Reconstitution and partial purification of an amiloride-sensitive, cation channel from rabbit kidney

The aim of the present study was to reconstitute and purify an epithelial potassium channel from rabbit kidney. Renal brush border membrane vesicles (BBMV) were found to contain a potassium conductance which was inhibited by amiloride, 5-(N-methyl-N-isobutyl)amiloride (MIA) and by barium. Membrane vesicle proteins were solubilized and reconstituted in proteoliposomes. Channel activity was assayed using Acridine orange and the voltage sensitive dye, 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Both methods yielded similar results which indicated the presence of an amiloride-sensitive, cation channel in the proteoliposomes. This channel was more permeable to K than to Na and its activity was increased in reconstituted proteoliposomes as compared to native brush border membranes. We conclude that rabbit BBMV possess an amiloride sensitive cation channel. Channel activity was successfully reconstituted in proteoliposomes and the protein was partially purified during reconstitution.     

Partial purification of alanine carrier from rabbit small intestine brush border membrane and its functional reconstitution into proteoliposomes

An alanine transport carrier was partially purified from brush border membranes of rabbit small intestine. The alanine carrier activity was not solubilized with 0.4% deoxycholate but recovered in the detergent-insoluble fraction. The detergent-insoluble proteins were reconstituted into proteoliposomes with soybean phospholipids. The reconstituted proteoliposomes were capable of uptake of alanine driven by an electrochemical potential of Na+. The initial rate of alanine uptake into the proteoliposomes was 90 pmoles/mg protein/sec, which was 15-fold higher than that observed with the native membrane vesicles. The uptake of alanine was effectively suppressed by various neutral amino acids but not by either cationic or anionic amino acids.     

Enzymic solubilization of the human intestinal brush border membrane enzymes.

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, gamma-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush bborder membrane vesicles by various enzymes (especially pancreatic proteases) have been studied. The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases. Electron microscopy of brush border membrane vesicles demonstrates "knob-like" structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of the enzymic activities with the removal of the particles. The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

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.     

Topological studies on the hydrolases bound to the intestinal brush border membrane. I. Solubilization by papain and triton X-100

Papain digestion of closed, right side out vesicles from pig, rat and rabbit jejunum brush border induces the release of the hydrolases bound to the membrane without grossly affecting the lipid bilayer limiting the vesicles. This observation definitely proves that intestinal hydrolases are surface components attached to the external side of the membrane. All proteins released by papain could be identified by electrophoresis and immunoelectrophoresis to already known intestinal hydrolases, with the exception of an unidentified substance strongly stained by the Schiff's reagent.The early observation that the aminopeptidase form released from pig brush border by Triton X-100 is different from that released by papain was extended to other hydrolases from pig, rat and rabbit. In some cases, the Triton-released form could be converted by further proteolytic digestion into a new form similar to that liberated by papain. These facts may be related to the existence of hydrophobic anchors retaining the intestinal hydrolases to the membrane surface.     

Enzymic solubilization of the human intestinal brush border membrane enzymes

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, γ-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush border membrane vesicles by various enzymes (especially pancreatic proteases) have been studied.The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases.Electron microscopy of brush border membrane vesicles demonstrates “knob-like” structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of th enzymic activities with the removal of the particles.The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

Cholesterol-transfer protein located in the intestinal brush-border membrane. Partial purification and characterization

Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.     

Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

A rapid method for the reconstitution of Na+-dependent neutral amino acid transport from bovine renal brush-border membranes.

1. A simple and rapid method for the reconstitution of Na+-dependent neutral amino acid transport activity from bovine renal brush border membranes is described. 2. The neutral detergent decanoyl-N-methylglucamide ('MEGA-10') was employed to solubilize the membrane protein. This obviated the necessity for a prolonged dialysis step. 3. The properties of amino acid transport in these vesicles were similar to those observed in native membranes. 4. This should be a useful procedure in the eventual identification and isolation of amino acid transport proteins.     

