Calmodulin binding to the intestinal brush-border membrane: comparison to other calcium-binding proteins

The intestinal brush-border membrane contains a high concentration of calmodulin bound to a 105,000 dalton (105 kDa) protein. Binding of radioiodinated calmodulin to this protein does not require calcium but is inhibited by trifluoperazine and excess unlabelled calmodulin. Recent evidence suggests that the 105 kDa protein in conjunction with calmodulin may be involved in the regulation of calcium transport across the brush-border membrane. In this report, we evaluated the binding of the 105 kDa protein to other radioiodinated calcium-binding proteins including the vitamin D-dependent intestinal calcium-binding protein. We observed that troponin C and S100 beta protein both bound strongly to the 105 kDa protein. The binding of S100 beta was inhibited by EGTA, but was little affected by trifluoperazine and excess unlabelled S100 beta, whereas that of troponin C was inhibited by trifluoperazine and excess unlabelled troponin C, but was little affected by EGTA. Both troponin C and S100 beta bound to a large number of proteins to which calmodulin did not bind. The vitamin D-dependent calcium-binding protein (calbindin) from chick intestine and rat kidney also bound to the 105 kDa protein, albeit more weakly than troponin C, S100 beta and calmodulin. The binding of the calbindins was increased by EGTA and was little affected by trifluoperazine and excess unlabelled calbindin. Parvalbumin, rat osteocalcin, and alpha-lactalbumin showed little binding to any brush-border membrane protein. Our results indicate that the 105 kDa calmodulin-binding protein of the intestinal brush border can bind to a variety of calcium-binding proteins all of which contain homologous regions thought to be the calcium-binding sites. Only the binding of troponin C resembles the binding of calmodulin, however, in being inhibited by trifluoperazine and excess unlabelled ligand. The functional significance of these observations in terms of regulating calcium transport across the brush-border membrane remains to be established.     

Biotin transport in rat intestinal brush-border membrane vesicles

Transport of biotin across rat intestinal brush-border membrane was examined using the brush-border membrane vesicle (BBMV) technique. Uptake of biotin by BBMV is the result of transport of the substrate into the intravesicular space with negligible binding to membrane surfaces. In the presence of a Na+ gradient (out greater than in), transport of biotin was higher with a transient 'overshoot' phenomenon. In comparison, transport of biotin in the presence of a choline gradient (out greater than in) was lower with no 'overshoot' phenomenon. In both jejunal and ileal BBMV, the transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in) but was linear in the presence of a choline gradient (out greater than in). Vmax of the Na+-dependent transport system was 0.88 and 0.37 pmol/mg protein per s and apparent Kt was 7.57 and 7.85 microM in jejunal and ileal BBMV, respectively. Structural analogues inhibited the transport process of biotin. Unlike the electrogenic transport of D-glucose, the transport of the anionic biotin was not affected by imposing a relatively positive intravesicular potential with the use of valinomycin and an inwardly-directed K+ gradient, suggesting that biotin transport is most probably an electroneutral process. This suggestion was further supported by studies on biotin transport in the presence of anions of different lipid permeability. The results of this study demonstrate that biotin transport across rat intestinal brush-border membrane is by a carrier-mediated, Na+-dependent and electroneutral process. Furthermore, transport of biotin is higher in the jejunum than the ileum.     

Spermine uptake by rat intestinal brush-border membrane vesicles

The uptake of spermine by isolated rat intestinal brush-border membrane vesicles was studied. Uptake was biphasic, with an initial rapid uptake followed by a prolonged slower phase. Spermine uptake was not affected by a Na+ electrochemical gradient. The equilibrium uptake of spermine was considerably dependent upon the medium pH. At pH 7.5 the degree of uptake was higher than that at pH 6.5 and was inversely proportional to the extravesicular osmolarity with a relatively high binding, which was estimated by extraporation to infinite extravesicular osmolarity (zero intravesicular space), while the uptake at pH 6.5 was not altered under the various medium osmolarities. A kinetic analysis of the initial uptake rate of spermine at 37 degrees C gave a Km of 24.2 microM and Vmax of 206.1 pmol/mg protein per min. Furthermore, the uptake at 4 degrees C was nonlinear, providing evidence for saturability. These findings suggest that spermine was associated with intestinal brush-border membrane vesicles in two ways, by binding to the outside and inside of membrane vesicles. The interaction of spermine and the apical membrane can be a contributory factor in the accumulation of this polyamine in the intestine of the intact animal.     

