Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

Isolation ofn-ethylmaleimide-labelled phlorizin-sensitived-glucose binding protein of brush border membrane from rat kidney cortex

A glucose receptor with high affinity for phlorizin from isolated brush border of rat kidney was labelled specifically withN-[¹⁴C]ethylmaleimide and then extracted from the membranes.After the solubilization of the brush borders with sodium dodecyl sulphate theN-[¹⁴C]ethylmaleimide-labelled receptor protein was isolated and was found to have a molecular weight of approximately 30 000 as determined by sodium dodecyl sulphate-polyacrylamide gel disc electrophoresis. The receptor protein eluted from the sodium dodecyl sulphate-containing gels migrates as a single band on sodium dodecyl sulphate-free polyacrylamide gels.The receptor protein can also be released from the brush borders with low concentrations of sodium deoxycholate. Under these conditions the molecular weight of theN-[¹⁴C]ethylmaleimide-labelled receptor protein is approximately 60 000 in contrast to the protein component solubilized with sodium dodecyl sulphate. Since this detergent is known to dissociate the brush border membrane into its protein components, our results suggest that the phlorizin- sensitive glucose receptor protein has a molecular weight of about 30 000.     

Labelling of intestinal brush border membrane proteins in vivo using diazotised [125I]iodosulfanilic acid

Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [¹²⁵I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [¹²⁵I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × $\text{g}$ from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins.     

Effect of massive proximal small bowel resection on intestinal brush border membrane proteins in the dog.

Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.     

Thyroxine-stimulated synthesis of microvillus membrane glycoproteins during culture of chick embryonic duodenum

The effect of thyroxine on biosynthesis of microvillus membrane glycoproteins has been investigated in organ culture of 18-day-old chick embryonic duodenum. Explants incorporate [³H]leucine and [³H]glucosamine continuously, and overall incorporation is enhanced by 10 nM thyroxine during 48 h of labeling; this increase in radioactivity is associated with vesicles released from the microvilli. Light microscope autoradiography, pulse labeling of brush border fragments, and pulse chase experiments reveal that [³H]glucosamine is incorporated into brush border at an increasing rate during culture, and that newly synthesized glycoproteins are discharged into the medium along with brush border enzymes (alkaline phosphatase and maltase). These results suggest that thyroxine stimulates biosynthesis of microvillus membrane glycoproteins, in addition to stimulating vesiculation of the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ³H-labeled vesicles and brush border fragments show that [³H]leucine and [³H]glucosamine are incorporated into proteins of high molecular weight. Two protein bands are identified as alkaline phosphatase and maltase. Thyroxine stimulates glycosylation of these enzymes, but does not change protein patterns. Radioactivity assay of alkaline phosphatase- and maltase-active gel slices suggests that thyroxine stimulation of these enzyme activities during culture is not correlated with de novo synthesis of these proteins.     

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.     

Identification of a 50,000 Mr protein from rabbit brush border membranes that binds Clostridium perfringens enterotoxin

A protein that binds Clostridium perfringens enterotoxin was extracted with NP-40 from rabbit intestinal brush border membranes. This protein was partially purified by affinity chromatography on enterotoxin-coupled CNBr-activated Sepharose 4B. The molecular weight of this protein was approximately 50,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity-purified samples containing this protein specifically inhibited biological activity of the enterotoxin on Vero (African green monkey kidney) cells. These studies suggest that this protein may be involved in the binding of the enterotoxin to rabbit intestinal epithelial cells.     

Biochemical characteristics of the outer membranes of plant mitochondria.

Like the outer membranes of liver mitochondria, those of plant mitochondria are impermeable to cytochrome c when intact and can be ruptured by osmotic shock. Isolated plant outer mitochondrial membranes are also similar to the corresponding liver membranes in terms of phospholipid and sterol content. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis experiments indicate that a single class of proteins (apparent molecular weight 30 000) comprises the bulk of the plant outer membrane protein. There are also considerable amounts of polysaccharide associated with these membranes, which may contribute to their osmotic stability.     