Accelerated transfer of neutral glycosphingolipids and ganglioside GM1 by a purified lipid transfer protein

The transfer of labeled neutral glycosphingolipids from sonicated phosphatidylcholine vesicles to erythrocyte ghosts is greatly stimulated by a nonspecific lipid transfer protein purified from beef liver. Globo-tetraglycosylceramide is transferred at a rate 40% of that for dipalmitoylphosphatidylcholine. II3-alpha-N-Acetylneuraminosyl-gangliotetraglycosylceramide is also transferred by the transfer protein, either from sonicated phosphatidylcholine vesicles or from ganglioside micelles to erythrocyte ghosts. The nonspecific lipid transfer protein catalyzes the net transfer of glycosphingolipids from brush border membrane vesicles (from rabbit intestine) to sonicated phosphatidylcholine/cholesterol vesicles.     

Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal proteases.

Isolated human intestinal brush border membranes were used as sources of enzyme to study their degradation by proteolytic enzymes. Human intestinal brush border hydrolases undergo degradation by two separate proteolytic systems. Sucrase and alkaline phosphatase are degraded by pancreatic proteases (e.g. chymotrypsin) at neutral pH, whereas trehalase is degraded by lysosomal extracts at acid pH. Both the membrane bound and membrane free isolated enzymes had similar sensitivity to proteolytic enzymes. Thus, initial removal from the membrane is not essential as a prerequisite to proteolysis. It is postulated that the brush border membrane of the intestine is subject to proteolysis by pancreatic enzymes from the external cell surface and by lysosomal proteases within the cell.     

Two Different Bacillus thuringiensis Delta-Endotoxin Receptors in the Midgut Brush Border Membrane of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)

Binding of three Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Ostrinia nubilalis larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of O. nubilalis gut epithelium. From competition binding experiments performed with I-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryIA(c). Immunological detection of ingested B. thuringiensis ICPs on gut sections of O. nubilalis larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to O. nubilalis, were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated crystal proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the epithelial microvilli of Malpighian tubules. In addition, CryIA(c) toxin also adheres to the basement membrane of the midgut epithelium.     

Molecular size of the renal sodium/phosphate symporter in native and reconstituted systems.

The size of the renal sodium/phosphate symporter was estimated with the radiation inactivation technique in isolated bovine brush border membrane vesicles and after reconstitution in proteoliposomes. The functional unit of the native phosphate carrier had a radiation inactivation size of 172 +/- 17 kDa. Identical values were obtained for the reconstituted carrier whether it was irradiated before or after the formation of the proteoliposomes (161 +/- 9 and 159 +/- 11 kDa, respectively). The sodium-independent uptake of phosphate was not affected significantly by radiation doses up to 10 Mrad. This activity is therefore not due to the reconstitution of a large phosphate-binding protein such as alkaline phosphatase. Furthermore, bromotetramisole, a specific inhibitor of phosphate binding to this enzyme, had no significant effect on the uptake of phosphate by the proteoliposomes.     

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. Relationship to its mechanism of action

Pretreatment of the D-deficient chick with 1,25-dihydroxyvitamin D3 increases de novo synthesis of phosphatidylcholine by a stimulation of CDP-choline: sn-1,2-diacylglycerol choline-phosphotransferase reaction. The time course of change in the incorporation of [3H]choline and [14C]ethanolamine into the brush border lipid fraction after 1,25-dihydroxyvitamin D3 treatment correlates closely with the time course of change in calcium uptake into the brush border membrane vesicles. Prior treatment with cycloheximide does not block this increase in phosphatidylcholine synthesis. In addition, 1,25-dihydroxyvitamin D3 administration increases the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction of the brush border to a great extent but does not increase the incorporation of [3H]palmitic acid into the phosphatidylcholine fraction. The incorporation of these 3H labeled fatty acids into diacylglycerol is not changed by 1,25-dihydroxyvitamin D3. These data indicate that 1,25-dihydroxyvitamin D3 enhances the synthesis of phosphatidylcholine independent of new protein synthesis, and also increases the incorporation of unsaturated fatty acids into phosphatidylcholine. From these results we suggest that changes in phospholipid metabolism in the enterocyte are the mechanisms by which 1,25-dihydroxyvitamin D3 acts to enhance calcium entry across the brush border membrane.     

