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D J Benos

Publications and source records attributed to D J Benos.

At least 145 records · Page 8Linked to original sources
PubMed1993

Single-channel characteristics of a purified bovine renal amiloride-sensitive Na+ channel in planar lipid bilayers.

We have purified an amiloride-inhibitable Na+ channel protein from bovine renal papillae using ion-exchange and immunoaffinity chromatography. In the present study, these purified Na+ channels were reconstituted into planar lipid bilayers, and their single-channel characteristics were studied. We observed both large- and small-conductance Na(+)-selective ion channels in planar lipid bilayers. Single-channel conductance for the large- and small-conductance channels saturated as a function of Na+ concentration. These relations could be fitted by a simple Langmuir isotherm with a Michaelis constant of 55 and 45 mM and a maximum open-state conductance of 56 or 8.4 pS, respectively. Both channels were perfectly cation selective, with a Na(+)-to-K+ permeability ratio of 6.7:1 for the large channel and 7.8:1 for the small channel, and their open single-channel current-voltage relations were linear when bathed with symmetrical Na+ solutions. The percent open time of the reconstituted large or small channels varied between 10 and 50% or 1 and 20%, respectively. After application of amiloride, both the large- and small-conductance Na+ channels were inhibited in a dose-dependent manner.

Amiloride↗
PubMed1993

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Duplicate Publications as Topic↗
PubMed1993

Regulation by phosphorylation of purified epithelial Na+ channels in planar lipid bilayers.

To determine the mechanism by which vasopressin increases apical membrane Na+ entry, we evaluated whether or not this hormone could recruit Na+ channels from a subapical membrane pool using specific polyclonal antibodies raised against high amiloride affinity bovine renal papillary Na+ channels. We also studied the effect of protein kinase A (PKA)-mediated phosphorylation on single-channel activity of highly purified Na+ channels incorporated into planar lipid bilayer membranes. PKA induced a significant increase in open-channel probability (Po) with no change in single-channel conductance. As shown previously and reconfirmed in the present work, PKA catalyzed the phosphorylation of a single subunit of this Na+ channel protein, namely, a 300-kDa polypeptide. On the other hand, protein kinase C, in combination with diacylglycerol, Ca2+, and phosphatidylserine, phosphorylated both the 130- and 55-kDa subunits of this purified Na+ channel, with a concomitant decrease in Po of both untreated and previously PKA-treated channels. We also found, in expression studies conducted in confluent monolayers of amphibian renal A6 cells, that vasopressin did not induce the apical insertion of new channel proteins. These observations support the hypothesis that vasopressin increases the apical Na+ permeability by activating Na+ channels already resident in the apical membrane by a direct phosphorylation mechanism rather than by cytoplasmic recruitment of latent Na+ channels.

Animals↗
PubMed1993

Culture-induced alterations in alveolar type II cell Na+ conductance.

Changes in Na+ transport in rat alveolar type II (ATII) cells during culture were quantified and related to alterations in spatial distribution of proteins antigenically related to amiloride-sensitive Na+ channels. Adult rat ATII cells were cultured for periods ranging from 24 to 96 h. When patch clamped in the whole cell mode, both freshly isolated and cultured ATII cells exhibited outwardly rectified Na+ currents. At 0 and 24 h in culture, these currents were equally inhibited by amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (inhibitory constant approximately 1 microM). These conductive pathways were equally permeable to Na+ and K+. Immunocytochemical localization at 0 or 24 h in culture revealed the presence of plasma membrane antigenic sites; after 48 h, the appearance of intracellular antigenic sites increased significantly. A single band of molecular mass 135 kDa in membrane proteins of freshly isolated ATII cells was recognized in Western blots; at 48 h in culture, two lower bands with molecular masses of 75 and 65 kDa were detected in either membrane or cytoplasmic proteins. Photolabeling with 2'-methoxy-5'-nitrobenzamil showed that the 135-, 75-, and 65-kDa bands contained amiloride-binding sites. These results suggest the presence of low amiloride affinity conductive pathways in freshly isolated and cultured ATII cells. Culturing ATII cells resulted in internalization and possible breakdown of these pathways and decreased Na+ transport.

Amiloride↗
PubMed1993

Glycosylation status of endogenous CFTR does not affect cAMP-stimulated Cl- secretion in epithelial cells.

