Rabbit Small Intestinal Brush Border Research Articles

Identification and characterization of rabbit small intestinal villus cell brush border membrane Na-glutamine cotransporter

Glutamine, the primary metabolic fuel for the mammalian small intestinal enterocytes, is primarily assimilated by Na-amino acid cotransporters. Although Na-solute cotransport has been shown to exist in the brush border membrane (BBM) of the absorptive villus cells, the identity of Na-glutamine cotransport in rabbit small intestinal villus cells was unknown. Na-dependent glutamine uptake is present in villus BBM vesicles. An intravesicular proton gradient did not stimulate this Na-dependent glutamine uptake, whereas Li+ did not significantly suppress this uptake. These observations in concert with amino acid substitution studies suggested that Na-glutamine cotransporter in the villus cell BBM was the newly identified cotransporter B0AT1 (SLC6A19). Quantitative real-time PCR identified the message for this cotransporter in villus cells. Thus a full-length cDNA of B0AT1 was cloned and expressed in MDA-MB-231 cells. This expressed cotransporter exhibited characteristics similar to those observed in villus cells from the rabbit small intestine. Antibody was generated for B0AT1 that demonstrated the presence of this cotransporter protein in the villus cell BBM. Kinetic studies defined the kinetic parameters of this cotransporter. Thus this study describes the identification, cloning, and characterization of the Na-amino acid cotransporter responsible for the assimilation of a critical amino acid by the absorptive villus cells in the mammalian small intestine.

Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid

To address the effect of the n-3 fatty acid, docosahexaenoic acid (22:6), on proteins that play a role in cholesterol absorption, CaCo-2 cells were incubated with taurocholate micelles alone or micelles containing 22:6 or oleic acid (18:1). Compared with controls or 18:1, 22:6 did not interfere with the cellular uptake of micellar cholesterol. Apical cholesterol efflux was enhanced in cells incubated with 22:6. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was decreased by 22:6. 22:6 decreased Niemann-Pick C1-Like 1 (NPC1L1) protein and mRNA levels without altering gene or protein expression of ACAT2, annexin-2, caveolin-1, or ABCG8. Peroxisome proliferator-activated receptor delta (PPARdelta) activation decreased NPC1L1 mRNA levels and cholesterol trafficking to the endoplasmic reticulum, suggesting that 22:6 may act through PPARdelta. Compared with hamsters fed a control diet or olive oil (enriched 18:1), NPC1L1 mRNA levels were decreased in duodenum and jejunum of hamsters ingesting fish oil (enriched 22:6). In an intestinal cell, independent of changes in ABCG8 expression, 22:6 increases the apical efflux of cholesterol. 22:6 interferes with cholesterol trafficking to the endoplasmic reticulum by the suppression of NPC1L1, perhaps through the activation of PPARdelta. Moreover, a diet enriched in n-3 fatty acids decreases the gene expression of NPC1L1 in duodenum and jejunum of hamster.

Identification of binding proteins for cholesterol absorption inhibitors as components of the intestinal cholesterol transporter

To identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2-azetidinone cholesterol absorption inhibitors. An integral membrane protein of M r 145.3±7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentration-dependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D-glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2-azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of M r 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes.

Uptake of Enalapril by Rabbit Small Intestinal Brush-Border Membrane Vesicles.

Uptake of the angiotensin-converting enzyme (ACE) inhibitor enalapril by rabbit small intestinal brush border membrane vesicles was examined. In the presence of an inward H+ gradient the uptake of this peptide mimetic drug was accelerated and an overshoot phenomenon was observed. The uptake was more stimulated by higher H+ gradient. Initial uptake rate was saturable in the presence of an inward H+ gradient, with apparent Km value of 4.2 mM. These findings suggest the involvement of the H+-coupled carrier-mediated transport in the uptake of enalapril. The uptake was inhibited by ACE inhibitor trandolapril but hardly or only slightly inhibited by aminocephalosporins.

