Role of intestinal brush border membrane aminopeptidase N in dipeptide transport

The kinetics of uptake of radioactive label from [U-14C]Gly, L-[4,5-3H]Leu and the dipeptide [14C]Gly-L-[4,5-3H]Leu by the brush border membrane vesicles of porcine small intestine have been studied. The effect of aminopeptidase N inhibitors and leucine-binding protein on accumulation rates has also been tested. Comparison of the kinetic parameters for uptake and hydrolysis of Gly-L-Leu makes it possible to conclude that the dipeptide transfer includes two conjugated steps, viz., hydrolysis catalysed by aminopeptidase N and transport of the resultant free amino acids by a specific carrier.     

Effects on intestinal nutrient uptake and brush border membrane enzymes in response to atherogenic diet in rhesus monkeys

Transport of nutrients and kinetic parameters (Vmax and Km) of brush border membrane (BBM) enzymes were studied in duodenum, jejunum, and ileum from atherogenic diet-fed monkeys. The Km remained unaltered while feeding of atherogenic diet resulted in higher Vmax of sucrase, maltase, and alkaline phosphatase and lower Vmax of gamma-glutamyltranspeptidase and leucine-aminopeptidase compared to controls. Na+-dependent D-glucose transport was higher in duodenum and jejunum and unaltered in ileum. In contrast to D-glucose transport, the transport of amino acids was decreased in all three intestinal segments from atherogenic diet-fed monkeys.     

Effect of cisplatin on brush border membrane enzymes and anti-oxidant system of rat intestine

Cisplatin (CP) is a widely used antineoplastic agent which exhibits gastrointestinal toxicity. The present work was done to study the effect of administration of CP on brush border membrane (BBM) enzymes and anti-oxidant system of rat intestine. Male Wistar rats were given a single intraperitoneal dose of CP (6 mg/kg body weight) and then sacrificed 1, 3, 5 and 7 days after this treatment. Control animals were given saline only. The administration of CP led to significant decline in the specific activities of BBM enzymes both in the mucosal homogenates and isolated membrane vesicles. Kinetic studies showed that the V(max) of the enzymes was decreased in BBM vesicles from CP treated rats while the K(m) remained unchanged. The activities of catalase, Cu-Zn superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase decreased while the activities of glutathione S-transferase and thioredoxin reductase increased in CP treated animals compared to the control group. Lipid peroxidation and total sulfhydryl groups were also altered upon CP treatment indicating the generation of oxidative stress. The maximum changes in all the parameters studied above were 3 days after administration of CP and then recovery took place on days 5 and 7. Thus, the administration of CP leads to significant alterations in the activities of BBM enzymes and the anti-oxidant status of rat intestine.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Abstract

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Alterations in brush border membrane enzymes,carbohydrate metabolism and oxidative damage to rat intestine by potassium bromate

The acute toxicity of potassium bromate (KBrO₃) on rat small intestine was studied in this work. Animals were given a single oral dose of KBrO₃ (100 mg/kg body weight) and sacrificed 12, 24, 48, 96 and 168 h after the treatment; control animals were not given KBrO₃. The administration of KBrO₃ resulted in a reversible decline in the specific activities of several BBM enzymes. Lipid peroxidation, protein oxidation and hydrogen peroxide levels increased while total sulfhydryl groups and reduced glutathione decreased in KBrO₃-treated rats indicating induction of oxidative stress in the intestinal mucosa. The activities of anti-oxidant and carbohydrate metabolic enzymes were also altered upon KBrO₃ treatment. The maximum changes in all the parameters were 48 h after administration of KBrO₃ after which recovery took place, in many cases almost to control values after 168 h. Histopathological studies supported the biochemical findings showing extensive damage to the intestine at 48 h and recovery at 168 h. These results show that a single oral dose of KBrO₃ causes reversible oxidative damage to the intestine.     

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

Molecular characteristics of small intestinal and renal brush border thiamin transporters in rats

The molecular characteristics of thiamin (T) transport were studied in the small intestinal and renal brush border membrane vesicles of rats, using [³H]T at high specific activity. The effects of various chemical modifiers (amino acid blockers) on T uptake were examined and their specificity assessed. Treatment with the carboxylic specific blockers 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, (1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and N-ethyl-5-phenylisoaxolium-3′-sulfonate (Woodward’s Reagent K) and with the sulfhydryl specific blocker p-chloromercuribenzene sulfonate inhibited T transport in both types of vesicles. Phenylglyoxal, but not ninhydrin, both reagents for arginine residues, and diethylpyrocarbonate, a reagent for histidine residues, specifically decreased T transport only in renal and small intestinal vesicles respectively. Similarly 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted, but not N-acetylimidazole, both of which are reagents for tyrosine residues. However, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibition was aspecific. Acetylsalicylic acid, a reagent for lysine and serine residues, decreased T transport, but the lysine effect was aspecific. Acetylsalicylic acid serine blockage also eliminated T/H⁺ exchange in small intestinal vesicles. Taken together, these results suggest that for T transport carboxylic and sulfhydryl groups and serine residues are essential in both renal and small intestinal brush border membrane vesicles. In addition, arginine and histidine residues are also essential respectively for renal and small intestinal transporters. Serine was essential for the T/H⁺ antiport mechanism.     