Purification of microvilli and an analysis of the protein components of the microfilament core bundle

A fast and convenient method for the purification of microvilli from chicken intestinal brush borders is described. The microvilli appear morphologically very similar to those found on intact brush borders. Removal of the microvillus membrane from the microvilli by Triton X-100 treatment reveals compact bundles of microfilaments with small regularly spaced projections along their length. SDS-polyacrylamide gel analysis of the protein components of the brush border, the microvilli and the microvillus core bundles shows that little or no tropomyosin, myosin or filamin is found in the microvillus, whereas polypeptide chains with mobilities characteristic for these proteins are present in the whole brush border. The majority of the microvillus core protein is actin, and the other major protein present has a polypeptide molecular weight of 95 000. Total actin from both brush borders and microvilli, characterized by isoelectric focussing analysis, contained about 40% β actin and 60% γ actin. The presence of both the β and γ cytoplasmic actins in the highly ordered parallel arrays of microfilaments of the microvilli is discussed in light of hypotheses for different functional roles of these two actin species.     

Histone H1 proteins act as receptors for the 987P fimbriae of enterotoxigenic Escherichia coli

The tip adhesin FasG of the 987P fimbriae of enterotoxigenic Escherichia coli mediates two distinct adhesive interactions with brush border molecules of the intestinal epithelial cells of neonatal piglets. First, FasG attaches strongly to sulfatide with hydroxylated fatty acyl chains. This interaction involves lysine 117 and other lysine residues of FasG. Second, FasG recognizes specific intestinal brush border proteins that migrate on a sodium-dodecyl sulfate-polyacrylamide gel like a distinct set of 32-35-kDa proteins, as shown by ligand blotting assays. The protein sequence of high performance liquid chromatography-purified tryptic fragments of the major protein band matched sequences of human and murine histone H1 proteins. Porcine histone H1 proteins isolated from piglet intestinal epithelial cells demonstrated the same SDS-PAGE migration pattern and 987P binding properties as the 987P-specific protein receptors from porcine intestinal brush borders. Binding was dose-dependent and shown to be specific in adhesion inhibition and gel migration shift assays. Moreover, mapping of the histone H1 binding domain suggested that it is located in their lysine-rich C-terminal domains. Histone H1 molecules were visualized on the microvilli of intestinal epithelial cells by immunohistochemistry and electron microscopy. Taken together these results indicated that the intestinal protein receptors for 987P are histone H1 proteins. It is suggested that histones are released into the intestinal lumen by the high turnover of the intestinal epithelium. Their strong cationic properties can explain their association with the negatively charged brush border surfaces. There, the histone H1 molecules stabilize the sulfatide-fimbriae interaction by simultaneously binding to the membrane and to 987P.     

Solubilization of brush borders of hamster small intestine and fractionation of some of the components

About 90% of the protein of hamster intestinal brush borders was solubilised in 0.25% (w/v) sodium dodecyl sulphate without total loss of biological activity. Detergent-polyacrylamide gel electrophoresis of the solubilised protein separated 10–15 bands and partially resolved maltase, lactase, sucrase-maltase, trehalase and alkaline phosphatase activities. The disaccharidases, which were associated with the higher molecular weight proteins, were preferentially solubilised with 0.1%. (w/v) Triton X-100, butanol or papain, whereas Tris and NaI extracted only the lower molecular weight proteins, possible derived from the core filaments.Electrophoresis of brush border proteins metabolically labelled with [¹⁴C] glucosamine suggested that many of the membrane-bound enzymes are glycoproteins. However, chromatography of a papain digest on Sephadex G-200 showed that the sucrase-maltase complex can be separated nearly free of carbohydrate without total loss of activity.The importance of characterizing membrane proteins solubilised by a number of techniques is discussed.     