Cystic fibrosis (CF) impairs Cl- secretion across epithelial tissues and is caused by mutations in an N-linked glycoprotein, the cystic fibrosis transmembrane conductance regulator (CFTR). We modified the glycosylation pattern of CFTR using inhibitors of oligosaccharide processing and determined their effects on both agonist-induced Cl- secretion and CFTR location in human colon (HT-29) cell lines. In both polarized and unpolarized HT-29 cells, immunoprecipitation of cell extracts using a monoclonal antibody against CFTR gave a single band at 170 kDa. Inhibitors of N-linked glycosylation reduced the molecular mass of this band: swainsonine by 10 kDa, deoxymannojirimycin by 30 kDa, and deoxynojirimycin by 10-20 kDa. However, the transepithelial Cl- current and conductance stimulated by adenosine 3',5'-cyclic monophosphate (cAMP)- or Ca(2+)-dependent secretagogues was not affected. In the polarized cells, CFTR was localized in the apical membrane domain. Treatment of the monolayers with glycoprocessing inhibitors did not affect CFTR's location. Thus, in human colonocytes that endogenously express CFTR, the extent of CFTR glycosylation does not influence the targeting of CFTR to the apical membrane domain or its function as an agonist-stimulated Cl- channel.

Cell Line↗
PubMed1993

Zn(II) transport and distribution in rat spermatids.

Zn(II) is an essential trace element. In spermatozoa, Zn(II) modulates metabolism and chromatin condensation. The mechanisms of uptake and distribution of this ion in sperm cells have not been explored. In rat spermatids, our results indicate that 1) 65Zn(II) binds with fast kinetics to a labile, presumably extracellular, compartment. This binding is temperature insensitive and not modified by metabolic inhibitors. 2) Entry of 65Zn(II) in the absence of externally added proteins occurs through a mediated transport system that allows exchange to reach steady state in approximately 15 min at 34 degrees C. 3) Upon entering the cells, 65Zn(II) binds tightly to cellular organelles. 4) Exchangeable Zn(II) bound to cytoplasmic proteins plus free intracellular Zn(II) appears to be < 20% of total exchangeable Zn(II). 5) The intracellular exchangeable Zn(II) compartment is decreased by metabolic inhibitors, showing a direct or indirect link between energy metabolism and cellular Zn(II) levels. 6) 65Zn(II) efflux from rat spermatids is a process with a rate constant of 0.16 +/- 0.13 min-1 at 34 degrees C. This exit rate of Zn(II) is likely to be affected by Zn(II) release from cytoplasmic binding sites or organelles.

Animals↗
PubMed1993

Fetal lung epithelial cells contain two populations of amiloride-sensitive Na+ channels.

Active Na+ transport by the alveolar epithelium plays a major role in reabsorption of the fetal lung fluid after birth. We characterized the biochemical and physiological characteristics of Na+ conductive pathways in distal fetal lung epithelial (FLE) cells isolated from 20-day-old rat fetuses. We demonstrated that a polyclonal antibody to Na+ channel protein (NaAb) binds to the plasma membranes of FLE cells. In Western blot studies, this NaAb and an anti-idiotypic monoclonal antibody to the amiloride-binding subunit of the Na+ channel protein recognized 150- and 90-kDa polypeptides in plasma membrane vesicles of FLE. 22Na+ flux measurements across plasma membrane vesicles of FLE revealed the existence of electrogenic Na+ transport, which was twice as high as the corresponding adult value. One hundred micromolars of amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride inhibited 30, 40, and 70% of the electrogenic Na+ transport across plasma membrane vesicles of FLE cells, respectively. The half-maximum inhibition of electrogenic Na+ transport by these substances occurred between 0.3 and 1 microM. [3H]benzamil equilibrium binding studies in membrane vesicles of FLE cells revealed the existence of two binding sites that had dissociation constant values of 19 and 1,525 nM, respectively. These data indicate the presence of both high- and low-amiloride affinity Na+ conductive pathways (channels) in FLE cells.

Amiloride↗
PubMed1993

Transport defects of rabbit inner medullary collecting duct cells in obstructive nephropathy.

