Brush Border Membrane Fraction Research Articles

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

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.

Identification and Characterization of Brush-Border Membrane-Bound Neutral Metalloendopeptidases From Rat Small Intestine

Neutral metalloendopeptidase enzymes were identified and partially characterized in the brush-border membranes of rat small intestinal mucosal cells using insulin B chain and glutaryl-trialanine-4-methoxy-beta-naphthylamide as substrates. Three different molecular species of endopeptidase were identified by disc gel electrophoresis. These enzymes were shown to be distinct from pancreatic endopeptidases on the basis of the following: enrichment in the brush-border membrane fraction, site of hydrolysis of peptide substrates, sensitivity to specific proteinase inhibitors, and the presence of brush-border membrane-associated endopeptidase activity in mucosal cells of Thirty-Vella loops. Hydrolysis of the substrates was shown to be a two-step process involving initial cleavage by endopeptidase with secondary hydrolysis of the peptide products by brush-border membrane aminopeptidase N. Hydrolysis of both substrates was maximum at a neutral pH and was strongly inhibited by metal chelating agents, phosphoramidone, and amastatin. Intestinal perfusion studies using glutaryl-trialanine-4-methoxy-beta-naphthylamide suggest that these enzymes play a physiologic role in protein digestion. It was concluded that neutral endopeptidases are integral components of the intestinal brush-border membrane and work in concert with aminopeptidase N to hydrolyze dietary protein. This process may be of nutritional importance in normal subjects and those with diminished exocrine pancreatic function.

Preparation of subcellular membranes from rat intestinal scrapings or isolated cells: Different Ca2+ binding, nonesterified fatty acid levels, and lipolytic activity

Basolateral, brush-border, and Golgi-enriched subcellular membrane fractions, prepared from homogenates of rat small intestinal mucosa obtained by scraping, had unusually high concentrations of nonesterified fatty acids. These fatty acids appear to be responsible for the large amount of calcium binding, an effect that previously was shown to be reduced in vitamin D deficiency. In contrast, basolateral and Golgi membranes prepared from isolated cells had low levels of nonesterified fatty acids and calcium binding. Intermediate levels were found with isolated cells that were not put through the usual washing procedures. Addition to homogenates of scrapings of a lipase inhibitor, diethyl-p-nitrophenyl phosphate, reduced calcium binding and nonesterified fatty acids to levels similar to those in membranes prepared from isolated cells. Phospholipase A activity was low in homogenates of isolated cells and high in scrapings; this was reduced in intestinal scrapings of vitamin D-deficient rats. Ileal membranes had more calcium binding than duodenal membranes, and ileal homogenates also had greater phospholipase A activity. Preparation of subcellular membranes from rat intestinal scrapirigs can result in altered lipid composition, probably due to lipolytic enzyme activity; in addition to increasing cation-binding, these high levels of fatty acids may affect other membrane properties and enzyme function.

Maintenance of epithelial surface membrane lipid polarity: a role for differing phospholipid translocation rates.

Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using 32P and either [3H]-glycerol or [2-3H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies, again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 X faster than into the brush border membrane and its relative composition was 2.5 X greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.

Subcellular fractionation and subcellular localization of aminopeptidase N in the rabbit enterocytes.

A fast and easy procedure is proposed for preparing concomitantly from the same sample of intestinal mucosa of A+ rabbits, four fractions high enriched in the brush-border and basolateral plasma membrane domains, rough endoplasmic reticulum, and smooth endoplasmic reticulum plus Golgi apparatus membranes, respectively. This is the first time the technique of flow fluorometry has been applied to characterize the brush-border and basolateral membrane fractions using polyclonal or monoclonal antibodies against antigens common to or specific for these two plasma membrane domains. This technique definitely proves the presence of aminopeptidase in at least 60% of the basolateral membrane vesicles, where its level is about 4.5% of that in the brush-border membrane vesicles. The endoglycosidase H-sensitive intermediate of glycosylation of aminopeptidase N in the steady state is accumulated in both the rough and smooth endoplasmic reticulum membranes. Although the rough membrane is more extensive it contains only about 40% of this transient form.

Distinction of three types of D-glucose transport systems in animal cells

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44–55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.

