the lipid environment surrounding the carrier, or in the transport driving force (Na + electrochemical gradient). Due to the rapid nature of glucose uptake, transport was assayed with the use of an apparatus that permitted uptake measurements as early as 1 s. Ethanol (340 m m) partially and acetaldehyde (44 m m) completely inhibited concentrative glucose uptake throughout the 1-min time course. Their inhibitory effects were reversible and irreversible, respectively. Kinetic measurements made during the initial rate of uptake (at 2 s) with various concentrations of glucose (0.05–8 m m) showed that ethanol and acetaldehyde both caused a decrease in V. Although ethanol did not substantially alter the transport K m , acetaldehyde increased the K m almost 50%. To determine whether ethanol or acetaldehyde directly interfered with glucose carrier function, uptake was measured in the presence of equilibrated Na +. Only acetaldehyde had a significant inhibitory effect under these conditions. Membrane permeability, as determined by efflux of preloaded 6-carboxyfluorescein dye, increased upon exposure of the vesicles to ethanol or acetaldehyde. Membrane fluidity measurements by fluorescence polarization showed that only acetaldehyde had a significant fluidizing effect. These results indicate that ethanol and acetaldehyde acted to perturb membrane integrity and inhibited glucose uptake indirectly by allowing the Na + gradient to dissipate. Acetaldehyde, which had a stronger inhibitory effect than ethanol, appeared also to directly inhibit carrier function.

Properties of Ca 2+ uptake and release by Golgi membrane vesicles from rat intestine

A previous study of energy-independent in vitro Ca 2+ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH) 2D 3 repletion ( R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA 74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology 78, 325–332). In the present study, properties of Ca 2+ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg 2+ and Sr 2+, but not by Na + or K +. Lowering the external [H +] or raising the internal [H +] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H +] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl 2 or SrCl 2 but not CaCl 2, NaCl, or KCl. Vesicles preloaded with K 2SO 4 failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl 2 was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca 2+ uptake maintained at equilibrium. Therefore, the remainder of the Ca 2+ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10 −6 m was found for this binding. The bound Ca 2+ could be rapidly released by external Mg 2+ or Sr 2+, but not Ca 2+, Na +, or K +. Release of bound Ca 2+ could also be induced by raising the [H +] of the external medium. Failure of external Ca 2+ to release bound Ca 2+ suggested that the release induced by external Mg 2+, Sr 2+, or H + was not due to competitive displacement of Ca 2+ from its binding sites. These results indicated that Ca 2+ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca 2+ to the vesicle. The effects of H +, Mg 2+, and Sr 2+ on Ca 2+ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.

Membrane lipids can modulate guanylate cyclase activity of rat intestinal microvillus membranes.

Studies of the temperature dependence (10-40 degrees C) of guanylate cyclase in rat intestinal microbillus membranes reveal a change in energy of activation (slope of the Arrhenius plot) at 30 +/- 1 degree C. The break point temperature corresponds to the lipid thermotropic transition in these membranes previously characterized by differential scanning calorimetry (range: 23-39 degrees C; peak temperature, 31 degrees C). The break point temperature for guanylate cyclase also corresponds to that of a number of other microbillus membrane enzymes and of D-glucose transport. These activities are defined as "intrinsic" membrane activities by this operational criterion. Treatment with the nonionic detergent Lubrol WX increased the guanylate cyclase activity 4- to 8-fold and removed the discontinuity in the Arrhenius plot.

Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes.

Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant, Km (1.5 x 10(5) M), similar to the inhibitory constant KI (3 X 10(-5) M), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabaiation rate constants reported for other tissues and species. The high dissociation rate constant 3.6 x 10(-2) sec-1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represents a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.

Thermotropic transitions in rat intestinal plasma membranes studied by differential scanning calorimetry and fluorescence polarization.

Thermotropic transitions in rat intestinal plasma membranes studied by differential scanning calorimetry and fluorescence polarization.

Biphasic recovery of vitamin D-dependent Ca2+ uptake by rat intestinal Golgi membranes

A previous study had demonstrated that rat intestinal epithelial cell Golgi, lateral-basal, and microvillus membrane vesicles translocated Ca2+, and that this process was markedly decreased in vitamin D-deficient rats. In the present study the kinetics of recovery of Ca2+ uptake by intestinal Golgi vesicles was examined in vitamin D-deficient rats after a single i.v. injection of 125 ng 1,25-dihydroxycholecalciferol (1,25-(OH)2D3). Calcium uptake was measured by determination of 45Ca2+ associated with Golgi membranes after collection by micropore filtration. Golgi membranes recovered rapidly (66% in 15 min, 83% in 30 min) but then quickly returned to prerepletion levels by 8 hr. This was followed by a second phase of recovery starting at 48 hr, with approximately 80% recovery by 96 hr which was maintained at least to 120 hr. By comparison, partial recovery of calcium uptake by microvillus membrane vesicles was first detected at 15 hr and full recovery did not occur until 72 hr; a biphasic pattern to microvillus membrane recovery was not apparent. The vitamin D-dependent recovery of Ca2+ uptake by Golgi membrane vesicles was also compared to studies with gut sacs which showed 50% recovery by 3 hr and 100% recovery by 6 hr; gut sac recovery was sustained for at least 120 hr. The recovery of Ca2+ uptake by Golgi was shown to be specific for 1,25-(OH)2D3. The first peak of recovery of Ca2+ uptake by intestinal Golgi membrane vesicles is one of the earliest reported effects of 1,25-(OH)2D3. The biphasic nature of recovery demonstrated by Golgi membranes suggests that there may be more than one mechanism of action of 1,25-(OH)2D3 on the intestine.

