Simultaneous isolation of brush border and basolateral membrane from rabbit enterocytes. Presence of brush border hydrolases in the basolateral membrane of rabbit enterocytes

By a slight modification of the procedure described by Gratecos et al. (Gratecos, D., Knibiehler, M., Benoit, V. and Sémériva, M. (1978) Biochim. Biophys. Acta 512, 508–524), the basolateral and brush border membranes of rabbit enterocytes have been purified concomitantly from the same aliquot of mucosa. The two types of membrane have been obtained with the same yield (15%) and enrichment of specific markers (18-fold).The presence in the basolateral membrane of hydrolases known to be specific of the brush border membrane has been confirmed by using immunological techniques.     

Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes

Glycosphingolipid/cholesterol-rich membranes ("rafts")can be isolated from many types of cells, but their existence as stable microdomains in the cell membrane has been elusive. Addressing this problem, we studied the distribution of galectin-4, a raft marker, and lactase, a protein excluded from rafts, on microvillar vesicles from the enterocyte brush border membrane. Magnetic beads coated with either anti-galectin-4 or anti-lactase antibodies were used for immunoisolation of vesicles followed by double immunogold labeling of the two proteins. A morphometric analysis revealed subpopulations of raft-rich and raft-poor vesicles by the following criteria: 1) the lactase/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (p < or = 0.01) than that of vesicles captured by anti-galectin-4 beads, 2) subpopulations of vesicles labeled by only one of the two antibodies were preferentially captured by beads coated with the respective antibody (p < or = 0.01), 3) the average diameter of "galectin-4 positive only" vesicles was smaller than that of vesicles labeled for lactase. Surprisingly, pretreatment with methyl-beta-cyclodextrin, which removed >70% of microvillar cholesterol, did not affect the microdomain localization of galectin-4. We conclude that stable, cholesterol-independent raft microdomains exist in the enterocyte brush border.     

Binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins

Transmissible gastroenteritis coronavirus (TGEV) is a porcine pathogen causing enteric infections that are lethal for suckling piglets. The enterotropism of TGEV is connected with the sialic acid binding activity of the viral surface protein S. Here we show that, among porcine intestinal brush border membrane proteins, TGEV recognizes a mucin-type glycoprotein designated MGP in a sialic acid-dependent fashion. Virus binding assays with cryosections of the small intestine from a suckling piglet revealed the binding of TGEV to mucin-producing goblet cells. A nonenteropathogenic mutant virus that lacked a sialic acid binding activity was unable to bind to MGP and to attach to goblet cells. Our results suggest a role of MGP in the enteropathogenicity of TGEV.     

Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes

Summary In vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to determine whether or not the newly synthesized aminopeptidase N transits through the basolateral membrane before it reaches the apical brush border, its final localization. The kinetics of the arrival of the newly synthesized enzyme in the Golgi complex, basolateral and brush border membrane fractions strongly suggest that on leaving the Golgi aminopeptidase N is transiently integrated into the basolateral domain before reaching the brush border.     

In vitro modification of cholesterol/ phospholipid ratio of enterocytes brush border membrane and its effect on l-leucine accumulation

• 1.1. Incubation of intestinal segments from chicken jejunum in liposome suspensions with different cholesterol/phospholipid ratios lead to change in the enterocytes brush border membrane cholesterol/phospholipid molar ratio.
• 2.2. The fatty acid composition, total amount and the ratios of the individual phospholipids of enterocytes brush border membrane were not affected.
• 3.3. The change in the cholesterol/phospholipid molar ratio had no effect on l-leucine accumulation in the enterocytes.

     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

Sucrase-isomaltase: a stalked intrinsic protein of the brush border membrane

The isolation and purification of sucrase-isomaltase from brush border membrane is described and the physicochemical properties of the pure enzyme are discussed. Our present understanding of the mode of association of the intrinsic membrane protein sucrase-isomaltase with the brush border membrane will be the central point of this contribution. The assembly of sucrase-isomaltase into phospholipid bilayers has been reported to result in a model membrane system which resembles the "native" brush border membrane as regards the mode of lipid-protein interaction. The physicochemical properties of this reconstituted model membrane will be compared to the in vivo situation as represented by brush border membrane vesicles routinely isolated from small intestinal brush borders. The biosynthetic mechanism will be discussed.     

