Intestinal absorption of dipeptides and beta-lactam antibiotics. II. Purification of the binding protein for dipeptides and beta-lactam antibiotics from rabbit small intestinal brush border membranes

By photoaffinity labeling of brush border membrane vesicles from rabbit small intestine with photoreactive derivatives of beta-lactam antibiotics and dipeptides, a binding protein for dipeptides and beta-lactam antibiotics with an apparent molecular weight of 127,000 was labeled. The labeled 127 kDa polypeptide could be solubilized with the non-ionic detergents Triton X-100, n-octyl glucoside or CHAPS. If the vesicles were solubilized prior to photoaffinity labeling, no clear incorporation of radioactivity into the 127 kDa polypeptide occurred indicating a loss of binding ability upon solubilization. By affinity chromatography of solubilized brush border membrane proteins on an agarose wheat germ lectin column, the binding protein for dipeptides and beta-lactam antibiotics of Mr 127,000 was retained on the column. With N-acetyl-D-glucosamine the photolabeled binding protein for beta-lactam antibiotics and dipeptides was eluted together with the brush border membrane-bound enzyme aminopeptidase N. Separation from aminopeptidase N and final purification was achieved by anion-exchange chromatography on DEAE-sephacel. Polyclonal antibodies against the purified binding protein were raised in guinea pigs. The photolabeled 127 kDa protein could be precipitated from solubilized brush border membranes with these antibodies. Incubation of brush border membrane vesicles with antiserum prior to photoaffinity labeling significantly reduced the extent of labeling of the 127 kDa protein. Treatment of brush border membrane vesicles with antiserum significantly inhibited the efflux of the alpha-aminocephalosporin cephalexin from the brush border membrane vesicles compared to vesicles treated with preimmune serum. These studies indicate that the binding protein for dipeptides and beta-lactam antibiotics of apparent molecular weight 127,000 in the brush border membrane of rabbit small intestinal enterocytes is directly involved in the uptake process of small peptides and orally active beta-lactam antibiotics across the enterocyte brush border membrane.     

Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco-2)

Escherichia coli heat stable enterotoxin (ST_a) and the newly identified endogenous ligand guanylin bind to an intestinal receptor and activate membrane bound guanylate cyclase. We compared ST_a binding and affinity crosslinking of ST_a receptors in human small intestine to those in the Caco-2 human colon carcinoma cell line. ST_a had similar kinetics of binding in human intestinal and Caco-2 brush border membranes. In both human intestine and Caco-2 brush border membranes, multiple specifically radiolabeled bands, including a 140–165 kDa band, were identified by affinity crosslinking. However, in human intestine the most prominent autoradiographic species was a 60 kDa band. A 60 kDa protein was also specifically immunoprecipitated from solubilized human brush border membranes using antisera raised against a cloned ST_a receptor fusion protein. Our observations of multiple crosslinked proteins in human intestine and Caco-2 cells could be explained by the existence of several members of a family of ST_a receptors and/or the existence of smaller ST_a binding proteins generated by the protease cleavage of a larger complete ST_a receptor. © 1993 Wiley-Liss, Inc.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N

A 120 kDa glycoprotein in the larval midgut membrane of the Iepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) δ-endotoxin, has been purified by a combination of protoxin affinity Chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin In the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.     

Intestinal receptors for adhesive fimbriae of enterotoxigenic Escherichia coli (ETEC) K88 in swine – a review

