A novel method of preparation of small intestinal brush border membrane vesicles by polyethylene glycol precipitation

A novel method of brush border membrane vesicle (BBMV) preparation from the small intestinal mucosa using polyethylene glycol (PEG) precipitation has been presented. This preparation is compared with calcium-precipitated BBMVs in marker enzyme enrichment, contamination by other subcellular membranes, transport of glucose, and lipid composition. PEG-precipitated BBMVs are comparable with calcium-precipitated membranes in all parameters except lipid composition and thiol content. PEG-precipitated membranes have more phosphatidylcholine and phosphatidylethanolamine and less lysophosphatidylcholine and lysophosphatidylethanolamine as compared to calcium-precipitated membranes. Diacylglycerol and triacylglycerol content are also high in PEG-precipitated membranes. Alteration in lipid composition indicate the possible activation of lipase and phospholipase by calcium during BBMV preparation, which is not seen in PEG precipitation. Thiol content is almost double in PEG-precipitated membranes as compared to calcium-precipitated membranes. These results indicate that PEG can be used for the preparation of BBMVs in native form from the intestine without any alteration in their structural components, and these membranes show comparable transport activity.     

Properties of Brush Border Vesicles Isolated from Rat Kidney Cortex by Calcium Precipitation

Brush border membrane vesicles were isolated from rat kidney cortex by differential centrifugation in the presence of 10 mM calcium. Their properties were compared to brush border vesicles isolated by free-flow electrophoresis. By the calcium precipitation method membrane vesicles were obtained in a shorter time with a similar enrichment of brush border marker enzymes (11- to 12-fold for alkaline phosphatase and maltase), with a similarly reduced activity of the marker enzyme for basal-lateral plasma membranes and an almost identical protein composition as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The transport properties of the two membrane preparations for D-glucose, L-phenylalanine, and phosphate are essentially the same; there is some indication for a lower sodium permeability of the vesicles prepared by the calcium precipitation method. The latter vesicles were also shown to exhibit sodium gradient stimulated uptake of L-glutamate.     

Estimation of apparent L-amino acid diffusion in porcine jejunal enterocyte brush border membrane vesicles

There is an overlap of carrier-mediated L-amino acid transport and apparent simple diffusion when measured in intestinal brush border membrane vesicles. Using L-threonine and L-glutamine as representative amino acids, this study was undertaken to estimate apparent simple diffusion of L-amino acids and to establish the effective dosage of HgCl2 for completely blocking carrier-mediated L-amino acid transport in porcine jejunal enterocyte brush border membrane vesicles. Jejunal mucosa was scraped from three pigs weighing 26 kg. Enterocyte brush border membrane vesicles, with an average enrichment of 24-fold in sucrase specific activity, were prepared by Mg2+-precipitation and differential centrifugation. In vitro uptake was measured by the fast filtration manual procedure. HgCl2 blocked the carrier-mediated initial transport of L-threonine and L-glutamine under Na+-gradient condition in a dose-dependent manner. At the minimal concentration of 0.165 micromol HgCl2 mg(-1) protein, carrier-mediated L-threonine and L-glutamine transport was completely inhibited. The apparent L-threonine and L-glutamine diffusion was estimated to be 8.6+/-0.7 and 12.4+/-1.0% of the total uptake at the substrate concentrations of 5 microM (L-threonine) and 50 microM (L-glutamine). Therefore, the treatment of porcine brush border membrane vesicles with a minimum of 0.165 micromol HgCl2 mg(-1) protein completely blocks carrier-mediated L-amino acid transport and enables the direct estimation of apparent L-amino acid diffusion in enterocyte brush border membrane vesicles.     

Purification of brush border membrane vesicles from horse kidney cortex using Percoll

A rapid method for preparation of brush border membrane vesicles from a large amount of horse kidney cortex is described. Self-orienting Percoll-gradient centrifugation minimized contamination by microsomal membranes. The characteristics of this preparation were checked by electron microscopy and measurement of L-alanine uptake.     

A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems

We have worked out a simplification of the procedure described by Schmitz et al. (Biochim. Biophys. Acta (1973) 323, 98--112) for the preparation of brush border membranes from small intestine. The procedure ultimately adopted is simple, rapid, does not necessarily require scraping and can be started from fresh or frozen material. It can be scaled up easily, allowing a quick production of large amounts of brush border membrane vesicles. These vesicles prove to be excellently suited for transport studies, as suggested by our measurements of D-glucose transport. Using these vesicles, the mode of choline transport across the brush border membrane was also investigated. Choline transport was found to occur by a saturable component with a Km of 83 +/- 4 micrometer (at 20 degrees C) and by a non-saturable component. It is independent of the presence of Na+ and appears to be non-electrogenic.     