NAD-glycohydrolase in renal brush border membranes

NAD is hydrolyzed during incubation with isolated renal brush border membranes (BBM). The specific enzymatic mechanisms have not been identified apart from the activity of ADP-ribosyltransferase, which accounts for a very small proportion of the total hydrolysis. In the present study, an NAD-glycohydrolase (NGH) was identified in the renal BBM using the cyanide-addition assay to monitor hydrolysis of NAD at the nicotinamide-ribose bond. The production of nicotinamide and ADP-ribose, the expected reaction products, was determined by thin-layer chromatography. The NGH was enriched ninefold in the BBM fraction and accounted for 36% of the total rate of NAD hydrolysis by BBM enzymes at pH 7.4. Assay of NGH in sealed BBM vesicles subjected to osmotic shock indicated that about 23% of the NGH is exposed on the cytoplasmic surface of the BBM. The enzyme was inhibited by nicotinamide in vitro and also when the nicotinamide was administered in vivo, suggesting, indirectly, that the enzyme may play a role in mediating the effects of nicotinamide on BBM phosphate transport.

696 ESTABLISHMENT OF AN ANIMAL MODEL OF OVALBUMIN - SENSITIZED MOUSE TO STUDY PROTEIN INTOLERANCE IN VITRO

Protein intolerance, such as cow's milk allergy, represents a common cause of enteropathy in infancy. However, no reliable laboratory test for the diagnosis is presently available. An animal model of ovalbundn (OVA) - sensitized mouse has been established to study the response of the small intestinal mucosa in organ culture. A decrease of γ-glutamyltranspeptidase (γ-GT), alkaline phosphatase (AlPase), sucrase (S), lactase (L) and glucoamylase (GA) activities was observed in the explants (T) cultured during 24 hours in presence of OVA. In contrast, a large increase of these enzymatic activities was noted in the culture media (M), the overall effect observed beeing a net stimulation of the total enzymatic activities of the culture system. During the same period, a 20% decrease in 3H-thymidine incorporation into DNA was noted (P< .005). Results (X ± S.E.M.) from 12 experiments are expressed as % of activity with respect to autocontrols cultured in presence of β-lactoglobulin. The enzymes accumulated mainly in the particulate fraction of the culture medium (brush border membrane fraction) suggesting an increased turn-over of some membrane components by a process of shedding or microvesiculation. This animal model represents a useful tool to evaluate the modifications of the small bowel mucosa induced by an allergen and to establish the criteria for an in vitro diagnosis of protein intolerance. * Supported by the Medical Research Council of Canada, Grant MT-3320

Evidence for mitochondrial origin of the HCO3(-)-ATPase in brush border membranes of rat proximal tubules.

High HCO3(-)-ATPase activity is known to exist in mitochondria of renal tubular cells. In brush border membrane (BBM) preparations of proximal tubules such an anion-stimulated enzyme was also found. However, these preparations always contained mitochondrial markers. The putative localization and the role of this ATPase in BBM is still controversial. Some authors consider the HCO3(-)-ATPase in the BBM to be a mitochondrial contamination; others attribute to this ATPase a key role in H+ transport in the proximal tubule. To reinvestigate this problem, BBMs from rat kidney cortex were isolated by a simple, rapid (1.5-h) Ca2+-precipitation method, yielding a BBM fraction enriched 12.4-fold with respect to the marker enzyme leucine aminopeptidase (LAP). There was no basolateral Na+-K+-ATPase and no mitochondrial succinate dehydrogenase detectable. Cytochrome c oxidase was drastically reduced to 7 +/- 1% of that observed in the homogenate (TH). The activity of HCO3(-)-ATPase in the BBM fraction was 19 +/- 4 IU/g protein, i.e., 27% that of the homogenate. As sonication of the TH exclusively increases the activity of HCO3(-)-ATPase, its relative activity was 7.5% and thus equal to that of the mitochondrial marker. In many BBM preparations no HCO3(-)-ATPase was detectable. In those BBM preparations in which traces of HCO3(-)-ATPase were found, this activity coincided with that of cytochrome c oxidase in the respective preparation. There was a constant activity ratio of cytochrome c oxidase/HCO3(-)-ATPase in the TH, BBM, and pellet 1. The activity of HCO3(-)-ATPase in BBM did not depend on the activity of LAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosaminoglycans in brush border membranes from rabbit small intestine.

A brush border membrane fraction was prepared from mucosae of rabbit small intestine by differential centrifugation. The membrane fraction was digested with pronase and a glycosaminoglycan fraction was isolated from the digest by fractionation with cetylpyridinium chloride. Individual glycosaminoglycans were quantitated by specific mucopolysaccharide-degrading enzymes and nitrous acid. The glycosaminoglycan composition was then calculated to be as follows: Hyaluronic acid, 34%; chondroitin sulfate A/C, 16%; dermatan sulfate, 11%; heparan sulfate, 39%.