Determining the structure of the intestinal receptor for enterotoxigenic Escherichia coli (ETEC) K88 fimbriae will make it possible to develop new strategies to prevent K88+ ETEC-induced disease in pigs. Putative K88 adhesin receptors have been identified in both intestinal brush border and mucus preparations as either glycoproteins or glycolipids. Proteins with sizes of 25, 35, 40–42, 60, and 80 kDa in the intestinal mucus and 16, 23, 35, 40–70, 74, 210, and 240 kDa in brush border membranes were reported to bind specifically to K88ab and K88ac fimbriae. The factors accounting for these variable results may include the variants of K88, ages, breeds, and phenotypes of pigs, and even the sampling sites in the small intestine. Of the reported K88 receptors, only three brush border receptors, i.e., a pair of mucin-type sialoglycoproteins (210 kDa or 240 kDa), an intestinal neutral glycosphingolipid (IGLad), and a 74-kDa transferrin glycoprotein (GP74), have fulfilled the criteria as phenotype-specific K88 fimbrial receptors. Inhibiting the attachment of ETEC to intestine by modifying the receptor attachment sites has been the key for developing novel approaches to preventing ETEC-induced diarrhea in pigs. These include: (1) receptor analogs from a variety of biological sources, (2) an enteric protected protease, (3) chicken egg-yolk containing anti-K88 fimbrial antibodies, and (4) some Lactobacillus isolates producing proteinaceous components or carbohydrates interacting with mucus components. Future studies should be directed to further characterize the carbohydrate and protein moieties of receptors recognized by the K88 adhesin variants and to identify the genes responsible for susceptibility to K88+ infections. Received: 29 February 2000 / Received revision: 4 May 2000 / Accepted: 5 May 2000     

Identification, purification, and characterization of a stilbenedisulfonate binding glycoprotein from canine kidney brush border membranes. A candidate for a renal anion exchanger

Canine renal brush border membrane proteins that bind stilbenedisulfonate inhibitors of anion exchange were identified by affinity chromatography. A 130-kDa integral membrane glycoprotein from brush border membrane was shown to bind specifically to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate immobilized on Affi-Gel 102 resin. The bound protein could be eluted effectively with 1 mM 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS). The 130-kDa protein did not bind to the affinity resin in the presence of 1 mM BADS or when the solubilized extract was covalently labeled with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This protein was labeled with [3H]H2DIDS, and the labeling was prevented by BADS. The 130-kDa protein did not cross-react with antibody raised against human or dog erythrocyte Band 3 protein. The 130-kDa protein was accessible to proteinase K and chymotrypsin digestion in vesicles but not to trypsin. The 130-kDa protein was sensitive to endo-beta-N-acetylglucosaminidase F treatment both in the solubilized state and in brush border membrane vesicles showing that it was a glycoprotein and that the carbohydrate was on the exterior of the vesicles. This glycoprotein was resistant to endo-beta-N-acetylglucosaminidase H treatment suggesting a complex-type carbohydrate structure. The protein bound concanavalin A, wheat germ agglutinin, and Ricinus communis lectins, and it could be purified using wheat germ agglutinin-agarose.     

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.     

Binding of Bacillus thuringiensis Cry1Ac Toxin to Aminopeptidase in Susceptible and Resistant Diamondback Moths (Plutella xylostella)

Bacillus thuringiensis Cry1Ac toxin bound to a 120-kDa protein isolated from the brush border membranes of both susceptible and resistant larvae of Plutella xylostella, the diamondback moth. The 120-kDa protein was purified by Cry1Ac toxin affinity chromatography. Like Cry1Ac-binding aminopeptidase N (EC 3.4.11.2) from other insects, this protein was eluted from the affinity column with 200 mM N-acetylgalactosamine. The purified protein had aminopeptidase activity and bound Cry1Ac toxin on ligand blots. Purified aminopeptidase was recognized by antibodies to the cross-reacting determinant found on phosphatidylinositol-specific phospholipase C-solubilized proteins. The results show that the presence of Cry1Ac-binding aminopeptidase in the brush border membrane is not sufficient to confer susceptibility to Cry1Ac. Furthermore, the results do not support the hypothesis that resistance to Cry1Ac was caused by lack of a Cry1Ac-binding aminopeptidase.     

Purification of a putative Na+/D-glucose cotransporter from pig kidney brush border membranes on a phlorizin affinity column

Phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, was derivatised to 3-aminophlorizin and subsequently coupled to Affi-Gel 15. Affinity chromatography of pig kidney brush border membranes solubilised in Triton X-100 allowed the purification of a 60 kDa protein on this resin. We consider this protein to be the Na+/D-glucose cotransporter, or part of it, for the following reasons: (i) binding of this protein to Affi-Gel 15 specifically requires phlorizin covalently attached to the resin and is lowered when phlorizin is replaced by phloretin; (ii) binding of the 60 kDa protein to a phlorizin affinity column requires the presence of Na+; (iii) polyclonal as well as monoclonal antibodies against the 60 kDa protein inhibit binding of phlorizin to brush border membranes from rabbit and pig kidney.     

Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger

The aim of this study was to identify and purify the Na+-H+ exchanger from rabbit renal brush border membranes by use of affinity chromatography. Triton-solubilized membranes were equilibrated with an affinity matrix consisting of the amiloride analogue A35 (5-N-(3-aminophenyl)amiloride) covalently coupled to Sepharose CL-4B beads through a triglycine spacer arm. The matrix was then washed extensively with buffer and sequentially eluted with buffer, buffer containing 5 mM amiloride, and 1% sodium dodecyl sulfate (SDS). Eluates were concentrated and subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained gel revealed a 25-kDa protein that was not visible in the initial solubilized brush border membrane extract, was not eluted from the affinity matrix by buffer alone, but was eluted with 5 mM amiloride. A subsequent elution with 1% SDS did not release any more of the 25-kDa protein, indicating that it had been completely eluted from the affinity matrix by amiloride. The presence of 5 mM amiloride during equilibration of the solubilized brush border extract with the affinity matrix completely blocked adsorption of the 25-kDa protein. The relative abundance of this protein correlated closely with Na+-H+ exchange activity when preparations of cortical brush border membrane vesicles, outer medullary brush border membrane vesicles, and cortical basolateral membrane vesicles were compared. Moreover, binding of the protein to the affinity matrix was inhibited by amiloride and amiloride analogues with a rank order identical to that for inhibition of Na+-H+ exchange activity. These findings strongly suggest that the 25-kDa protein is a structural component of the Na+-H+ exchanger.     

Intestinal mucins: the binding sites forsalmonella typhimurium

Mucus-bacterial interactions in the gastrointestinal tract and their impact on subsequent enteric infections are poorly delineated. In the present study, we have examined the binding ofSalmonella typhimurium to rat intestinal mucus and characterized a mucus protein (Mucus-Rs) which specifically binds to S. typhimurium. Both virulent (1402/84), and avirulent (SF 1835) S. typhimurium were observed to bind to crude mucus, however, the virulent strain showed 6 fold more binding as compared to avirulent strain. Fractionation of crude mucus on sepharose CL-6B resolved it into three major peaks. Maximal bacterial binding was observed with a high mol. wt. glycoprotein corresponding to neutral mucin. SDS-PAGE of purified protein (termed Mucus-Rs) under non reducing conditions showed it to be a homogenous glycoprotein (mol. wt. 250 kDa), while under reducing conditions, three bands corresponding to mol. wt. of 118,75 and 60 kDa were observed. Pretreatment of Mucus-Rs with pronase, trypsin and sodium metaperiodate markedly inhibited bacterial binding. GLC analysis of Mucus-Rs showed it to contain Mannose, Glucose, Galactose, Glucosamine, Galactosamine and Sialic acid as main sugars. Competitive binding in the presence of various sugars and lectins indicated the involvement of mannose in the mucus-bacterial interactions. The Mucus-Rs binding was highly specific for S. typhimurium; no significant binding was seen with E.coliand V. cholerae. Thus, we conclude that S. typhimurium specifically binds to a 250 kDa neutral mucin of intestinal tract. This binding appears to occur via specific adhesin-receptor interactions involving bacterial pili and mannose of neutral mucin.     

Lithocholate binding by Y and Y' proteins in bovine small intestine.