Inhibitory effects of angiotensin-converting enzyme inhibitor on cefroxadine uptake by rabbit small intestinal brush border membrane vesicles.

Effects of angiotensin-converting enzyme (ACE) inhibitors, captopril, enalapril maleate and quinapril, on the uptake of aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush border membrane vesicles were examined. These ACE inhibitors significantly inhibited the uptake of cefroxadine, which is transported by H+/dipeptide transporter in the membrane, in the order of captopril < enalapril < quinapril in the presence of an inward H+ gradient. Inhibitory effect of quinapril was more marked than that of aminocephalosporin cephradine, while in the absence of an inward H+ gradient inhibition by these ACE inhibitors was much less. Dixon plot analysis showed that the inhibition by enalapril and quinapril in the presence of an inward H+ gradient occurred in a competitive manner and estimated inhibition constants of these two drugs to be 5.3 mM and 0.46 mM, respectively. These results suggested the strong affinity of these ACE inhibitors, especially quinapril, on the H+/dipeptide transporter.

A comparative study of sterol absorption in different small-intestinal brush border membrane models

We reported previously that the absorption of cholesterol and long-chain cholesteryl esters by rabbit small-intestinal brush border membranes (BBMV) is protein-mediated (Thurnhofer, H., and H. Hauser. 1990. Biochemistry. 29:2142-2148; Compassi, S., M. Werder, D. Boffelli, F. E. Weber, H. Hauser, and G. Schulthess. 1995. Biochemistry. 34: 16473-16482). Evidence is presented for similar cholesterol transport activities in rabbit, pig, and human BBMV. As BBMV are subject to a number of limitations and the influence of these on sterol absorption is unknown, it is desirable to verify results obtained with this model system in other brush border membrane models more closely related to the in vivo situation. Sterol absorption in intact enterocytes parallels the absorption measured in BBMV, provided that both model systems are normalized to equal sucrase activity. The parallel behavior of the two brush border membrane models lends support to our previous conclusion that the brush border membrane takes up free and esterified cholesterol in a facilitated and energy-independent process. The absorption of sterols in small-intestinal segments mounted in the Ussing chamber is shown to be a complex process in which the diffusion of the bile salt micelles to the brush border membrane is rate-limiting. All brush border membrane models share the disadvantage of being unstable and subject to degradation. The seriousness of the problem increases apparently with the complexity of the model, i.e., in the order BBMV-->enterocytes-->intestinal segments. One main conclusion of this study is that no brush border membrane model is sufficient and satisfactory, therefore conclusive work in lipid absorption can never be based on a single brush border membrane model.

Characteristics of uptake of cefroxadine by rabbit small intestinal brush border membrane vesicles.

Characteristics of transport of an oral aminocephalosporin, cefroxadine, in rabbit small intestinal brush border membrane vesicles were examined. Uptake rate of cefroxadine was saturable in the presence of an inward H+ gradient, and kinetic parameters were similar to those of cephradine. However, the uptake rate was almost linear with the concentration in the absence of an inward H+ gradient up to 5 mM. Overshoot phenomenon was observed in the presence of an inward H+ gradient at 37 degrees C, but it disappeared with decrease of temperature. The Arrhenius plot of uptake rate constant showed a break point at approximately 30 degrees C. Cefroxadine uptake was optimum in the vicinity of pH 5.5 at 37 degrees C, but the dependence on extravesicular pH disappeared at 15 degrees C. The uptake of cefroxadine in the presence of an inward H+ gradient was markedly inhibited by other aminocephalosporins such as cephalexin, but the inhibition was only slight in the absence of an inward H+ gradient. Alkyl alcohols such as n-hexyl alcohol also inhibited H(+)-coupled uptake of cefroxadine at the concentration range at which the alcohols increased the membrane fluidity, and overshoot phenomenon diminished, suggesting that H(+)-coupled transport of cefroxadine is sensitive to the alcohol-induced increase in membrane fluidity. On the other hand, the alcohols rather stimulated its uptake in the absence of an H+ gradient.