Maturation of Sucrase-Isomaltase Complex in Human Fetal Small and Large Intestine during Gestation

Sucrase-isomaltase complex is expressed in human small intestine throughout gestation and in the large intestine between 12 and 30 wk. The molecular form of the enzyme was studied in the brush-border membrane fractions by the immunoblotting method. Before 30 wk of gestation, the enzyme is present only as the high molecular weight prosucrase-isomaltase, while from 30 wk until birth the two subunits are also present. The fetal enzyme, as its proform and as its two subunits, has a faster mobility in sodium-dodecylsulfate polyacrylamide gel electrophoresis, than the adult enzyme (removal of sialic acid residues from fetal enzymes emphasizes this difference). The colonic and the small intestinal fetal enzymes are identical.

Distributions of post-proline cleaving enzyme-, converting enzyme-, trypsin- and chymotrypsin-like activities in various nephron segments and in brush-border membranes isolated from rat kidney.

Distributions of post-proline cleaving enzyme-, converting enzyme-, chymotrypsin- and trypsin-like activities, were investigated in nephron segments and in brush-border membrane fraction isolated from rat kidney. These activities were found to be higher in the proximal tubule than in other segments, and were hardly detectable in the cortical collecting tubule. In the proximal tubule, trypsin-like activity showed the highest value in the pars convoluta, while the other peptidases showed their highest activities in the pars recta. Post-proline cleaving enzyme- and trypsin-like activities were also high in the glomerulus. In addition, post-proline cleaving enzymeand converting enzyme-like activities were found in the brush-border membrane, while chymotrypsin- and trypsin-like activities were found in the cell component (s) other than the brush-border membrane. From these distributions of the peptidases, it is speculated that peptides are degraded both in the glomerulus and in the proximal tubule, and that in the proximal tubule the peptides are degraded both in the brush-border membrane and in other cell component (s).

Activities and subcellular localizations of enzymes implicated in gastroduodenal bicarbonate secretion.

Analytical subcellular fractionation of tissue whole homogenates and microanalysis of organelle marker enzymes were used to study the activity and subcellular localization of enzymes implicated in HCO3 secretion in rat duodenal and gastric antral mucosae. The following organelles, characterized by their marker enzymes, were located in the density gradients: cytosol (lactate dehydrogenase), plasma membrane (5'-nucleotidase), peroxisomes (catalase), mitochondria (succinate dehydrogenase), endoplasmic reticulum (Tris-resistant alpha-glucosidase), lysosomes (N-beta-acetylglucosaminidase), and brush-border membrane (Zn2+-resistant alpha-glucosidase and alkaline phosphatase). Compared with gastric antrum, rat duodenal mucosa contained over twice the activity of HCO3-ATPase and of Na+-K+-ATPase but less than one-tenth the activity of carbonic anhydrase. Duodenal HCO3-ATPase activity was observed in both mitochondrial and brush-border membrane fractions, whereas antral HCO3-ATPase activity was confined to mitochondria. Na+-K+-ATPase activity was found largely in the basolateral membrane (duodenum) and plasma membrane (antrum). In both tissues carbonic anhydrase activity was localized to the cytosolic fraction. These observations offer further evidence that differing biochemical mechanisms underlie HCO3 secretion by gastric and duodenal epithelia.

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

1. 1. Intestinal brush border membrane (bbm) fractions have been isolated from fetal and neonatal mice. 2. 2. The existence of discordant developmental patterns of intestinal enzymatic activity derived from total homogenate and bbm fraction was confirmed. 3. 3. 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. 4. 4. The phenomenon of solubility for glucoamylase and alkaline phosphatase is already present two days before birth.

Digestive and absorptive functions along dog small intestine: comparative distributions in relation to biochemical and morphological parameters

1. 1. The digestive (hydrolytic enzymes) and absorptive (sugar and amino acid transport) functions of dog small intestine have been evaluated in different segments and analysed in relation to morphometric and biochemical parameters. 2. 2. The dog small intestine is a cylinder of decreasing diameter in which the underlying mueosa thins down from duodenum to ileum, though maintaining its cellular homogeneity as revealed by measuring the mucosal weight, the total DNA and protein content and the protein content of the brush border membrane. 3. 3. Sucrase, gamma-glutamyltranspeptidase, leueylnaphthylamidase and alkaline phosphatase specific activities, measured both in homogenates of the mucosa and purified brush border membrane fractions, were found distributed along proximo-distal gradients of activity. However, different patterns were obtained which are specific for the enzyme considered. 4. 4. Kinetic parameters, V max and K m, were estimated for sucrase and alkaline phosphatase in purified brush border membrane fractions. It appeared that V max correlated well with the observed distribution of catalytic sites along the small intestine. 5. 5. Sugar (glucose) and amino acid (alanine and leucine) transport capacities were also distributed according to specific proximo-distal gradients but passive and facilitated diffusions were not affected. Only the active, Na +-dependent component of transport was sensitive to position along the small intestine and we postulated that this Actaptation should involve variations in carrier densities. 6. 6. It is therefore concluded that absorbo-digestive functions are intrinsic characteristics of the brush border membrane which are regulated according to the position along the small intestine.

