Treatment Of Brush Border Membranes Research Articles

Inhibition of gentamicin binding to rat renal brush-border membrane by megalin ligands and basic peptides

Our previous studies showed that coadministration of cytochrome c and a 20-residue basic peptide, N-WASP181–200 (NISHTKEKKKGKAKKKRLTK, p I = 10.87) inhibits renal accumulation of gentamicin. In this study, we examined effects of ligands of megalin, an endocytic receptor involved in renal uptake of gentamicin, and basic peptides including N-WASP180–200 and its mutant peptides on gentamicin binding to isolated rat renal brush-border membrane (BBM). Gentamicin binding to BBM was inhibited by megalin ligands, basic peptide fragments of cytochrome c, and N-WASP181–200 in a concentration-dependent manner. Klotz plot analysis showed that N-WASP181–200 inhibited the binding of gentamicin in a competitive manner. By substituting glycines for lysines in N-WASP181–200 at positions 9 and 15, the inhibitory effect on gentamicin binding to BBM was reduced, which may be related to a decrease in the α-helix content in the peptide. Gentamicin binding to BBM treated with trypsin, in which megalin completely disappeared, was significantly but not completely decreased compared with the native BBM. In addition, treatment of BBM with trypsin led to a decrease in the inhibitory effect of N-WASP181–200 on gentamicin binding. These observations support that megalin ligands and basic peptides including N-WASP181–200 decrease renal accumulation of gentamicin by inhibiting its binding to BBM of proximal tubule cells, partly interacting with megalin. In addition, the α-helix conformation may play an important role in the inhibitory effect of N-WASP181-200 on the binding of gentamicin to BBM.

Horseradish peroxidase binding to intestinal brush-border membranes of Cyprinus carpio. Identification of a putative receptor.

Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush-border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K(d) = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin-type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3-kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization.

Affinity identification of organic anion transporters in brush-border membrane vesicles from rat kidney

The inhibitory properties of bromoacetyl-p- aminohippuric acid as the affinity probe of the organic anion transport system were studied. Bromoacetylated p- aminohippurate was shown to be able to inhibit irreversibly the p- aminohippurate (PAH) uptake in brush-border membrane vesicles. The inhibition depends on both the time of treatment and the affinity probe concentration. The treatment of brush-border membrane with 1 mM bromoacetyl-p- aminohippurate for 1.5 h results in 100% irreversible inhibition of PAH transport but no changes were observed in the activity of alkaline phosphatase, γ-glutamyltranspeptidase or maltase. The affinity labelling of the organic anion transporters was performed with bromoacetyl-p- amino[ 3 H] hippuric acid . It was shown, by means of SDS-polyacrylamide gel electrophoresis, that the probe bound covalently to the brush-border membrane proteins with molecular masses of 28 kDa, 63 kDa, 98 kDa, and > 150 kDa . The data obtained with SITS and probenecide as the organic anion transport inhibitors indicate that brush-border membrane proteins of 28 kDa, 63 kDa, 98 kDa may correspond to the organic anion transport system.

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.

Uptake of cholesterol by small intestinal brush border membrane is protein-mediated

Absorption of cholesterol by small intestinal brush border membrane from either mixed micelles or small unilamellar vesicles is protein-mediated. It is a second-order reaction. The kinetic data are consistent with a mechanism involving collision-induced transfer of cholesterol. With micelles as the donor particle, there is net transfer of cholesterol while with small unilamellar vesicles as the donor, cholesterol is evenly distributed between the two lipid pools at equilibrium. The cholesterol absorption by brush border membrane from both mixed micelles and small unilamellar vesicles reveals saturation kinetics. Proteolytic treatment of brush border membrane with papain releases about 25% of the total membrane protein. As a result, the cholesterol uptake by brush border membrane changes from a second-order reaction to a first-order one. The reaction mechanism changes from collision-induced cholesterol uptake to a mechanism involving diffusion of monomeric cholesterol through the aqueous phase. The protein(s) released into the supernatant by papain treatment of brush border membrane exhibit(s) cholesterol exchange activity between two populations of small unilamellar vesicles. The supernate-protein(s) bind(s) the spin-labeled cholesterol analogue 3-doxyl-5 alpha-cholestane.

Binding of Escherichia coli heat-stable enterotoxin to rat intestinal cells and brush border membranes.

The association of heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli 431 with isolated rat intestinal epithelial cells and brush border membranes was characterized. Specific binding of strain 431 125I-STa to a single class of specific high-affinity receptors was saturable and temperature dependent and reached a maximum between 5 and 10 min. A 1,000-fold excess of unlabeled 431 STa competitively displaced 90 to 95% of radiolabeled enterotoxin bound to brush border membranes. In contrast, specific binding of 431 125I-STa to intestinal cells ranged from 40 to 65%. The number of STa-specific receptors on rat intestinal cells determined by Scatchard analysis was 47,520 +/- 14,352 (mean +/- standard error of the mean) per cell, with affinity constants (KaS) of 2.55 X 10(11)and 4.32 x 10(11) liters/mol determined for intestinal cells and brush border membranes, respectively. Villus intestinal cells appeared to possess about twice as many STa receptors as did crypt cells. Dissociation of specifically bound 431 125I-STa from intestinal cells and brush border membranes was minimal (2 to 5%). In addition, neither the rate nor the extent of dissociation was increased by a 1,000-fold excess of unlabeled homologous 431 Sta. Binding experiments with 431 125I-STa and brush border membranes showed that purified unlabeled STas from enterotoxigenic E. coli strains 667 (class 1 porcine enteropathogen), B-41 (bovine enteropathogen), and human strains 213C2 (Mexico) and 153961-2 (Dacca, Bangledesh) exhibited patterns of competitive inhibition similar to those of homologous unlabeled 431 STa (class 2 enteropathogen). A lipid extract which contained gangliosides and glycolipids exhibited dose-dependent competitive inhibition of heat-labile enterotoxin binding to brush border membranes but did not inhibit binding of 431 125I-STa. Purified heat-labile enterotoxin from strain 286C2 did not inhibit binding of 431 STa to brush border membranes. Pronase treatment of brush border membranes reduced binding of 431 125I-STa by about 30%, suggesting that the STa receptor was a protein or a glycoprotein. The putative STa receptor was radiolabeled with 431 125I-STa and solubilized with sodium deoxycholate. One major radioactive band was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. These data suggested that STas bind essentially irreversibly to a specific receptor on the cell surface of intestinal cells before activation of guanylate cyclase.

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes

Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the 32P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of 32P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent K m for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10 −8 m cAMP. The K a values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.