Relationship of alcohol-induced changes in Mg(2+)-ATPase activity of rabbit intestinal brush border membrane with changes in fluidity of its lipid bilayer.

We examined the effects of seven n-alkyl alcohols (from n-butyl to n-undecyl alcohol), isoamyl alcohol and benzyl alcohol on the activity of membrane enzyme Mg(2+)-ATPase of the rabbit small intestinal brush border membrane. Their relationships with the changes in the fluidity of the membrane lipid bilayer were examined through studies on the fluorescence anisotropies of diphenylhexatriene (DPH) and its ionic derivatives. Good linear correlations were found both between the partition coefficients of the alcohols and their concentrations causing similar decreases in the activity of Mg(2+)-ATPase and between their partition coefficients and the alcohol-induced changes in fluorescence anisotropies. Within the concentration range of the alcohols tested, including isoamyl alcohol and benzyl alcohol, the decreases in activity of the membrane enzyme Mg(2+)-ATPase clearly corresponded with the decreases in fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane. From these findings, one possible explanation is that inhibition of this enzyme by the alcohols is due to perturbation of the lipid bilayer of the brush border membrane.

Characterization of Lipid Exchange Proteins Isolated from Small Intestinal Brush Border Membrane

Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 +/- 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an integral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.

Absorption of Monoacylglycerols by Small Intestinal Brush Border Membrane

The absorption of monoacylglycerol by small intestinal brush border membrane is a passive process, i.e., the movement of monoacylglycerol from small unilamellar phospholipid vesicles as donor particles through the aqueous medium and the incorporation into the outer monolayer of the lipid bilayer of the brush border membrane are passive processes involving diffusion of the lipid along a concentration gradient. Small unilamellar vesicles of egg phosphatidylcholine containing 1 mol% of radiolabeled hexadecylglycerol were used as donor, and rabbit small intestinal brush border membrane vesicles or intact enterocytes isolated from pig jejunum, as acceptor. Hexadecylglycerol was employed as a lipase-resistant model compound for monoacylglycerols. Both acceptor membranes behave similarly in terms of hexadecylglycerol absorption: the kinetics of hexadecylglycerol absorption are biphasic. The initial fast phase is due to the movement of hexadecylglycerol from the donor particle through the aqueous medium to the outer lipid monolayer of the acceptor membrane, and the second slow phase probably involves the flip-flop motion of hexadecylglycerol from the outer to the inner monolayer of the acceptor membrane. The values for the pseudo-first-order rate constants of the initial fast phase for hexadecylglycerol absorption are relatively large and primarily determined by the high solubility (cmc) of hexadecylglycerol in aqueous media. The pseudo-first-order rate constants depend linearly on the protein (lipid) concentration of the acceptor membrane, indicating that the on rate of the hexadecylglycerol into the brush border membrane is rate limiting. The mechanism of the hexadecylglycerol absorption involves mainly monomer diffusion and probably collision-induced transfer.

Intestinal absorption of beta-lactam antibiotics and oligopeptides. Functional and stereospecific reconstitution of the oligopeptide transport system from rabbit small intestine.