Intestinal absorption of dipeptides and beta-lactam antibiotics. II. Purification of the binding protein for dipeptides and beta-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 beta-lactam antibiotics and dipeptides, a binding protein for dipeptides and beta-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 beta-lactam antibiotics of Mr 127,000 was retained on the column. With N-acetyl-D-glucosamine the photolabeled binding protein for beta-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 alpha-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 beta-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 beta-lactam antibiotics across the enterocyte brush border membrane.     

Proline transport across the intestinal microvillus membrane may be regulated by membrane physical properties.

There is now abundant evidence that integral membrane protein function may be modulated by the physical properties of membrane lipids. The intestinal brush border membrane represents a membrane system highly specialized for nutrient absorption and, thus, provides an opportunity to study the interaction between integral membrane transport proteins and their lipid environment. We have previously demonstrated that alterations in this environment may modulate the function of the sodium-dependent glucose transporter in terms of its affinity for glucose. In this communication we report that membrane lipid-protein interactions are distinctly different for the proline transport proteins. Maximal transport rates for L-proline by either the neutral brush border or imino transport systems are reduced 10-fold when the surrounding membrane environment is made more fluid over the physiological range that exists along the crypt-villus axis. Furthermore, in microvillus membrane vesicles prepared from enterocytes isolated from along the crypt-villus axis a similar gradient exists in the functional activity of these transport systems. This would imply that either the functional activity of these transporters are regulated by membrane physical properties or that the synthesis and insertion of these proteins is coordinated in concert with membrane physical properties as the enterocyte migrates up the crypt-villus axis.     

Two Different Bacillus thuringiensis Delta-Endotoxin Receptors in the Midgut Brush Border Membrane of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae)

Binding of three Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Ostrinia nubilalis larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of O. nubilalis gut epithelium. From competition binding experiments performed with ¹²⁵I-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryIA(c). Immunological detection of ingested B. thuringiensis ICPs on gut sections of O. nubilalis larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to O. nubilalis, were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated crystal proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the epithelial microvilli of Malpighian tubules. In addition, CryIA(c) toxin also adheres to the basement membrane of the midgut epithelium.     

Identity of neutral alpha-glucosidase AB and the glycoprotein processing enzyme glucosidase II. Biochemical and genetic studies

We have previously partially purified, characterized, and chromosomally mapped a human isozyme of alpha-glucosidase which is active at neutral pH. This isozyme appears as a doublet of enzyme activity on native gel electrophoresis and was termed neutral alpha-glucosidase AB. We now report genetic and biochemical evidence that neutral alpha-glucosidase AB is synonymous with the glycoprotein processing enzyme glucosidase II. We have found that a mutant mouse lymphoma line which is deficient in glucosidase II is also deficient in neutral alpha-glucosidase AB, as defined electrophoretically and quantitatively (less than 0.5% of parental). In contrast, both mutant and parental cell lines exhibited several lysosomal hydrolases which are processed by glucosidase II. We have also further purified the human neutral alpha-glucosidase A component of neutral alpha-glucosidase AB 740-fold from placenta in order to compare its biochemical properties with those described for rat liver and pig kidney glucosidase II. Both glucosidase II and neutral alpha-glucosidase AB are high-molecular mass (greater than 200,000 dalton) anionic glycoproteins which bind to concanavalin A, have a broad pH optima (5.5-8.5), and have a similar Km for maltose (4.8 versus 2.1 mM) and the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (35 versus 19 microM). Similar to human neutral alpha-glucosidase AB, purified rat glucosidase II migrates as a doublet of enzyme activity on native gel electrophoresis. Although rat glucosidase II has been reported to have a subunit size of 67 kDa, pig glucosidase II has been found to have a subunit size of 100 kDa, like the 98-kDa major protein in purified human neutral alpha-glucosidase A. Although we have not demonstrated that neutral alpha-glucosidase AB is microsomal nor that it hydrolyzes the natural substrate of glucosidase II, we believe that the genetic evidence is compelling for and the biochemical data consistent with the hypothesis that neutral alpha-glucosidase AB and glucosidase II are synonymous. These and previous results would localize glucosidase II to the long arm of human chromosome II.     

Purification and characterization of chick intestine brush border membrane. Effects of 1alpha(OH) vitamin D3 treatment

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1alpha-hydroxyvitamin D3 treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.     

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.