Ca+2 transport across intestinal brush border membranes of the cichlid teleost Oreochromis mossambicus

Summary Brush border membranes were isolated from tilapia (Oreochromis mossambicus) intestine by the use of magnesium precipitation and differential centrifugation. The membrane preparation was enriched 17-fold in alkaline phosphatase. The membranes were 99% right-side-out oriented as indicated by the unmasking of latent glyceraldehyde-3-phosphate dehydrogenase and acetylcholine esterase activity by detergent treatment. The transport of Ca⁺² in brush border membrane vesicles was analyzed. A saturable and a nonsaturable component in the uptake of Ca⁺² was resolved. The saturable component is characterized by a K _m much lower than the Ca⁺² concentrations predicted to occur in the intestinal lumen. The nonsaturable component displays a Ca⁺² permeability too high to be explained by simple diffusion. We discuss the role of the saturable component as the rate-limiting step in transmembrane Ca⁺² movement, and suggest that the nonsaturable component reflects a transport mechanism operating well below its level of saturation.The authors wish to thank Tom Spanings for his superb organization of fish husbandry, and Maarten de Jong (Dept. of Physiology, Faculty of Medicine, University of Nijmegen) for making the automated stopped-flow apparatus available to us.     

The effect on amino acid transport of trypsin treatment of rat renal brush border membranes

Trypsin treatment of isolated rat renal brush border membrane vesicles which preferentially releases l-leucine aminopeptides (EC 3.4.11.2) decreases their ability to take up a variety of amino acids under Na⁺-gradient conditions. Such treatment did not alter the osmotic properties of the vesicles nor affect their fragility. A linear correlation could be demonstrated between the l-leucine aminopeptidase activity of the membranes and the initial rate of uptake of l-leucine and l-proline. Velocity of uptake-concentration dependence studies with these substrates indicate that the major effect of trypsinization is to decrease the maximum velocity (V_max1) of the low-K_m high-affinity system with little effect on the V_max2 of the high-K_m low-affinity transport process and no effect on the apparent Michaelis constants of either. Although the data indicate that l-leucine aminopeptidase activity and uptake of l-leucine and l-proline are affected in parallel, they should not be construed to imply a role of the enzyme in the transport process, especially in view of the global decrease in the uptake of various amino acids and sugars.     

A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a mediterranean teleost (Boops salpa)

The uptake of d-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na⁺ electrochemical gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{C}{}_{\mathrm{<mtext>in</mtext>}}$). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN^?, increases the Na⁺-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na⁺ gradient, the presence of Cl^? on the external side of the vesicles. In the absence of Cl^?, amino acid uptake is not stimulated by the Na⁺ gradient and is not influenced by an electrical membrane potential generated by SCN^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) or by a K⁺ diffusion potential ($\text{C}{}_{\mathrm{<mtext>in</mtext>}}\text{>C}{}_{\mathrm{<mtext>out</mtext>}}$). This Cl^? requirement differs from the Na⁺ requirement, since a Cl^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na⁺ gradient.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

A rapid method for the isolation of kidney brush border membranes

A simple rapid method for the preparation of purified brush border membranes from rabbit kidney proximal tubules is described. The method is based on hypotonic lysis, Ca²⁺ aggregation of contaminants and differential centrifugation. In contrast to most other published methods, the brush border membranes are free of contamination by basolateral membranes.     

Labelling of intestinal brush border membrane proteins in vivo using diazotised [125I]iodosulfanilic acid

Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [¹²⁵I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [¹²⁵I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × $\text{g}$ from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins.     

Na+-dependent,electroneutral l-ascorbate transport across brush border membrane vesicles from guinea pig small intestine

In brush border vesicles from guinea pig small intestine l-ascorbate transport is Na⁺-dependent and electroneutral (in the presence of Na⁺, as shown by its lack of response to either positive or negative Δψ across the membrane).l-Ascorbate transporter has the kinetic characteristics of a mobile carrier (K_m for l-ascorbate, 0.3 mM). d-Isoascorbate (erythorbate) seems to be another, but poorer, substrate of the same transporter.l-Ascorbate transport is subjected to heterologous inhibition by d-glucose.     

l- andd-alanine transport in brush border membrane vesicles from lepidopteran midgut: Evidence for two transport systems

Summary In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when K_out>K_in and K_out=K_in.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.     