The kinetic mechanism of the glutamate-aspartate carrier in rat intestinal brush-border membrane vesicles: the role of potassium

The sodium dependent transport system for L-glutamate and L-aspartate localized in the apical part of rat enterocytes has previously been kinetically characterized (Prezioso, G., and Scalera, V. (1996). Biochim. Biophys. Acta 1279, 144–148). In this paper the mechanism by which the potassium cation specifically activates the L-glutamate–sodium cotransport process is investigated. Potassium has been found to act as an activator when it is present inside the membrane vesicles, while its presence outside is ineffective, and the effect is saturable. The kinetic parameters with respect to sodium and glutamate have been compared in the presence and in the absence of the activator. The results indicate that the ordered sodium–sodium glutamate mechanism is not altered by potassium, and that the activation is probably exerted on both the rate determining steps of the transport process. It is proposed that (1) a specific binding site for potassium is present on the inside hydrophilic part of the membrane carrier, (2) the binding of the effector accelerates the intramembrane rearrangement steps of both the disodium glutamate–carrier complex and the free carrier, (3) the affinity of the carrier is lowered with respect to sodium whereas it is increased for glutamate, and (4) K⁺ antiport is not performed by this carrier.     

Phosphate transport in intestinal brush-border membrane

In the small intestine of the rabbit the process of Na⁺-dependent uptake of phosphate occurs only at the brush-border of duodenal enterocytes. Li⁺ can replace Na⁺. The process is activated when either K⁺, Cs⁺, Rb⁺, or choline is present in the intravesicular space. The presence of membrane-permeable anions is essential for maximum rates of phosphate transport. We conclude that the mechanism of the phosphate carrier is electrogenic at pH 6–8, probably two Na⁺ moving with each H₂PO ₄ ^– . This. will lead to the development of a positive charge within the vesicle. The variation of theK _m for H₂PO ₄ ^– with pH is thought to be the consequence of the affinity of the carrier protein for H₂PO ₄ ^– increasing as the pH increases. Polyclonal antibodies against membrane vesicles isolated from rabbit duodenum, jejunum, and ileum were prepared. The antibodies raised against the ileum and jejunum both activated the phosphate transport process, while the anti-duodenum antibody preparation inhibited phosphate transport.     

Uptake characteristics of polyamines into rat intestinal brush-border membrane.

The uptake characteristics of polyamines, such as spermine, spermidine and putrescine, have been investigated using brush-border membrane vesicles isolated from the small intestine of rats. The uptake of these polyamines into the membrane vesicles was high and the order of uptake was spermine greater than spermidine greater than putrescine at medium pH 7.5, respectively. The medium pH considerably affected the uptake of these polyamines and the amount of uptake increased remarkably with an increase of the medium pH (pH 7.5 or 8.0 greater than pH 5.5). An inward Na+ gradient did not stimulate the uptake rate of any of these polyamines. We have also examined the binding behaviour to the membrane lipid, phospholipids and total lipid, and there was a good correlation in the binding properties, pH-dependency and uptake activity, between the liposomes and brush-border membrane vesicles. These results suggest that the uptake of the polyamine into the vesicles consisted of rapid binding to the outside intestinal surface and slower binding to the inside membrane after permeation. Furthermore, findings from experiments concerning the mutual inhibition among these polyamines and concerning the effect of other polycations, having 2-5 amines in number, on the uptake of spermine, suggest that the number of amino groups in the polyamine molecules plays an important role in the uptake process into the brush-border membrane vesicles.     

Folate binding and transport by rat kidney brush-border membrane vesicles

[3H]Pteroylglutamic acid (PteGlu) uptake was studied using brush-border membrane vesicles isolated from rat kidney. Results on the uptake of [3H]PteGlu by brush-border membrane vesicles incubated in media of increasing osmolarities demonstrated that uptake was contributed by two components, intravesicular transport and membrane binding. Both the components of the uptake exhibited similar pH dependence, with maxima at pH 5.6, and were found to be saturable mechanisms with Km values of 6.7.10(-7) and 11.2.10(-7) M, respectively. These studies show that PteGlu is transported by isolated rat kidney brush-border membrane vesicles in a manner consistent with a saturable system and that a binding component may be functionally associated with this.     