The H(+)-dependent uptake system responsible for the enteral absorption of oligopeptides and orally active beta-lactam antibiotics was functionally reconstituted into liposomes. Membrane proteins from rabbit small intestinal brush border membrane vesicles were solubilized with n-octyl glucoside and incorporated into liposomes using a gel filtration method. At protein/lipid ratios of 1:10 and 1:40, the uptake of the orally active alpha-amino-cephalosporin, D-cephalexin into proteoliposomes was stimulated by an inwardly directed H+ gradient and was protein-dependent. In these proteoliposomes the binding protein for oligopeptides and beta-lactam antibiotics of Mr 127,000 could be labeled by direct photoaffinity labeling with [3H]benzylpenicillin revealing an identical binding specificity as in the original brush border membrane vesicles. The uptake system for beta-lactam antibiotics and oligopeptides showed a remarkable stereospecificity; only D-cephalexin was taken up by intact brush border membrane vesicles, whereas the L-enantiomer was not taken up to a significant extent. This stereospecificity for uptake was also seen after reconstitution of solubilized brush border membrane proteins into liposomes demonstrating a functional reconstitution of the peptide transporter. Both enantiomers however, bound to the 127-kDa binding protein as was shown by a decrease in the extent of photoaffinity labeling of the 127-kDa protein in the presence of both enantiomers. After reconstitution of subfractions of brush border membrane proteins obtained by wheat germ lectin affinity chromatography into proteoliposomes, only liposomes containing the 127-kDa binding protein showed a significant uptake of D-cephalexin whereas the L-enantiomer was not transported. The uptake rates for D-cephalexin into proteoliposomes correlated with the content of 127-kDa binding protein in these liposomes as was determined by specific photoaffinity labeling with [3H]benzylpenicillin. The purified 127-kDa binding protein was also reconstituted into liposomes and its ability for specific binding of substrates as well as stereospecific uptake of cephalexin could be restored. These results indicate that the binding protein for oligopeptides and beta-lactam antibiotics of Mr 127,000 mediates the stereospecific and H(+)-dependent transport of orally active beta-lactam antibiotics across the enterocyte brush border membrane. We therefore suggest that this 127-kDa binding protein is the intestinal peptide transport system (or a component thereof).

Lipid asymmetry in rabbit small intestinal brush border membrane as probed by an intrinsic phospholipid exchange protein

All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.

Intestinal absorption of dipeptides and β-lactam antibiotics. II. Purification of the binding protein for dipeptides and β-lactam antibiotics from rabbit small intestinal brush border membranes

By photoaffinity labeling of brush border membrane vesicles from rabbit small intestine with photoreactive derivatives of β-lactam antibiotics and dipeptides, a binding protein for dipeptides and β-lactam antibiotics with an apparent molecular weight of 127 000 was labeled. The labeled 127 kDa polypeptide could be solubilized with the non-ionic detergents Triton X-100, n-octyl glucoside or CHAPS. If the vesicles were solubilized prior to photoaffinity labeling, no clear incorporation of radioactivity into the 127 kDa polypeptide occurred indicating a loss of binding ability upon solubilization. By affinity chromatography of solubilized brush border membrane proteins on an agarose wheat germ lectin column, the binding protein for dipeptides and β-lactam antibiotics of M r 127 000 was retained on the column. With N- acetyl- d- glucosamine the photolabeled binding protein for β-lactam antibiotics and dipeptides was eluted together with the brush border membrane-bound enzyme aminopeptidase N. Separation from aminopeptidase N and final purification was achieved by anion-exchange chromatography on DEAE-sephacel. Polyclonal antibodies against the purified binding protein were raised in guinea pigs. The photolabeled 127 kDa protein could be precipitated from solubilized brush border membranes with these antibodies. Incubation of brush border membrane vesicles with antiserum prior to photoaffinity labeling significantly reduced the extent of labeling of the 127 kDa protein. Treatment of brush border membrane vesicles with antiserum significantly inhibited the efflux of the α-aminocephalosporin cephalexin from the brush border membrane vesicles compared to vesicles treated with preimmune serum. These studies indicate that the binding protein for dipeptides and β-lactam antibiotics of apparent molecular weight 127 000 in the brush border membrane of rabbit small intestinal enterocytes is directly involved in the uptake process of small peptides and orally active β-lactam antibiotics across the enterocyte brush border membrane.

Mechanisms of linoleic acid uptake by rabbit small intestinal brush border membrane vesicles.