Urinary obstruction markedly reduces collecting duct Na+ reabsorption. To define the cellular mechanisms of this derangement in Na+ reabsorption in inner medullary collecting duct (IMCD) of obstructed kidneys, suspensions of intact IMCD cells and inner medulla plasma membranes (IMPM) were prepared from 24 h obstructed and untreated control kidneys. Oxygen consumption (QO2) studies revealed marked reductions in both amiloride-sensitive and ouabain-sensitive QO2 but not ouabain-insensitive QO2 in intact IMCD cells from obstructed, compared with control animals, indicating a reduction in oxygen-dependent transport activities of both the Na+ channel and the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Amiloride-sensitive conductive 22Na+ uptake in intact IMCD cells from obstructed kidneys was significantly decreased by 45% at 10 s, 30 s, and 1-5 min (10 s: 2.42 +/- 0.63 vs. 4.49 +/- 0.64 nmol Na+ flux/mg protein, n = 7, P < 0.05; 1 min: 4.65 +/- 0.7 vs. 8.27 +/- 0.98 nmol Na+ flux/mg protein, n = 7, P < 0.05), indicating decreased activity of amiloride-sensitive Na+ channels in these cells. However, immunoblots of IMPM with antibodies to Na+ channel proteins did not show significant differences in content of Na+ channel proteins between membranes from obstructed and control groups. Ouabain-sensitive Na(+)-K(+)-ATPase activity in IMPM of obstructed kidneys was also reduced (61.1 +/- 18.1 vs. 152.6 +/- 25.8 nmol ATP degradation.min-1.mg protein-1, n = 6, P < 0.02), and immunoblots with monoclonal antibodies against the alpha 1- and beta-subunits of rabbit Na(+)-K(+)-ATPase showed a 51 +/- 7% reduction of both subunits in IMPM from obstructed kidneys (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Functional reconstitution of a chloride channel protein from bovine trachea.

We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Biochemical evidence for the presence of an amiloride binding protein in adult alveolar type II pneumocytes.

An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.

Affinity Labels↗
PubMed1992

Putative immunolocalization of the mechanoelectrical transduction channels in mammalian cochlear hair cells.

Hair cells bear an apical bundle of stereocilia arranged in serried rows. Deflection of the bundle controls the opening and closing of mechanoelectrical transduction channels, thereby altering the conductance across the apical plasma membrane. Two locations for these channels have been proposed in the bundle, either near the bases of the stereocilia or towards their tips. One hypothesis that is consistent with the latter possibility suggests that fine extracellular filaments, which run between the tips of the shorter stereocilia and the sides of the taller stereocilia behind, operate the channels. Determining the precise position of the channels is essential to test this hypothesis. We have therefore attempted to localize them immunocytochemically. Because hair-cell transduction is amiloride sensitive, the channels may have an amiloride-binding site associated with them. We have therefore used a polyclonal antibody raised against another amiloride-sensitive ion channel to hunt for them. This antibody recognizes a 62-64 kDa band in immunoblots of cochlear tissue, and produces discrete labelling in the hair bundle. This is most concentrated just below the tips of the shorter stereocilia, coinciding with a region of specialization in the closely apposed membranes of the short and tall stereocilia but not with either end of the tip link.

Amiloride↗
PubMed1992

Cellular differentiation is required for cAMP but not Ca(2+)-dependent Cl- secretion in colonic epithelial cells expressing high levels of cystic fibrosis transmembrane conductance regulator.

The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored. Thus, the protein encoded by this gene, designated "cystic fibrosis transmembrane conductance regulator" (CFTR), somehow participates in the Cl- secretory response. In this paper we have correlated CFTR gene expression with cAMP and Ca(2+)-dependent Cl- secretion in unpolarized (parental) and polarized (Cl.19A) clones of the human colonic adenocarcinoma cell line HT-29. These cell lines were found to express equally high levels of CFTR mRNA at 4 days post-passage. In addition, protein expression (determined by immunoprecipitation) was also identical. The cAMP-generating agonist forskolin had little effect on 125I efflux from the unpolarized cells. In contrast, this agonist increased 125I efflux 3-fold in polarized cells. The lack of response in the unpolarized cells was not due to the inability of forskolin to raise cAMP levels. Neurotensin, a Ca(2+)-mobilizing agonist, stimulated 125I efflux from both cell lines. In the polarized cells, the magnitude of this response was attenuated at 8 days post-seeding. At this time, the undifferentiated line attained some cAMP responsiveness. This latter effect was paralleled by the appearance of monolayers within areas of the multicell layer. Cell-attached patch-clamp recording from apical membrane patches of polarized cells revealed the presence of a forskolin-stimulated 8-pS Cl- channel; no channel activity was observed in forskolin-stimulated unpolarized cells. Ca(2+)-activated Cl- channels were found in both cell lines. In agreement with the 125I efflux data, the single-channel activation response to [Ca2+]i was smaller in the polarized cell line. From these studies, we can conclude that CFTR expression, measured both at the mRNA and protein level, does not correlate with the colonocyte's ability to secrete chloride ions in response to a cAMP-generating agonist. Cyclic AMP-dependent Cl- secretion requires cellular polarization; specifically, the delineation of an apical membrane. Differences in the cellular location of CFTR during differentiation are likely to explain our results. In contrast, Ca(2+)-stimulated Cl- secretion occurred independently of cellular polarization but was reduced when the cells formed tight junctions.