Identification and isolation of the binding substance for Clostridium perfringens enterotoxin on Vero cells

Abstract To identify the binding substance for Clostridium perfringens enterotoxin (CPE), the CPE-binding substances metabolically labelled with [³H]leucine on CPE-susceptible (Vero) and resistant (L-929) cells were analyzed by solubilization, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The CPE-binding substance was found on Vero cells, but not on L-929 cells. The molecular weight of the CPE-binding substance was found to be 60 000 on SDS-PAGE. The CPE-binding substances were isolated from Vero cells and Balb/c mouse intestinal brush border membranes by affinity chromatography on CPE-coupled Sepharose 4B. They were homogeneous substances with molecular weights of 60 000 on SDS-PAGE and inhibited to the same extent the binding reaction of ¹²⁵I-labeled CPE with Vero cells. These results suggests that the CPE-binding substances are the receptors of CPE on these cells.     

Characterization of bile acid binding to rat intestinal brush border membranes.

Studies were performed to characterize the binding1 of bile acids to intestinal brush border membranes. Total 14C-taurodeoxycholate binding was: 1) similar for brush borders prepared from jejunum and ileum, 2) linear with respect to monomer concentration, 3) uninhibited by a structural analog, and 4) not depressed by boiling or trypsin. A linear relationship existed between binding and the number of hydrogen bonds formed by a bile acid and the slope of the line corresponded to delta deltaF of 300 cal/mol. The binding of bile acids to the 105,000 x g supernatant fraction of sonicated brush borders was similar to the binding of phospholipid liposomes using gel chromatography. These data suggest that: 1) the kinetics and characteristics of binding of bile acid to ileal brush borders do not reflect the kinetics and characteristics of active ileal transport previously obtained in whole tissue preparations, but instead reflect the kinetics and characteristics of passive jejunal transport; 2) a determinant of binding is hydrogen bonding with water; 3) isolated intact brush borders are relatively polar membranes; and 4) binding to solubilized brush borders may represent partitioning between the aqueous phase and membrane lipid.     

Protein turnover in intestinal mucosal villus and crypt brush border membranes

Relative turnover rates of intestinal brush border proteins have been studied by double labelled technique. Brush borders were isolated from cells at all levels along the villus, and from the crypts. Proteins with large molecular weight (>150,000) demonstrated more rapid turnover compared with other brush border proteins at all levels along the villus. This rapid turnover was not seen in crypt brush borders. These findings support the concept of protein turnover in intestinal brush borders, and demonstrate differences between the proteins in rapidly growing crypt cells and non growing villus cells.     

The effect of vitamin D3 metabolites on membrane proteins of chick duodenal brush borders.

Chick intestinal brush border proteins were examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Following injection of 25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃, a large molecular weight protein present in the vitamin D-deficient brush borders diminishes and a larger protein appears. This change occurs before calcium binding protein can be detected by Chelex assay and prior to the increase in total alkaline phosphatase but correlates closely with increased intestinal calcium absorption in response to the metabolites. The two brush border proteins have been solubilized with n-butanol and partially characterized. The vitamin D-deficient protein has a molecular weight of about 200,000 and has alkaline phosphatase activity but no detectable calcium binding activity. The protein which appears in response to metabolites has a molecular weight of 230,000, binds calcium, and also has alkaline phosphatase activity.     

Intramolecular crosslinking of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (rat kidney) is a heterodimeric glycoprotein (subunit molecular weights 52,000 and 25,000). In addition to its single-chain biosynthetic precursor (Mr 78,000), glycosylated high molecular weight forms (Mr 85,000-95,000) have been reported in various rat tissues as well as during in vitro translation of its mRNA. Studies reported here suggest that these might be attributed to the anomalous behavior of intramolecularly crosslinked species. Thus, chemical crosslinking of the purified enzyme (as well as enzyme on the renal brush border membranes) by bifunctional reagents such as dimethyl suberimidate and by an active site-directed reagent, diazotized p-amino-hippurate, produces stable heterodimers which exhibit molecular weights identical to that of the native enzyme when subjected to gel filtration. However, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crosslinked species exhibit apparent Mr values of 85,000 to 110,000, depending upon the crosslinking agent used. Protein glycosylation alone does not account for such anomalous electrophoretic behavior; the extent and the regions of the enzyme involved in formation of crosslinks appear to exert considerable constraints upon their conformation even in denaturing media.     