Oxalate uptake in intestinal and renal brush-border membrane vesicles (BBMV) in vitamin B6-deficient rats

Acute, subclinical, and chronic pyridoxine deficiency did not modify the oxalate influx in rat intestinal BBMV but elevated the oxalate reabsorption by renal tubular cells. The Na+ and K+ ions did not affect oxalate uptake in either intestinal or renal BBMV. Although thiol group blocking agents did not affect intestinal uptake of oxalate they significantly altered oxalate translocation across the renal tubular cells. Following pyridoxine deficiency the rat kidneys appear to be more specific for inducing oxalate lithiasis as compared to oxalate influx through the intestine.     

Membrane-potential-dependent uptake of tryptamine by rat intestinal brush-border membrane vesicles.

The effect of membrane potential on the uptake of tryptamine, an organic cation, by rat intestinal brush-border membrane vesicles was studied. In the presence of an outwardly directed H(+)-gradient, the initial uptake of tryptamine was stimulated remarkably and the overshoot phenomenon was observed. In contrast, the uptake was depressed by an inwardly-directed H(+)-gradient. The effect of H(+)-gradient on the uptake of tryptamine was maintained in the presence of FCCP, whereas it vanished when voltage-clamped vesicles were used. Moreover, the uptake of tryptamine was linearly augmented with increase of the valinomycin-induced inside-negative K+ diffusion potential. These results suggest that tryptamine is taken up into intestinal brush-border membrane vesicles depends upon the ionic diffusion potential. The effect of several indole derivatives and amine compounds on the uptake of tryptamine was also examined. The uptake of tryptamine was inhibited by all amine compounds used, but anionic and zwitterionic compounds had no effect, suggesting that these amines interact on brush-border membrane and cause an inhibitory effect.     

Phospholipid topology and flip-flop in intestinal brush-border membrane

The topological distribution of the two major phospholipids of brush-border membrane, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), has been investigated using brush-border membrane vesicles from rabbit small intestine. Bee venom phospholipase A2 and phosphatidylcholine exchange protein from bovine liver were used as membrane probes. It is shown that the brush-border membrane retains its integrity under conditions of phospholipase hydrolysis and intermembrane phospholipid exchange. Kinetic analysis of the data of phospholipase hydrolysis and phospholipid exchange at temperatures under 10 degrees C shows that both PC and PE occur in two pools: a minor (about 25%) more readily accessible pool and a major one (about 75%) less readily available. The rate of PC exchange between these two pools is relatively fast. The half-time derived under conditions of phospholipase hydrolysis is of the order of 20 min. Under conditions of phospholipid exchange the exchange rates may be even faster. The difference in exchange kinetics observed with the two methods of probing is probably due to changes in membrane properties such as the bilayer fluidity induced by the probing process itself. It is proposed that the two pools represent the transverse distribution of the phospholipids. The two major phospholipids of brush-border membranes, PC and PE, would be distributed mainly on the inner (cytoplasmic) side of the brush-border membrane. The phospholipid exchange between the brush-border vesicles and unilamellar phosphatidylcholine vesicles in the presence of phosphatidylcholine exchange protein reveals that significant quantities of phospholipid are taken up by brush-border membrane independently, i.e., in a separate process independent of the exchange protein-catalyzed phosphatidylcholine exchange.     

Immunocytochemical detection of GLUT2 at the rat intestinal brush-border membrane.

We have proposed a new model of intestinal sugar absorption in which high sugar concentrations promote rapid insertion of the facilitative transporter GLUT2 into the brush-border membrane so that absorptive capacity is precisely regulated to match dietary intake during the assimilation of a meal. However, location of GLUT2 at the brush border by immunocytochemistry has been problematical. We report that control of rapid GLUT2 trafficking and the use of an antibody to a sequence within the large extracellular loop of GLUT2 permits localization of GLUT2 at the brush border. To reveal brush-border GLUT2 fully, it is necessary to digest the sugar chain at the glycosylation site close to the antigenic site. In this way, we have demonstrated by immunocytochemistry PKC-dependent changes in the regulation of brush-border GLUT2 in rat jejunum that correspond to those seen by Western blotting. The functional and immunocytochemical data are now reconciled.     