Isolation and characterization of epididymal epithelial cell plasma membranes

Sperm maturation and storage occur in a unique milieu created in large part by the epididymal epithelium. To learn more about the interaction of the epididymal epithelial cell with both luminal and systemic environments, we now report on the preparation and characterization of epididymal epithelial cell plasma membranes. A preparation enriched for epididymal epithelial cell plasma membranes was isolated from collagenase-digested epididymal tubule fragments by hand-Dounce homogenization, differential centrifugation, and sucrose gradient centrifugation. The final membrane fraction was enriched 11-fold for the plasma membrane marker 5'-nucleotidase; 2.6-fold for the lysosomal marker acid phosphatase, and 3-fold for the Golgi marker thiamine pyrophosphatase. No enrichment was observed for mitochondrial or endoplasmic reticulum enzyme markers. Specific and saturable transferrin-binding activity was also detected in the final preparation. Electron microscopy revealed the presence of vesicles and sheets of membranes as well as an occasional Golgi apparatus. The plasma membrane fraction was used to generate monoclonal antibodies. Of 102 wells exhibiting growth, 12 were positive by immunofluorescent staining of frozen sections. Ten of these recognized determinants in epithelial cells, and 2 stained peritubular smooth muscle cells. Most of the epithelial cell-specific antibodies stained brush border alone or in combination with the basolateral plasma membrane. Three antibodies stained the Golgi apparatus. Most antibodies were specific for particular epididymal regions, 3 also recognized determinants in the kidney, and 1 stained residual bodies in the testis.     

Use of percollTM 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 X g on a PercollTM (Pharmacia) self-forming gradient. The PercollTM was removed by ultracentrifugation for 30 min at 100 000 X g, utilizing a batch rotor in the Beckman airfugeTM. (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 PercollTM 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 Beta-gluconridase and (Na+ + K+)-ATPase in the PercollTM preparations. (4) Comparative lipid analyses of P2 and final PercollTM preparations showed that levels of total phospholipid and free fatty acids were several-fold higher in the PercollTM 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 PercollTM is essential for determining small quantitative changes which might occur in the membrane lipid composition under experimental conditions were intestinal absorptive function is altered.     

Cimetidine transport in rat renal brush border and basolateral membrane vesicles

The transport of cimetidine by rat renal brush border and basolateral membrane vesicles has been studied in relation to the transport system of organic cation. Cimetidine inhibited [3H]tetraethylammonium uptake by basolateral membrane vesicles in a dose dependent manner, and the degree of the inhibition was almost the same as that by unlabeled tetraethylammonium. In contrast, cimetidine inhibited the active transport of [3H]tetraethylammonium by brush border membrane vesicles more strongly than unlabeled tetraethylammonium did. In agreement with the transport mechanism of tetraethylammonium in brush border membranes, the presence of an H+ gradient ([H+]i greater than [H+]o) induced a marked stimulation of cimetidine uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was inhibited by unlabeled tetraethylammonium. These results suggest that cimetidine can share common carrier transport systems with tetraethylammonium in renal brush border and basolateral membranes, and that cimetidine transport across brush border membranes is driven by an H+ gradient via an H+-organic cation antiport system.     

Isolation and reconstitution of the intestinal Na+/glucose cotransporter

The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.     

Simultaneous preparation of basolateral and brush-border membrane vesicles from sea bass intestinal epithelium

A method is described for simultaneous preparation of brush-border and basolateral sea bass enterocyte membranes using simple differential centrifugation and discontinuous sucrose gradient density centrifugation techniques. Basolateral membranes were purified with a Na+/K(+)-ATPase yield of about 11% of the original activity, with an enrichment factor of 12. The yield of maltase-glucoamylase, a specific marker of brush-border membranes, was also about 11% of the original activity, with 15-fold enrichment. The characteristics of these membrane preparations were determined. Electron microscopy analysis showed that these two membrane preparations were uniform in size and vesicular in nature. Orientation studies revealed that the luminal membrane vesicles were right-side out and 43% of the antiluminal membrane vesicles were sealed inside out. Investigation of D-glucose and L-leucine uptake showed that these two plasma membrane preparations retained their transport properties.     