We examined the initial transport of a long-chain unsaturated fatty acid, linoleic acid, by brush border membrane vesicles isolated from rabbit small intestine. This preparation allowed us to examine the transport of linoleic acid across the brush border membrane without the effect of the unstirred water layer or cytosol binding proteins. Linoleic acid was solubilized in a 2 mM taurocholate solution which did not compromise the functional integrity of the vesicles. Linoleic acid uptake in the range of 1 to 100 microM followed passive diffusion kinetics. Time course study showed that linoleic acid uptake reached maximal levels during the initial 15 seconds. Although the amount of linoleic acid accumulated in the vesicles diminished over the next 30 minutes, the molar quantity was still twentyfold higher than that of D-glucose (6.5 vs 0.33 nmol/mg protein). Uptake of D-glucose by the vesicles demonstrated typical osmotic responsiveness. We found no osmotic effect on linoleic acid uptake. Hypotonic lysis of membrane vesicles loaded with linoleic acid released 40% of the fatty acid. We concluded that a major portion of the accumulated fatty acid was bound to or incorporated into the membrane itself while ca. 40% did traverse the membrane and accumulated in the intravesicular space as nonmicellar aggregates. The known inhibitors of anion transport, diisothiocyanatostilbene and isothiocyanatostilbene did not change the transport of linoleic acid. We conclude that, in the absence of an unstirred layer or cytosol proteins, linoleic acid transport at up to 100 microM concentration is passive with rapid accumulation both by the cell membrane and the lumen of vesicles.

60 ENTEROPATHOGENIC E. COLI (EPEC) DISRUPT SMALL INTESTINAL BRUSH BORDER MEMBRANCE Na+/H* EXCHANGE

Despite a highly specific morphological lesion of the small intestinal brush border membrane, the mechanisms whereby EPEC produce diarrhoea are unknown; mucosal invasion and toxin production are not thought to be important factors. We have therefore studied the effects of the rabbit EPEC strain, RDEC-1, upon Na+/H+ exchange at 60 sec in rabbit small intestinal brush border membrane vesicles (BBMV), following incubation at 37°C with RDEC-1. Before incubation, BBMV showed a 3.4-fold enhancement of Na+ uptake in the presence of an outwardly directed H+ gradient (p<0.0001), indicating intact Na+/H+ exchange. However, after 60 min incubation with RDEC-1; H+-stimulated Na+ uptake was markedly reduced to 43% of original enhancement (p<0.01) but reduced to only 90% in control BBMV (NS).

Ramipril inhibition of rabbit ( Oryctolagus cuniculus) small intestinal brush border membrane angiotensin converting enzyme

1. 1. Rabbit small intestinal brush border membranes possessed prominent angiotensin converting enzyme (ACE) activity. 2. 2. Intestinal ACE was located on the lumen surface, as verified by ACE co-enrichment with brush border membrane marker enzymes. 3. 3. Hydrolysis kinetics of rabbit intestinal ACE were comparable to the lung, utilizing the substrate (N-[3-(2- furyl) acryloyl]- l- phenylalanylglycylglycine ; the V max = 543 ± 51 μmol/min/g and K m = 0.62 ± 0.09 mmol/l. 4. 4. Intestinal brush border ACE activity was strongly inhibited by the antihypertensive drug Ramipril, which yielded an ic 50 value of 5 nmol/l; the ACE activity remained completely inhibited during 15 days after a single dose of 10 μmol/l Ramipril.

Further characterization of an early expressed glycoprotein of the rabbit small intestinal brush border. Its interaction with some hydrolases.