Terbium as a fluorescent probe for analysis of the nature of Ca2+-binding sites of rat intestinal mucosal membranes.

Journal Article Terbium as a Fluorescent Probe for Analysis of the Nature of Ca2+-Binding Sites of Rat Intestinal Mucosal Membranes Get access Takao OHYASHIKI, Takao OHYASHIKI Department of Physiological Chemistry, School of Pharmacy, Hokuriku UniversityKanagawa-machi, Kanazawa, Ishikawa 920–11 Search for other works by this author on: Oxford Academic PubMed Google Scholar Kenzo CHIBA, Kenzo CHIBA Department of Physiological Chemistry, School of Pharmacy, Hokuriku UniversityKanagawa-machi, Kanazawa, Ishikawa 920–11 Search for other works by this author on: Oxford Academic PubMed Google Scholar Tetsuro MOHRI Tetsuro MOHRI Department of Physiological Chemistry, School of Pharmacy, Hokuriku UniversityKanagawa-machi, Kanazawa, Ishikawa 920–11 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 86, Issue 5, November 1979, Pages 1479–1485, https://doi.org/10.1093/oxfordjournals.jbchem.a132666 Published: 01 November 1979 Article history Received: 14 April 1979 Published: 01 November 1979

Functional interactions of lipids and proteins in rat intestinal microvillus membranes.

Interactions of lipids and proteins in isolated rat intestinal microvillus membranes were examined by studying the temperature dependence of enzyme activities and of D-glucose transport in relation to the membrane lipid thermotropic transition observed by fluorescence polarization (26 +/- 2 degrees C) and differential scanning calorimetry (23--39 degrees C). Two groups of activities were defined. Enzymes of the first group, comprising lactase, maltase, sucrase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, all yielded a single slope on the Arrhenius plot in the range 10--40 degrees C and did not appear to experience functionally the effects of the lipid thermotropic transition. Each activity of the second group, comprising calcium- and magnesium-dependent adenosine triphosphatases, p-nitrophenylphosphatase, and D-glucose transport, showed a change in the slope of the Arrhenius plot in the range 25--30 degrees C, corresponding to the lower region of the lipid transition. The terms "extrinsic" and "intrinsic" activities could be applied to these groups. Delipidation of the particulate p-nitrophenylphosphatase removed the discontinuity in the Arrhenius plot. Subsequent relipidation with a variety of lipids restored a break point, but the temperature corresponded to the original discontinuity (25--29 degrees C) rather than to the phase transition temperature of the exogenous lipid added.

Sugar hydrolases and their arrangement on the rat intestinal microvillus membrane.

The arrangement of the sugar hydrolases, sucrase-isomaltase, maltase, and lactase on the microvillus membrane of rat intestine was investigated by immunological technique. The enzymes were purified essentially free of each other to near homogeneity and antisera of high specificity were obtained against each. Microvillus membranes were prepared routinely in high purity from rat intestine and contained an average 61% protein, 20% lipid, and 19% carbohydrate, with the sugar hydrolases comprising an estimated 20--25% of the membrane protein. The immunoreactivity of membrane-bound sucrase-isomaltase, maltase, and lactase was investigated with antisera demostrating specific reactivity to each, when tested in the presence of other membrane extractives. The membrane-bound enzymes were found in each case to combine with antibody in amounts equivalent to that required to effect precipitation of comparable units of the free enzymes from solution. Preloading membrane vesicles with antibodies to any two of the enzymes did not affect either the immunoreactivity or extractability (by papain or Triton X-100) of the third. The antibody-binding studies indicated an arrangement of these enzymes independent of each other on the membrane surface, in a manner allowing each to maintain a high degree of molecular freedom.

Identification of glycoproteins as receptors for cholera toxin in rat intestinal microvillus membranes

Identification of glycoproteins as receptors for cholera toxin in rat intestinal microvillus membranes

Fluorescence polarization studies of rat intestinal microvillus membranes.

Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin.

Factors influencing absorption and excretion of drugs. VI. Effect of sodium cholate on in situ rat intestinal absorption of buformin.

The effect of sodium cholate on the absorption of strongly basic drug, buformin, was studied in the in situ rat small intestinal loops. In cases of either jejunum or ileum, the absorption of buformin significantly increased above the critical micelle concentration of sodium cholate. Since sodium cholate had little effect on the physico-chemical property such as the apparent partition coefficient of buformin, it became evident that the enhancement of buformin absorption was not caused by the intraluminal effect of the bile salt. The absorption of buformin was also enhanced in the jejunal and ileal loops pretreated with sodium cholate. On the absorptive surface, sodium cholate did not increase the affinity of buformin to the intestinal mucosal surface. In addition, the release of membrane components such as protein, total phosphorus, and phospholipid from the intestine was accelerated to greater extent by sodium cholate. From these results, it is suggested that sodium cholate alters the composition of the in situ rat intestinal membrane by producing a release of membrane components from the intestinal tissues, resulting in an increase in the permeability of the small intestine to buformin.

Interaction of transferrin with iron-binding sites on rat intestinal epithelial cell plasma membranes.

SummaryFollowing oral administration of 59Fe to rats, over 50% of the isotope in the intestinal mucosa was bound to the plasma membrane of the epithelial cells. Labeled plasma membranes incubated in a medium that contained either plasma or transferrin released a significantly greater quantity of 59Fe than labeled membranes incubated in a control medium. These results suggest that transferrin interacts with iron-receptor sites on the epithelial cell plasma membrane.