Plasma membranes from rat intestinal epithelial cells at different stages of maturation. I. Preparation and characterization of plasma membrane subfractions originating from crypt cells and from villous cells.

To determine the mechanism of the maturation of the brush border membrane in intestinal epithelial cells, purification of the plasma membrane from undifferentiated rat crypt cells and of the basal-lateral membrane from villous cells has been performed. The method is based on density perturbation of the mitochondria to selectively disrupt their association with the membrane. With both cell populations, two membrane subfractions displaying the same respective density on sucrose gradient have been obtained with an overall yield of 15--20% and a 10-fold enrichment of the plasma membrane markers 5'-nucleotidase and (Na+ + K+)-dependent, ouabain-sensitive ATPase chosen to follow their purification. The four fractions were constituted by sheets and apparently closed vesicles of various sizes. Each fraction was characterized by a distinct protein composition and different levels of enzyme activities. The cells, used for the preparation of the membranes, were isolated as a villus to crypt gradient. This separation and that of the membranes, led to the conclusion that the (Na+ + K+)-dependent ATPase is localized principally in the plasma membrane of all cells whatever their state of maturation, while 5'-nucleotidase is predominantly located in the basal-lateral membrane of the villous cells and may serve as a specific marker for the purification of this membrane. Finally it has been shown that aminopeptidase, dissacharidases and alkaline phosphatase do not appear simultaneously in the maturation process of the cells, alkaline phosphatase being absent from the crypt cells and aminopeptidase being the first to be synthesized. This enzyme seems to appear in the crypt cells membrane before being integrated into the mature brush border membrane.     

The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.     

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.     

Improved stability of rabbit and rat intestinal brush border membrane vesicles using phospholipase inhibitors

The initial rates of Na(+)-dependent D-aspartate and D-glucose uptakes were shown to decline from the time of resuspension of brush border membrane vesicles isolated from rabbit and rat jejunum by standard divalent cation precipitation procedures. The former were however more stable than the latter and followed quite closely the decrease in the intravesicular volume, thus suggesting that the loss of transport activity may involve both nonspecific opening of the vesicles and either direct or indirect specific inactivation of the transporters. Uptake rates for both substrates did tend to stabilize at 6-24 h from resuspension, however this final 'next day' uptake activity was too low to be of practical use in kinetic studies. Freezing aliquots of rabbit jejunal vesicles in liquid N2 until the time of assay resulted in complete stabilization of D-glucose uptake. A modified homogenate buffer designed to inhibit a broad spectrum of phospholipase activities resulted in a partial stabilization of glucose transport by rabbit jejunal vesicles with, on average, an over 6-fold enrichment in the 'next day' stable specific activity of uptake as compared to unfrozen vesicles. The modified homogenate buffer also improved the stability and the 'next day' specific activities of D-glucose uptake in rat jejunal brush border vesicles and D-aspartic acid uptake in rabbit jejunal vesicles. It also completely stabilized the intravesicular volume in the latter preparation. An evaluation of the kinetic parameters of Na(+)-dependent D-glucose transport in rabbit vesicles prepared from either the standard homogenate media and frozen in liquid N2 or the modified media and allowed to stabilize overnight, revealed a single transport system with a Km of 0.31-0.32 mM as the best model to fit the data. As such the modifications to the homogenate media do not appear to effect the functional properties of D-glucose transport in the membrane. While being less efficient in stabilizing the vesicles than the rapid freezing protocol, it is shown that the modified homogenate should however be preferred when dealing with slowly permeant ions like choline since it provides in this case the only alternative to reliable measurement of uptake rates across a stable and equilibrated vesicle preparation.     

Effect of parathyroid hormone on Na+-dependent phosphate transport and cAMP-dependent 32P phosphorylation in brush border vesicles from isolated perfused canine kidneys

Concentrative uptake of 32Pi induced by the dissipation of a Na+ gradient (overshoot) was demonstrated in brush border membrane vesicles obtained from isolated perfused canine kidneys. Na+-dependent 32Pi transport was decreased in brush border vesicles from isolated kidneys perfused with parathyroid hormone (PTH) for 2 h compared to uptake measured in vesicles from kidneys perfused without PTH. Cyclic AMP-dependent 32P phosphorylation of a 62,000 Mr protein band was demonstrable on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of membrane suspensions from kidneys perfused +/- PTH. Evidence that perfusion with PTH resulted in cAMP-dependent phosphorylation in isolated kidneys from parathyroidectomized dogs (decreased cAMP-dependent 32P phosphorylation of the 62,000-Mr band in brush border vesicles) was obtained after 2-h perfusion with PTH. Decreased 32P phosphorylation was not observed if membranes were allowed to dephosphorylate prior to 32P phosphorylation in vitro. We conclude that brush border vesicles from isolated perfused canine kidneys can be used to study the action of PTH on Na+-Pi cotransport in brush border membranes and on cAMP-dependent phosphorylation of the membrane. It is strongly suggested that PTH effects changes in Na+-dependent 32Pi transport in isolated brush border vesicles and changes in 32P phosphorylation of vesicles via a direct action on the renal cortical cell rather than as a consequence of extrarenal actions of the hormone.     