Mouse 23 B 921 monoclonal antibody recognized a rabbit brush-border antigen with an apparent molecular mass 140 kDa (140-kDa Ag) which, unlike most hydrolases, is expressed in the poorly differentiated crypt cells of the small intestine. Immunoelectron microscopy of brush-border vesicles showed that the 23 B 921 bound to an epitope localized on the outside of the membrane. As is the case with hydrolases the external domain of the 140-kDa Ag constitutes the main part of the molecule, which can be released by papain treatment of brush-border vesicles. The presence of a small hydrophobic domain, anchoring the molecule into the membrane and responsible for its amphipatic character, was shown by its affinity for Triton-X114 micelles. The topological organization in the membrane of 140-kDa Ag seemed to be identical to that of hydrolases. Unlike hydrolases, however, the native structure of the antigen was found from its sensitivity to proteolysis to be very dependent on its integration into the lipid bilayer. Nevertheless, detergent-extracted 140-kDa Ag can be purified by immunoaffinity chromatography although it cannot be stocked for more than 48 h even in the presence of protease inhibitors. The carbohydrate moiety of 140-kDa Ag, bearing the human blood group A determinant, amounts to 20% of the total molecular mass. The existence of some privileged relationship was established between 140-kDa Ag and hydrolases: in the membrane, hydrolases protect the 140-kDa Ag from papain action; after detergent extraction, 140-kDa Ag is strongly associated with several hydrolases particularly disaccharidases.

Effect of hydrogen ion-gradient on carrier-mediated transport of glycylglycine across brush border membrane vesicles from rabbit small intestine.

Glycylglycine (Gly-Gly) transport across rabbit small intestinal brush border membrane vesicles was studied in the presence and absence of a Na+ gradient or a pH gradient. The transport was found to be entirely independent of Na+ but significantly stimulated by lowering extravesicular pH (pHo). The maximum stimulation was seen at pHo 5.5, where the uptake rate was about 2-times higher than the control value. FCCP, a protonophore, abolished the stimulating effect of low pHo, and low pHo conditions without a pH gradient did not stimulate the uptake rate. Overshoot uptake of Gly-Gly was observed when a pH gradient of 2 pH units was imposed across the vesicular membrane. Valinomycin-induced inside-negative K+ diffusion potential also had a stimulating effect on the uptake, and fluorescence measurements of vesicular suspensions containing diS-C3-(5) revealed the occurrence of depolarization of the vesicular membranes when Gly-Gly was added to the suspensions. Kinetic study showed that a pH gradient caused a decrease of Kt for Gly-Gly without affecting Vmax. All the data obtained indicate that Gly-Gly transport is independent of Na+, dependent on a H+ gradient, and electrogenic, suggesting the mechanism of cotransport with H+.

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 CaCl 2 aggregation and sequential centrifugation followed by purification of the brush border pellet (P 2) 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 P 2 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 P 2 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 P 2 pellets obtained after CaCl 2 and MgCl 2 aggregation showed that CaCl 2 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.

Rabbit small intestinal brush border membrane. Preparation and lipid composition

1. 1. Rabbit intestinal brush border membrane vesicles were prepared either from frozen or fresh tissue and the lipid composition was analysed. 2. 2. Lipids extracted from membranes prepared by the Ca 2+-precipitation method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98–112; Kessler, M., Acuto, O., Storelli, C., Murer, H., Müller, M. and Semenza, G. (1978) Biochim. Biophys. Acta 506, 136–154) had exceptionally high levels of lysophospholipids and free fatty acids. An intrinsic, Ca 2+-activated phospholipase A is responsible for the lipid decomposition. 3. 3. It was necessary to modify the preparation of brush border membranes. Essentially, EGTA is used to keep the free Ca 2+ concentration low so that phospholipases are inactivated, and Mg 2+ instead of Ca 2+ is employed to aggregate selectively contaminating membranes. The modified procedure gives membranes suitable for lipid analysis. 4. 4. The molar ratio of neutral, phospholipid and glycolipid is about 1 : 1 : 1. The major neutral lipids are free cholesterol and fatty acids. The major phospholipids are phosphocholine-containing (approx. 45%) and phosphatidylethanolamine (approx. 40%). The remainder (15–20%) is made up of acidic phospholipids, mainly phosphatidylserine and phosphatidylinositol. The major glycolipid is ceramide monohexoside.