Asymmetric distribution of the Na+/H+ antiporter in the renal proximal tubule epithelial cell.

The subcellular distribution of the Na+/H+ antiporter in renal proximal tubule cells was studied with differential and density gradient centrifugation. Enzyme markers for basolateral membranes [Na+/K+)-ATPase), brush border membranes (maltase), and a variety of intracellular organelles (NADPH cytochrome c reductase, thiamine pyrophosphatase, acid phosphatase, and succinate cytochrome c reductase) were simultaneously assayed in sucrose density gradients. Basolateral membranes (median rho = 1.150) were well separated from brush border membranes (median rho = 1.165) by this technique. Markers for other cellular organelles had intermediate or bimodal distributions. To determine the cellular location of the Na+/H+ antiporter, Na+-dependent collapse of preformed pH gradients was assayed in the sucrose density gradient fractions using acridine orange. Na+/H+ antiporter activity paralleled the distribution of the brush border membrane fractions; activity in the peak basolateral membrane fraction was less than 5% of that in the peak brush border fraction. To determine whether antiporter activity was potentially detectable in all cell fractions, nigericin was added to each fraction and K+/H+ exchange was assayed with acridine orange. Activity was present in all sucrose density gradient fractions. In addition, there was no alteration in Na+/H+ exchange activity measured in brush border membranes after mixing with cell sol or basolateral membranes, showing that neither inhibitors nor activators of the Na+/H+ antiporter were present in any of the cell fractions. These controls confirmed the finding that Na+/H+ antiporter activity was absent from basolateral membranes. The presence of the Na+/H+ antiporter in brush border membranes and its absence from basolateral membranes is consistent with its playing an important role in the vectorial transport of H+ from blood to tubular lumen in the renal proximal tubule.

Distribution and biosynthesis of aminopeptidase n and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N d dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidyl N of brush border mebrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidaseV had a more uniform distribution profile with relatively high in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cell. Both enzymes were primarily associated with brush border membranesin all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity / mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fol activation of precursor enzyme forms occurs during transfer from the Golgi membraanes to the brush border membranes.

Isolation of basolateral and brush-border membranes from the rabbit kidney cortex: Vesicle integrity and membrane sidedness of the basolateral fraction

A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na + + K +)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35–50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na + + K +)-ATPase activity and [ 3H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na + + K +)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na + and K +. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation.

Alterations in brush border membrane proteins and membrane-bound enzymes of the tapeworm, Hymenolepis diminuta, during development in the definitive host

During growth and maturation of the tapeworm, Hymenolepis diminuta, significant decreases occur in the brush border membrane-bound alkaline phosphatase, phosphodiesterase. 5′-nucleotidase, adenosine triphosphatase and ribonuclease activities. These decreases are accompanied by qualitative and quantitative changes in the polypeptide profiles of the brush border membrane fraction. Gradients of enzymatic activities and polypeptide profiles are also demonstrable when mature tapeworms are cut into pieces and the brush border membrane of each piece analyzed individually. In fully developed tapeworms the enzymatic activities and polypeptide profiles of membrane preparations reflect mainly the contributions of the more mature proglottids; these proglottids constitute most of the tapeworm biomass. The most anterior sections of these fully developed worms are biochemically similar to young, developing worms.

Nonpancreatic hydrolysis of N-benzoyl-L-tyrosyl-p-aminobenzoic acid (PABA peptide) in the rat small intestine.

Factors affecting the intestinal hydrolysis of orally administered N-benzoyl-L-tyrosyl-p-aminobenzoic acid (PABA peptide) and urinary recovery of p-aminobenzoic acid have been studied in rats, after diversion of the pancreatic and biliary secretions. The exclusion of pancreatic secretions from the small intestine lowered, but did not completely abolish, the urinary recovery of PABA, whereas diversion of the bile had no significant effect. A particulate-bound enzyme capable of splitting PABA peptide was found to be present throughout the rat small intestine, and its activity remained unchanged in the absence of biliopancreatic secretions. The intestinal PABA peptide hydrolase could be solubilized with papain and Triton from a partially purified brush-border membrane fraction, and it eluted in gel filtration as a high-molecular-weight peak, well separated from the other known peptidases of the intestinal brush-border membrane.