Adenocarcinoma↗
PubMed1992

Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive G alpha i-3 protein.

We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the alpha i-3 subunit of the Gi-3 protein. We also showed that a 700-kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the alpha i-3 subunit was a member of this Na+ channel complex. G alpha i structure and function were identified by Western blotting with specific G alpha i subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and G alpha i proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the alpha i-3 subunit of the Gi-3 protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize G alpha i-3 on Western blots of Golgi membranes which contain alpha i-3 but not Na+ channel proteins, nor do they immunoprecipitate alpha i-3 from solubilized Golgi membranes; however, alpha i-3 is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both alpha i-3 and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, alpha i-3, but not alpha i-2, stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory alpha i-3 protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation.

Adenosine Diphosphate Ribose↗
PubMed1992

Immunopurification and structural analysis of a putative epithelial Cl- channel protein isolated from bovine trachea.

We have purified to homogeneity a 38-kDa protein (called p38) from bovine tracheal epithelium. This protein, when reconstituted into liposomes, mediates stilbene disulfonate-sensitive 125I- conductive uptake. On nonreduced or partially reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this protein associates into a doublet of 62-64 kDa. In some experiments a multimer of 141 kDa was also observed. Rabbit polyclonal anti-P38 antibodies have been produced and used to immunopurify the native transporter. Upon reconstitution of the immunoaffinity-purified protein into liposomes, a 260-fold enhancement of 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate and valinomycin-sensitive 125I- uptake was observed as compared to proteoliposomes containing unseparated material. On Western blots of total solubilized tracheal membrane proteins or semipurified fractions, the antibody recognized the 62-64-kDa doublet much better than the original 38-kDa antigen. Similar protein bands were detected in T84 and CFPAC cells as well. However, if apical membrane proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, the antibody recognized major bands at 140 and approximately 240 kDa. Upon partial reduction, immunolabeling of these proteins diminished with the concomitant appearance of the 62-64-kDa doublet. Upon complete reduction, the appearance of 32- and 38-kDa proteins was evident with the disappearance of the 62-64-kDa doublet. We hypothesize that the native Cl-channel is a heteromer containing at least four subunits connected by S-S bridges.

Animals↗
PubMed1992

Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity.

High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.

Amiloride↗
PubMed1992

Identification of a membrane protein from T84 cells using antibodies made against a DIDS-binding peptide.

The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4'-diisothiostilbene-2,2'-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 1989]. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Antibodies against the cystic fibrosis transmembrane regulator.

Rabbit polyclonal antibodies have been raised against high-performance liquid chromatography purified synthetic peptides corresponding to two discrete regions of the cystic fibrosis transmembrane regulator (CFTR) protein: the R-domain (residues 785-796) and the extreme COOH-terminus (residues 1467-1480). Antibodies (Ab) to each of these peptides were affinity purified either by passage over a peptide-derivatized agarose matrix (Ab 785) to produce monospecific polyclonal antibodies or by protein A affinity chromatography to purify the immunoglobulin G1 fraction free of other serum proteins (Ab 1467). These antibodies recognize a candidate CFTR protein in the colonic cell line T84, as determined by Western blot analysis and by immunoprecipitation and labeling of the precipitate with [gamma-32P]ATP in the presence of protein kinase A. Both antibodies precipitated CFTR-related polypeptides from four mammalian cell types (HeLa, Bsc-40, HEp-2, and Chinese hamster ovary cells) transfected with the full-length CFTR cDNA clone using a vaccinia T7 protein expression system. Similar results were observed using a yeast CFTR expression system. In each case the Mr values of the bands observed were consistent with that expected for the CFTR protein. These antibodies should be useful probes for the immunocytochemical localization, immunoaffinity purification, and ultimately the functional characterization of the CFTR protein.

Animals↗