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.     

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.     

A new method for the rapid isolation of basolateral plasma membrane vesicles from rat liver. Characterization, validation, and bile acid transport studies

Basolateral plasma membrane vesicles were prepared from rat liver by a new technique using self-generating Percoll gradients. The method is rapid (total spin time of 2.5 h) and protein yields were high (0.64 mg/g of liver). Transmission electron microscopy studies and measurements of marker enzyme activities indicated that the preparation was highly enriched in basolateral membranes and substantially free of contamination by canalicular membranes or subcellular organelles. High total recoveries for protein yield and marker enzyme activities during the fractionation procedure indicated that enzymatic activity was neither lost (inactivation) nor increased (activation). Thus, the pattern of marker enzyme activities found in the membrane preparation truly reflected substantial enrichment in membranes from the basolateral surface. Analysis of freeze-fracture electron micrographs suggested that approximately 75% of the vesicles were oriented "right-side-out." In order to assess the functional properties of the vesicles, the uptake of [3H]taurocholate was studied. In the presence of a Na+ gradient, taurocholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot"). In the absence of a gradient but in the presence of equimolar Na+ inside and outside of the vesicle, taurocholate uptake was faster than in the absence of Na+. These findings support a direct co-transport mechanism for the uptake of taurocholate and Na+. Kinetic studies demonstrated that Na+-dependent taurocholate uptake was saturable with a Km of 36.5 microM and a Vmax of 5.36 nmol mg-1 protein min-1. The high yield, enzymatic profile and retention of transport properties suggest that this membrane preparation is well suited for studies of basolateral transport.     

Mechanism of neomycin stimulation of D-glucose uptake in rabbit intestinal brush border membrane

In order to study the effect of the antibiotic neomycin on the intestinal epithelium, D-glucose was used as a probe molecule and its transport into rabbit brush border membrane vesicles was measured by a rapid filtration method. Treatment of the epithelium with neomycin sulfate prior to the preparation of the brush border membrane enhanced the D-glucose uptake, whereas neutral N-acetylated neomycin did not. This action of neomycin was related to its polycationic character and not to its bactericidal action. No significant difference could be demonstrated between the protein content or disaccharidase-specific activities of the brush border fractions from treated or non-treated intestines. Electrophoretic protein patterns of SDS-solubilized membrane were not significantly different after neomycin treatment. To gain more information on the mechanism involved in the stimulation of D-glucose transport, experiments were conducted on phosphatidyl glycerol artificial membranes and the results compared with those obtained with brush border membrane. At a concentration of 10(-7) M, neomycin decreased the nonactin-induced K+ conductance by a factor of approx. 100. The membrane conductance was linearly dependent on the neomycin concentration and the conductance in 10(-2) M KCl was 10 times that in 10(-3) M KCl. The valence of neomycin was estimated, from the slope of these curves, to be between 6 and 4. In contrast, acetylated neomycin had no effect on the nonactin-induced K+ membrane conductance. Therefore, the effect of neomycin on artificial membrane is related to its 4 to 6 positive charges. It is proposed that the stimulation of sugar transport in brush border membrane is related to screening of the membrane negative charges by the positively-charged neomycin. Accumulation of anions at the membrane surface then occurs and their diffusion into the intravesicular space would increase the transmembrane potential which, in turn, stimulates the entry of D-glucose.     

The basolateral membrane of rat enterocyte: its purification from brush border contamination

Basolateral membranes obtained by self-orienting Percoll-gradient centrifugation were treated with 5 mM CaCl2 to minimize the cross-contamination by brush border membranes. From marker enzyme-specific activities it was calculated that in this preparation the basolateral/brush border membrane ratio was 22.6. A low L-glucose permeability across basolateral membrane vesicles together with ATP-dependent sodium uptake was observed.