Identification and characterization of two huge protein components of the brush border cytoskeleton: evidence for a cellular isoform of titin

Two extremely high molecular weight proteins were found to be components of the intestinal epithelial cell brush border cytoskeleton. The largest brush border protein, designated T-protein, migrated on SDS gels as a doublet of polypeptides with molecular weights similar to muscle titin T I and T II. The other large brush border protein, designated N-protein, was found to have a polypeptide molecular weight similar to muscle nebulin. In Western analysis, a polyclonal antibody raised against brush border T-protein reacted specifically with T-protein in isolated brush borders and cross-reacted with titin in pectoralis and cardiac muscle samples. T-protein was distinguished from the muscle titins by an anti-cardiac titin mAb. A polyclonal antibody raised against N-protein was specific for N-protein in brush borders and cross-reacted with nothing in pectoralis muscle. Immunolocalization in cryosections of intestinal epithelia and SDS-PAGE analysis of fractionated brush borders revealed that both T-protein and N-protein are concentrated distinctly in the brush border terminal web region subjacent to the microvilli, but absent from the microvilli. EM of rotary-replicated T-protein samples revealed many of the molecules to be long (912 +/- 40 nm) and fibrous with a globular head on one end. In some of the molecules, the head domain appeared to be extended in a fibrous conformation yielding T-protein up to 1,700-nm long. The brush border N-protein was found as long polymers with a repeating structural unit of approximately 450 nm. Our findings indicate that brush border T-protein is a cellular isoform of titin and suggest that both T-protein and N-protein play structural roles in the brush border terminal web.     

Isolation and characterization of a small intestinal surfactant-like particle containing alkaline phosphatase and other digestive enzymes

Digestive brush-border enzymes in particulate form have been reported in the intestinal lumen in vivo and in medium from organ explants in vitro. It has been suggested that these particles derive from membrane shedding of the apical brush border. This study describes the isolation and characterization of particles derived from the 105,000 x g supernatant fraction of intestinal luminal washings and from light scrapings of the mucosa itself after fat feeding of rats. These fractions were separated in a continuous NaBr gradient, producing a visible band of 1.07-1.08 g/liter density and resulting in a 15-fold enrichment of intestinal alkaline phosphatase in the band fraction. Other brush-border hydrolases were represented in the banded fraction, but at specific activities only 1/5th to 1/36th that of the brush border. The major phospholipid in the fraction was phosphatidylcholine (58 +/- 15%), containing 75% saturated fatty acids. In contrast, the major brush-border phospholipid was phosphatidyl-ethanolamine. These characteristics showed that the particles derived from the lumen and mucosal surface were not identical to fragments of the brush border. Electron microscopy of the banded fraction revealed partially coiled membrane fragments. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, some proteins (e.g. surfactant protein B, collagenous protein 4) were found in common between the intestinal particles and rat pulmonary surfactant. These data suggest the production of a particle secreted by rat intestine that differs from brush-border membranes and that shares some morphological and biochemical similarities with pulmonary surfactant.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 2. Stabilization of an active conformation

Conformation-dependent fluorescein isothiocyanate (FITC) labeling of the pig renal Na+/glucose symporter was investigated with specific monoclonal antibodies (MAb's). When renal brush border membranes were pretreated with phenyl isothiocyanate (PITC), washed, and then treated at neutral pH with FITC in the presence of transporter substrates Na+ and glucose, most of the incorporated fluorescence was associated with a single peak after resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular mass of the FITC-labeled species ranged from 79 to 92 kDa. Labeling of this peak was specifically reduced by 70% if Na+ and glucose were omitted. Na+ could not be replaced by K+, Rb+, or Li+. FITC labeling of this peak was also stimulated after incubation of membranes with MAb's known to influence high-affinity phlorizin binding, and stimulation was synergistically increased when MAb's were added in the presence of Na+ and glucose. Substrate-induced or MAb-induced labeling correlated with inactivation of Na+-dependent phlorizin binding. MAb's recognized an antigen of 75 kDa in the native membranes whereas substrate-induced FITC labeling was accompanied by loss of antigen recognition and protection from proteolysis. These findings are consistent with a model in which MAb's stabilize a Na+-induced active conformer of the Na+/glucose symport system.