Isolation and characterization of brush border membrane vesicles from pig small intestine

1. Brush border membrane vesicles (BBMV) were isolated from swine mid-intestine by a MgCl2 precipitation and sucrose density gradient centrifugation. 2. Transport of D-glucose and L-alanine were Na+-stimulated and into an osmotically sensitive space. 3. Estimates of kinetic parameters for Na+-dependent D-glucose transport were: apparent Kt = 1.8 mM and Jmax = 16.8 nmol/mg protein/min. 4. Results of experiments with the delta pH sensitive fluorescent probe 9-aminoacridine indicated independent mechanisms for Na+-dependent glucose transport and Na+/H+ exchange. 5. This study demonstrates that pig BBMV provide a useful model for investigating intestinal membrane transport.     

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.     

Binding of intrinsic factor to ileal brush border membrane in the rat

The rat ileal brush border membrane binds both free [¹²⁵I]-intrinsic factor (IF) and the IF-[⁵⁷Co]cobalamin (cbl) complex. This binding is observed with IF isolated from rat stomach, but not from IF isolated from hog, canine and human stomachs. The binding of rat-IF[⁵⁷Co]cbl can be blocked with free rat IF but not with hog IF. The IF-cbl complex binds at a higher affinity (Ka=0.15 × 10⁹ M^?1) compared to that of free IF (Ka=0.9 × 10⁹ M^?1). Rat IF-cbl also binds efficiently to human and canine ileal membranes. While antibody to the canine ileal receptor blocks the binding of rat, human or hog IF-[⁵⁷Co]cbl to human and canine ileal membranes, it does not affect the binding of rat IF-[⁵⁷Co]cbl to rat ileal membranes. These findings demostrate that the rat ileal receptor is different from canine and human ileal receptors.     

Functional characterization of purified zinc transporter from renal brush border membrane of rat

Major zinc binding protein purified from renal brush border membrane (BBM) (R. Kumar, R. Prasad, Biochim. Biophys. Acta 1419 (1999) 23) was reconstituted into liposomes and its functional characteristics were investigated. Physical incorporation of the major zinc binding protein into the proteoliposomes was checked by SDS-PAGE, which showed a single band on silver staining. The structural integrity of the proteoliposomes was assessed by phase contrast microscopy, which revealed the proteoliposomes as globular structures and intact boundaries. Further structural integrity/leakiness of the proteoliposomes was checked by monitoring efflux of Zn(2+) from the pre-loaded proteoliposomes in the presence of either 2 mM Ca(2+) or Cd(2+) or Zn(2+). It was observed that even after 2 h of the initiation of efflux, 85-95% of Zn(2+) was retained in the proteoliposomes, thereby indicating that proteoliposomes were not leaky and maintained structural integrity during the uptake study. Zinc uptake into the proteoliposomes followed Michaelis-Menten kinetics with affinity constant (K(m)) of 1.03 mM and maximal velocity (V(max)) of 1333 nmol/mg protein per min. The uptake process followed first-order kinetics with a rate constant (k) of 1. 09x10(-3) s(-1). The specificity of zinc transport system was determined by studying the interaction of divalent cations viz. Ca(2+) and Cd(2+) with the zinc uptake. It was observed that Cd(2+) competitively inhibited the zinc uptake process with inhibitory concentration (K(i)) of 2.9 mM. Kinetic analysis of inhibitory effect of Cd(2+) on zinc uptake revealed an increase in K(m) to 1.74 mM without influencing V(max). Zn(2+) uptake into the proteoliposomes was found to be temperature sensitive and Arrhenius plot showed a breakpoint at 27 degrees C. The apparent energies of activation (E(a)) were found to be 7.09 and 2.74 kcal/mol below and above the breakpoint, respectively. The initial velocity of Zn(2+) uptake increased with the increase in outwardly directed proton gradient ([H](i) greater than [H](o)). The Zn(2+) uptake was inhibited by DCCD, thereby suggesting the involvement of -COOH groups in the translocation of Zn(2+) across the lipid bilayer. The ratio of acidic to basic amino acids (1.26) strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding to zinc. The findings of the present study confirms that 40 kDa major zinc binding glycoprotein purified from renal BBM is a zinc transporter involved in the influx of Zn(2+) into the epithelial cells of the renal tubular system.     