Transport of glycyl-L-proline in intestinal brush-border membrane vesicles of the suckling rat: characteristics and maturation

Transport of the dipeptide glycine-L-proline (Gly-L-Pro) in the developing intestine of suckling rats and its subsequent maturation in adult rats was examined using the brush-border membrane vesicles (BBMV) technique. Uptake of Gly-L-Pro by BBMV was mainly the result of transport into the intravesicular space with little binding to membrane surfaces. Transport of Gly-L-Pro in BBMV of suckling rats was: (1) Na+ independent; (2) pH dependent with maximum uptake at an incubation buffer pH of 5.0; (3) saturable as a function of concentration (apparent Km = 21.5 +/- 7.9 mM, Vmax = 8.6 +/- 1.5 nmol/mg protein per 10 s); (4) inhibited by other di- and tripeptides; and (5) stimulated and inhibited by inducing a negative and positive intravesicular membrane electrical potential, respectively. Similarly, transport of Gly-L-Pro in intestinal BBMV of adult rats was saturable as a function of concentration (apparent Km = 17.4 +/- 8.6 mM, Vmax = 9.1 +/- 2.1 nmol/mg protein per 10 s) and was stimulated and inhibited by inducing a relatively negative and positive intravesicular membrane potential, respectively. No difference in the transport kinetic parameters of Gly-L-Pro was observed in suckling and adult rats, indicating a similar activity (and/or number) and affinity of the transport carrier in the two age groups. These results demonstrate that the transport of Gly-L-Pro is by a carrier-mediated process which is fully developed at the suckling period. Furthermore, the process is H+-dependent but not Na+-dependent, electrogenic and most probably occurs by a Gly-L-Pro/H+ cotransport mechanism.     

Characterization of binding between the rat small intestinal brush-border membrane and dietary proteins in the sensory mechanism of luminal dietary proteins.

Dietary proteins are recognized by the gastrointestinal tract to display physiological functions, however, the sensory mechanism of the intestinal mucosa is not known. We examined binding properties between the rat small intestinal brush-border membrane (BBM) and proteins by using a surface plasmon resonance biosensor. BBM and solubilized BBM prepared from the rat jejunum bound to casein immobilized on the sensor surface, but not to bovine serum albumin. The ileal BBM showed less binding to casein than the jejunal BBM. Solubilized BBM binding to immobilized alpha-casein was slightly inhibited by aminopeptidase inhibitors, but still more inhibited by addition of casein with the inhibitors. Guanidinated casein inhibited the solubilized BBM binding to alpha-casein more strongly than casein (casein sodium and alpha-casein) inhibited. Trypsinization of solubilized BBM abolished its binding activity to alpha-casein. These results indicate that some membrane protein, but not aminopeptidases, contained in BBM interacts with dietary proteins, and that guanidinated casein has a higher affinity for BBM than intact casein. These binding intensities for proteins were closely correlated to physiological responsiveness, and are possibly involved in a sensory system for dietary protein in the intestine.     

Identification of microsomal triglyceride transfer protein in intestinal brush-border membrane

Microsomal triglyceride transfer protein (MTP) is a heterodimeric complex consisting of a unique large 97-kDa protein and the multifunctional 58-kDa protein disulfide isomerase (PDI). It plays an essential role in the assembly of lipoproteins by shuttling lipids between phospholipid membranes. Based on cell fractionation, early studies have suggested the endoplasmic reticulum (ER) as the exclusive site of MTP. Focusing on the plasma membrane in this study, our attempts with immunoelectron microscopy and specific antibodies surprisingly revealed that labeling was not exclusively confined to the microsomes of rat absorptive cells. Immunogold labeling was also detected over the microvillus membrane of enterocytes. Western blot analysis and biochemical activity measurement confirmed MTP protein expression in brush-border membrane vesicles (BBMV) isolated from the intestinal epithelial cells of various species. Furthermore, MTP was coexpressed in microvilli membrane with PDI that is crucial to maintain the structure and activity of the MTP complex. The treatment of Caco-2 cells with nocodazole and colchicine blocked the appearance of MTP in the apical membrane. Similarly, the addition of BMS-197636, a known inhibitor of MTP transfer activity, suppressed the latter. In conclusion, the present studies suggest that MTP is present in the brush-border membrane of the enterocyte. Understanding the possible physiological role of MTP in this location may reveal additional functions.     

D-glucose uptake in intestinal brush-border membrane vesicles of rachitic rats

We have previously reported the metabolic consequences of feeding rats Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio. Using isolated brush-border membrane vesicles prepared from the jejunum, ileum and duodenum of control and rachitic rats, we have demonstrated a marked decrease of Na+-dependent D-glucose uptake at jejunum-ileum level of rachitic rats. At duodenum level Na+-dependent D-glucose transport was not influenced by rickets. A lack of any significant difference between the two animal groups was observed studying the facilitated transport of D-glucose, the diffusion of L-glucose and the Na+-dependent uptake of phenylalanine and aspartate.     