Isolation of brush border membranes in vesicular form from the intestinal spiral valve of the small dogfish (Scyliorhinus canicula)

A simple, rapid method for the preparation of purified brush border membranes in vesicular form from rabbit kidney proximal tubules has been applied with closely similar results to the intestinal spiral valve of the small dogfish (Scyliorhinus canicula). Since the dogfish belongs to one of the most ancient species of fish, it may be suggested that the method is generally applicable to all species later evolved which possess a brush border membrane at the mucosal surface of the cells of the intestine or kidney.     

Use of Percoll™ in the isolation and purification of rabbit small intestinal brush border membranes

(1) Intestinal absorption is altered under a variety of circumstances in health and disease and to determine a possible relationship between intestinal absorptive function and intestinal brush border membrane composition, we undertook the isolation and purification of rabbit jejunal and ileal brush borders, to allow further studies of their lipid composition under varied experimental conditions. (2) A modification of an established method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98–112) utilized CaCl₂ aggregation and sequential centrifugation followed by purification of the brush border pellet (P₂) at 27 000 × g on a Percoll™ (Pharmacia) self-forming gradient. The Percoll™ was removed by ultracentrifugation for 30 min at 100 000 × g, utilizing a batch rotor in the Beckman airfuge™. (3) Pure brush border membrane vesicles were obtained and characterized by specific marker analysis and electron microscopy. Comparative marker analyses performed on P₂ and final Percoll™ preparations from animals showed that the purification achieved was 8–11-fold greater when compared to the original homogenates. Verification of purity was also demonstrated by the absence of DNA and very low levels of β-gluconridase and (Na⁺ + K⁺)-ATPase in the Percoll™ preparations. (4) Comparative lipid analyses of P₂ and final Percoll™ preparations showed that levels of total phospholipid and free fatty acids were several-fold higher in the Percoll™ preparations on a per mg protein basis. (5) A comparison of the activity of enzyme markers and the levels of total free fatty acids in P₂ pellets obtained after CaCl₂ and MgCl₂ aggregation showed that CaCl₂ aggregation gave the more consistently reproducible results. (6) Although standard procedures of membrane preparations not involving density gradient separation provide membranes of reasonable purity for the estimation of lipid components, we consider the final purification step of density gradient separation using Percoll™ is essential for determining small quantitative changes which might occur in the membrane lipid composition under experimental conditions where intestinal absorptive function is altered.     

Effect of oxidants on small intestinal brush border membranes and colonic apical membranes--a comparative study

This study compares composition of the rat small intestinal brush border membranes (BBM) and colonic apical membranes (CAM) and their susceptibility to in vitro exposure to various oxidants. Differences were observed between BBM and CAM in their lipid composition, sugar content, alkaline phosphatase (ALP) activity and cholesterol/phospholipid ratio. BBM and CAM were exposed to superoxide generated by xanthine+xanthine oxidase (X-XO) or peroxides such as tertiary butyl hydroperoxide (tBuOOH) and hydrogen peroxide (H(2)O(2)) and alterations in ALP activity, peroxidation parameters and membrane lipids were analyzed. Exposure of BBM and CAM to superoxide resulted in decrease in ALP activity and increase in peroxidation parameters such as protein carbonyl content, malondialdehyde and conjugated diene. Superoxide exposure also resulted in lipid alterations specifically in certain phospholipids. These alterations were prevented either by superoxide dismutase or by allopurinol. Peroxides did not have any significant effect. These results suggest that both BBM and CAM are susceptible to superoxide, which can bring about peroxidation and degradation of membrane lipids specifically, certain phospholipids.     

Sugar hydrolases of the infant rat intestine and their arrangement on the brush border membrane

Lactase and maltase, the predominant sugar hydrolases associated with the intestinal brush border membrane of the suckling rat, were purified essentially free of the other to near homogeneity (lactase at specific activity 23, maltase at specific activity 58), and their specific physicochemical properties determined. Antisera prepared to each showed by immunodiffusion a single common precipitin line with pure enzyme and solubilized proteins of the brush border membrane. Brush border membranes were purified 26–35-fold from infant rat intestine. Membranes prepared from 10-day-old rats contained 32% protein, 43% lipid and 25% carbohydrate with lactase and maltase estimated to comprise in excess of 10% and 2%, respectively, of the membrane protein.Immunotitration curves of lactase and maltase showed equivalent antibody binding by the membrane-bound and free enzyme forms. Furthermore, antibody binding to one enzyme did not affect the immunotitration curve or the extractability (by papain or Triton X-100) of the other membrane-bound enzyme. It was concluded that the lactase and maltase molecules are attached singly on the external membrane surface in a spatially independent manner with their antigenic sites as freely available to antibody binding as exhibited by their papain-solubilized counterparts.