Cytosolic proteins may play an important role in the intracellular transport of bile acids in enterocytes. The lithocholate binding properties of cytosolic protein from bovine small intestine were studied. Lithocholate binding was observed in the Y (45-50 kDa), Y' (30-35 kDa), and Z fractions (10-15 kDa) following gel filtration of cytosol. A Y protein with glutathione S-transferase activity (46 kDa) was purified by S-octyl-glutathione affinity chromatography and chromatofocusing (eluted at pH 7.5) of the Y fraction. Two Y' bile acid binding proteins with dihydrodiol dehydrogenase activity were partially purified from the Y' fraction by chromatofocusing and hydroxyapatite-HPLC. The lithocholate binding affinity of Y' protein (Kd < 0.35 microM) was higher than that of Y protein (Kd = 2 microM) and was comparable to that of Z protein (Kd = 0.2 microM). The binding affinity of Y protein was higher for bilirubin (Kd = 2.5 microM) than that for BSP (Kd = 200 microM). This was comparable to the binding affinity of bovine hepatic Y protein. These data indicate that Y' and Z proteins participate in the intracellular transport of bile acids from the brush border to the basolateral pole in enterocytes.     

Partial purification of Cry 1A toxin-binding proteins from Helicoverpa armigera larval midgut

Toxicity of insecticidal endotoxins produced by Bacillus thuringiensis correlates with the presence of specific proteins in the midgut of susceptible larvae. This study was aimed at identifying and purifying Cry 1A binding proteins from Helicoverpa armigera, an important crop pest of India. B. thuringiensis strain HD 73 which produces Cry 1Ac toxin, specific for H. armigera was used in this study. Toxin-binding proteins from insect larvae were detected by employing a toxin overlay assay using both radiolabelled as well as unlabelled toxin. Detergent-solubilized fractions of larval brush border membranes were subjected to soybean agglutinin (SBA) chromatography, from which N-acetylgalactosamine (NAG)-containing proteins were eluted. Analysis of the SBA-purified proteins indicated that four proteins of approximately 97, 120, 170 and 200 kDa could bind to Cry 1Ac toxin, and three proteins of 97, 170 and 200 kDa proteins could bind to Cry 1Ab. Furthermore, in the presence of excess Cry 1Ab toxin, the labelled Cry 1Ac toxin could bind only to 170 and 200 kDa proteins, implying that Cry 1Ab can also bind the 120 kDa protein. This study therefore demonstrates that in H. armigera, midgut proteins of 97, 120, 170 and 200 kDa have the ability to bind both Cry 1Ab and Cry 1Ac. Furthermore, while the 170 and 200 kDa proteins have higher affinity for Cry 1Ac, the 97 kDa has higher affinity for Cry1 Ab. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of intestinal and renal Na+-glucose cotransporter by naringenin

Reduction in glucose uptake constitutes a possible means of controlling diabetic hyperglycemia. Using purified intestinal brush border membrane vesicles and everted intestinal sleeves, we have demonstrated that naringenin, a flavonoid present in citrus fruits and juices, significantly inhibited glucose uptake in the intestine. In addition, naringenin also elicited inhibitory actions towards glucose uptake in renal brush border membrane vesicles. Naringin, a glycoside of naringenin, was totally inactive in these aspects. Naringenin exhibited moderate inhibitory action on glucose uptake in rabbit intestinal brush border membrane vesicles, and showed strong inhibitory action in rat everted intestinal sleeves. The IC(50) values were 205.9 and 2.4 micromol/l, respectively. Lineweaver-Burk analysis demonstrated that naringenin inhibited glucose uptake in rat everted intestinal sleeves in a competitive manner with a K(i) value of 1.1 micromol/l. Glucose uptake activities in both the intestinal and renal brush border membrane vesicles of diabetic rats were significantly higher than in normal rats. Naringenin (500 microM) reduced glucose uptake by more than 60% in both the intestinal and renal brush border membrane vesicles of diabetic rats to a level similar to that of the normal rats. The IC(50) values of naringenin in the renal brush border membrane vesicles of normal and diabetic rats were 323.9 and 166.1 micromol/l, respectively. These results suggest that inhibition of intestinal glucose uptake and renal glucose reabsorption explains, in part at least, the in vivo antihyperglycemic action of naringenin and its derivatives. The possible application of these natural compounds in controlling hyperglycemia warrants further investigations.     