Identification of identical binding polypeptides for cephalosporins and dipeptides in intestinal brush-border membrane vesicles by photoaffinity labeling

The uptake of a photolabile derivative of the orally effective cephalosporin cephalexin, N-(4-azidobenzoyl)cephalexin, was investigated in brush-border membrane vesicles. The compound was taken up into the intravesicular space and inhibited the active uptake of cephalexin in a concentration-dependent manner. Therefore, this probe interacts with the transport system shared by alpha-aminocephalosporins and dipeptides. Photoaffinity labeling of brush-border membrane vesicles from rat small intestine with N-(4-azido[3,5-3H]benzoyl) derivatives of the cephalosporin cephalexin and the dipeptide glycyl-L-proline resulted in the covalent incorporation of radioactivity into membrane polypeptides with apparent molecular weights of 127,000, 100,000, 94,000 and 86,000, the polypeptide of molecular weight 127,000 being predominantly labeled. The specificity of labeling was demonstrated by a decrease in the labeling of the polypeptide of apparent molecular weight 127,000 in the presence of beta-lactam antibiotics and dipeptides, whereas glucose, taurocholate or amino acids had no effect on the labeling pattern. These data demonstrate an interaction of cephalosporins and dipeptides with a common membrane protein of molecular weight 127,000, which could be a component of the intestinal transport system(s) responsible for the uptake of orally effective cephalosporins and dipeptides.     

Characterization of chicken cystatin binding to rat renal brush-border membranes

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K_d = 3.67–4.07 μM) and high capacity (B_max = 2.32–2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K_i = 0.7 μM) among other megalin/cubilin ligands tested. The presence of Ca^+ 2 ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.     

Peptide transport in rabbit intestinal brush-border membrane vesicles studied with a potential-sensitive dye

Peptide transport in purified rabbit intestinal brush-border membrane vesicles has been studied using a potential-sensitive fluorescent dye, di-S-C3(5). Transport of dipeptides is accompanied by an increase in the fluorescence of the dye in the presence and absence of Na+, indicating electrogenic, Na+-independent peptide transport. Dipeptides containing D-amino acids also increase the fluorescence, showing that these peptides too possess significant affinity for the peptide transport system. beta-Alanylglycylglycine and prolylglycylglycine, very much like the dipeptides, increase the fluorescence even in the absence of Na+ which demonstrates the Na+-independent, electrogenic transport of tripeptides. However, concentrations needed for half-maximal fluorescence changes are higher for tripeptides than for dipeptides suggesting different affinities for the carriers. The studies, in addition, provide evidence for the existence of more than one carrier system for translocation of small peptides in rabbit intestinal brush-border membrane.     

31P-NMR study of pig intestinal brush-border membrane structure: effect of zinc and cadmium ions

³¹P-NMR experiments on intact pig small intestine brush-border membrane vesicles (BBMV) and detergent-solubilized membranes gave direct insights into the organization of the phospholipids (PL) and their interaction with zinc and cadmium ions. Various endogenous PL were identified from well resolved BBM micelle spectra. These experiments revealed a strong interaction of Zn²⁺ and Cd²⁺ with the negatively charged phosphatidylinositol and phosphatidylserine. In BBM micelles, a progressive time-dependent PL degradation occurred in the absence of ions and indicated the presence of active phospholipases. The presence of zinc inhibited the degradation process whereas cadmium had the opposite influence. ³¹P spectra of BBMV were carefully characterized. Neither zinc nor cadmium affected the PL bilayer structural organization. A degradation of PL, monitored by the increase of the inorganic phosphate (P _i) signal, also occurred in vesicles but to a lesser extent than in micelles. A 2/3 internal, 1/3 external PL asymmetry was observed in the absence and presence of ions. Offprint requests to: P. Ripoche     

N-linked neutral sugar chains of aminopeptidase N purified from rat small intestinal brush-border membrane.

Neutral oligosaccharides, which accounted for 74% of the total N-linked sugar chains released by hydrazinolysis of rat small intestinal aminopeptidase N, were investigated on a structural basis. They are mainly composed of complex-type sugar chains with tri- and tetraantennary structures, and small amounts of high mannose type sugar chains (7% of the total neutral sugar chains) are also included. The unique feature of the complex-type sugar chains is the most of them contain terminal N-acetylglucosamine residues and blood group H antigenic determinants in their outer chain moieties, and bisecting N-acetylglucosamine residues in their trimannosyl cores. Both the type 1H and type 2H determinants are found, but the former is mainly expressed at the distal portions of the outer chain moieties of the oligosaccharides.