Comparative study of some intestinal brush border membrane enzymes during perinatal development of the mouse

Intestinal brush border membrane (bbm) fractions have been isolated from fetal and neonatal mice. The existence of discordant developmental patterns of intestinal enzymatic activity derived from total homogenate and bbm fraction was confirmed. It originates chiefly from two phenomena: (a) variations in the state of purity of brush border fractions, and (b) loss of brush border membrane enzyme activities in supernatant that increases with age. The phenomenon of solubility for glucoamylase and alkaline phosphatase is already present two days before birth.     

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

A retinyl ester hydrolase activity intrinsic to the brush border membrane of rat small intestine.

Retinol esterified with long-chain fatty acids is a common dietary source of vitamin A. Hydrolysis of these esters in the lumen of the small intestine is required prior to absorption. Bile salt-stimulated retinyl esterase activity was present with purified rat intestinal brush border membrane, with the maximum rate of ester hydrolysis at approximately pH 8, the physiological luminal pH. Taurocholate, a trihydroxy bile salt, stimulated hydrolysis of short-chain fatty acyl retinyl esters more than hydrolysis of long-chain fatty acyl esters. Deoxycholate, a dihydroxy bile salt, primarily stimulated hydrolysis of long-chain esters. Calculated Kms of 0.74 microM for retinyl palmitate (16:0) hydrolysis and 9.6 microM for retinyl caproate (6:0) hydrolysis suggested the presence of two separate activities. Consistent with that, the activity responsible for retinyl caproate hydrolysis could be inactivated to a greater degree than retinyl palmitate hydrolysis by preincubation of the brush border membrane at 37 degrees C for extended times. Brush border membrane from animals who had undergone common duct ligation 48 h prior to tissue collection showed little ability to hydrolyze retinyl caproate but retained 70% of retinyl palmitate hydrolytic activity, compared to sham-operated controls. Thus, two distinguishable retinyl esterase activities were recovered with purified brush border membranes. One apparently originated from the pancreas, was stimulated by trihydroxy bile salts, and preferentially hydrolyzed short-chain retinyl esters, properties similar to cholesterol ester hydrolase, known to bind to the brush border. The other was intrinsic to the brush border, stimulated by both trihydroxy and dihydroxy bile salts, and preferentially hydrolyzed long-chain retinyl esters, providing the majority of activity of the brush border against dietary retinyl esters.     

Proteases of swine small intestine enterocytes. Kinetics of substrate hydrolysis and inhibition of aminopeptidase N from brush border vesicles

The kinetics of hydrolysis of L-leucine p-nitroanilide and some p-nitrophenylalanine dipeptides by vesicular aminopeptidase N from the porcine small intestine brush border membrane was studied. It was shown that the catalytic properties of the vesicular enzyme are very similar to those known for its solubilized counterpart. Both enzymes are inhibited by o-phenanthroline, ZnCl2 and puromycin with Ki = 10(-5)-10(-6) M. The data obtained offer new possibilities for investigating the role of aminopeptidase N in the amino acid and peptide transport across the enterocyte membrane.     

Kinetics of leucine transport in brush border membrane vesicles from lepidopteran larvae midgut.

The kinetics of K(+)-leucine cotransport in the midgut of lepidopteran larvae was investigated using brush border membrane vesicles. Initial rate (3 s) of leucine uptake was determined under experimental conditions similar to those occurring in vivo, i.e. in the presence of delta psi much greater than 0 (inside negative) and a delta pH of 1.4 units (7.4in/8.8out). Leucine and K+ bind to the carrier according to a sequential mechanism, and the binding of one substrate changed the dissociation constant for the other substrate by a factor of 0.15. Both trans-K+ and trans-leucine were mixed-type inhibitors of leucine uptake. Moreover, a portion of total leucine uptake was K+ independent, and it was competitively inhibited by trans-leucine. We interpret the trans inhibitory effects to mean that the partially loaded K+ only form is virtually unable to translocate across the membrane, whereas the binary complex carrier, leucine, can isomerize from the trans to the cis side of the membrane. However, the K(+)-independent leucine uptake occurs with a Keq greater than 1, i.e. the efflux route through the partially loaded leucine only form is slower than the rate of isomerization of the unloaded carrier from trans to cis side. Taken together, these results suggest a model in which transport occurs by an iso-random Bi Bi system. Since K+ does not act as a pure competitive activator, this model is different from that proposed for most of the Na(+)-linked solutes transport agencies and may be related to the broadening of the cation specificity of the amino acid transporters in lepidopteran larvae.