Identification and characterization of Aedes aegypti aminopeptidase N as a putative receptor of Bacillus thuringiensis Cry11A toxin

Bacillus thuringiensis subsp. israelensis, which is used worldwide to control Aedes aegypti larvae, produces Cry11Aa and other toxins during sporulation. In this study, pull-down assays were performed using biotinylated Cry11Aa toxin and solubilized brush border membrane vesicles prepared from midguts of Aedes larvae. Three of the eluted proteins were identified as aminopeptidease N (APN), one of which was a 140 kDa protein, named AaeAPN1 (AAEL012778 in VectorBase). This protein localizes to the apical side of posterior midgut epithelial cells of larva. The full-length AaeAPN1 was cloned and expressed in Eschericia coli and in Sf21 cells. AaeAPN1 protein expressed in Sf21 cells was enzymatically active, had a GPI-anchor but did not bind Cry11Aa. A truncated AaeAPN1, however, binds Cry11Aa with high affinity, and also Cry11Ba but with lower affinity. BBMV but not Sf21 expressed AaeAPN1 can be detected by wheat germ agglutinin suggesting the native but Sf21 cell-expressed APN1 contains N-acetylglucosamine moieties.     

Identification and partial purification of K88ab Escherichia coli receptor proteins in porcine brush border membranes

Six receptor proteins, with molecular masses ranging from 94 to 27 kDa, that bind to Escherichia coli K88ab fimbriae were recovered from brush border membranes and were detected after solubilization with Triton X-114. The recovery of these receptor proteins in the aqueous phase suggests their peripheral localization. The 63-, 60- and 33-kDa K88ab binding proteins were recovered using gel-filtration chromatography of the aqueous phase. Electronic Publication     

Isolation and reconstitution of the intestinal Na+/glucose cotransporter

The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.     

Purification and characterization of a major zinc binding protein from renal brush border membrane of rat.

In spite of the fact that zinc is an essential trace element, mechanisms that contribute to zinc homeostasis in mammals are poorly understood. An attempt has been made to identify and purify zinc binding components from renal brush border membrane (BBM), which could be involved in the binding of zinc and the subsequent translocation across the BBM. A 40 kDa major zinc binding protein has been identified and purified from renal BBM, which showed a dissociation constant (Kd) of 211 microM and maximal binding (Bmax) of 207 nmol/mg protein. 8 g zinc atoms could interact with 1 mol of protein. Specificity of the protein for zinc was checked by metal displacement and UV-absorption assay. It was found that only Cd2+ could displace the zinc bound to the protein. Other metals tested (Cu2+, Mg2+, Ca2+) did not show any interaction with the protein. These data indicated that purified protein is highly specific and has a high affinity for zinc. The carbohydrate content was found to be 7.85 mg% in the purified protein. Immunofluorescence localization of this protein in kidney sections revealed that this major zinc binding protein is exclusively localized in the proximal convoluted tubules. These results suggested that the 40 kDa major zinc binding transmembrane glycoprotein is highly specific for zinc and has a high affinity for zinc.     

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.     

Different molecular sizes for Na(+)-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesicles

We compared several features of Na(+)-dependent phosphono[14C]formic acid (PFA) binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles. From kinetic analyses, we estimated an apparent Km for PFA binding of 0.86 mM, an order of magnitude greater than that for phosphate and the high-affinity phosphate transport system. A hyperbolic Na(+)-saturation curve for PFA binding and a sigmoidal Na(+)-saturation curve for phosphate transport were demonstrated; based on these data, we estimated stoichiometries of 1:1 for Na+/PFA and 2:1 for Na+/phosphate. By radiation inactivation analysis, target sizes for brush border membrane protein(s) mediating Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport corresponded to molecular masses of 555 +/- 32 kDa and 205 +/- 36 kDa, respectively. Similar analysis of the phosphate-inhibitable component of Na(+)-dependent PFA binding gave a target size of 130 +/- 28 kDa. We also demonstrated that phosphate deprivation, which elicits a 2.6-fold increase in brush border membrane Na(+)-dependent phosphate transport, had no effect on either Na(+)-dependent PFA binding or on the target size for PFA binding. However, phosphate deprivation appeared to increase the target size for phosphate transport (from 255 +/- 32 to 335 +/- 75 kDa (P less than 0.01]. In summary, we present evidence for several differences between Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles and suggest that PFA may not interact exclusively with the proteins mediating Na(+)-phosphate co-transport.