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PubMed1991

Photoaffinity labeling of soluble auxin-binding proteins.

The photoaffinity labeling agent azido-IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analogue of the endogenous auxin indole-3-acetic acid, was used to search for auxin-binding proteins in the soluble fraction of Hyoscyamus muticus cells. Azido-IAA became covalently attached to three polypeptides with a high specific activity. The labeling was specific for IAA and not due to random tagging. Two polypeptides with molecular masses of 31 and 24 kDa in the 0-30% ammonium sulfate fraction were labeled after UV photolysis at 0 degree C but not at -196 degrees C, and appeared to have a high affinity indole-binding site(s) for which active, non-indole auxins were not good ligands. A third polypeptide with a molecular mass of 25 kDa present in the 50-60% ammonium sulfate fraction labeled exclusively at -196 degrees C and had a significant affinity for active auxins but not for inactive indoles. The azido-IAA labeling pattern, pI, competition results, and immunoprecipitation all indicate that the 31- and 24-kDa polypeptides are related to the basic form of endo-1,3-beta-glucanase (EC 3.2.1.39). Azido-IAA labeling polypeptides equivalent to the 31- and 24-kDa species were apparently also present in the cell wall. The low pH optimum for binding of azido-IAA to the 25-kDa polypeptide suggests the location of the active protein in a compartment such as the vacuole or a transport vesicle rather than in the cytosol.

Affinity Labels↗
PubMed1982

Demonstration of specific high affinity binding sites in plasmid DNA by photoaffinity labeling with an ethidium analog.

We have used photoaffinity labeling of pBR322 DNA with 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride to demonstrate high affinity ethidium-binding sites. Plasmid equilibrated with as little as 1 drug/DNA molecule was photoactivated, freed of uncomplexed drug by ethanol precipitation, and subjected to restriction analysis. There was highly specific, rather than random, blockage of HhaI sites (d(GCGC)) at low drug concentrations. Furthermore, the same 7 new digestion fragments were generated at drug to nucleotide ratios ranging from 1:100 to 1:8000. All the new DNA fragments had chain lengths greater than the largest HhaI fragment (393 base pairs). At higher ligand concentrations closely approximating those needed for equilibrium binding studies, detection of the high affinity sites was greatly masked. Drug binding to HhaI restriction fragments which had been prepared prior to the action of drug did not induce new bands. Furthermore, the larger DNA fragments from drug-labeled plasmid were resistant to HhaI digestion over a wide range of enzyme concentrations. These findings suggest that ligand binding can be highly selective even between sites which have the same tetranucleotide sequence. Therefore, selective drug binding must be dictated not only by local base sequence preference, but also by other long range parameters.

Affinity Labels↗
PubMed1991

Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.

Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.

Affinity Labels↗
PubMed1994

Photoaffinity labeling of the ATP binding domain of Rubisco activase and a separate domain involved in the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Photoaffinity labeling of Rubisco activase with 2- and 8-N3ATP was used to identify the adenine binding domain for ATP. Rubisco activase hydrolyzed both of these analogs of ATP and used their hydrolysis to support a low rate of Rubisco activation. When irradiated with ultraviolet light, these and other azido-substituted adenine nucleotides covalently modified Rubisco activase at two distinct binding sites. Competition binding experiments with ATP and ADP showed that one of the sites was the ATP binding domain. The other site was not a nucleotide binding domain per se but would bind adenine nucleotides if an azido moiety was present on the base. Tryptophan and other indoles prevented azidoadenine nucleotides from labeling this domain but afforded little protection to the ATP binding domain. The ability to selectively protect each of the two binding sites made it possible to localize the adenine binding domain for ATP to the region of Rubisco activase from N68-D74 and the other binding domain to a region near the N-terminus from Q10 to D14. Modification of the region from Q10 to D14 by photoaffinity labeling prevented Rubisco activase from promoting activation of Rubisco without affecting ATP hydrolysis. These data suggest that a specific region of Rubisco activase near the N-terminus may be a site of interaction with Rubisco. Binding of azidoadenine nucleotides in this region appears to be fortuitous and may involve base-stacking with the species-invariant Trp at position 16 and hydrogen bonding of the azido moiety.

Adenosine Triphosphate↗
PubMed1986

Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells.

Photoactive radioactive analogues of vinblastine were used to photoaffinity label membranes of Chinese hamster lung drug-sensitive (DC-3F), multidrug-resistant sublines selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX), and revertant (DC-3F/ADX-U) cells. A radiolabeled doublet (150-180 kDa) consisting of a major and minor band which was barely detectable in parental drug-sensitive cells was increased up to 150-fold in the drug-resistant variants but only 15-fold in the revertant cells. Photoaffinity labeling in the presence of 200-fold excess vinblastine reduced radiolabeling of the 150-180-kDa species up to 96%, confirming its Vinca alkaloid binding specificity. The radiolabeled doublet comigrated with a Coomassie Blue stained polypeptide doublet in the drug-resistant cells and was immunoprecipitated with polyclonal antibody which is specific for the 150-180-kDa surface membrane glycoprotein in multidrug-resistant cell lines. The identification of this Vinca alkaloid acceptor in multidrug-resistant plasma cell membranes suggests the possibility of a direct functional role for the 150-180-kDa surface membrane protein in the development of multidrug resistance.

Affinity Labels↗
PubMed1974

Photoaffinity labeling of the ouabain-binding site on (Na+ plus K+) adenosinetriphosphatase.

An ethyl diazomalonyl derivative of cymarin was synthesized in order to photoaffinity label the cardiac glycoside-binding site on (Na(+) + K(+)) adenosinetriphosphate (EC 3.6.1.3). When a noncovalent complex of the enzyme and this cardiac glycoside derivative was photolyzed, a covalent bond was formed between the ligand and the larger of the two polypeptide subunits of the enzyme. Several control experiments demonstrate that this photochemical reaction occured while the ligand was bound to the site at which it inhibits the enzyme activity. Another specific inhibitor, tentatively identified as the ethyl chloromalonyl derivative of cymarin, produced similar photoaffinity labeling of the larger subunit, demonstrating that the photolytic dissociation of the diazo group may not be responsible for the photochemical reaction. Since the cardiac glycoside-binding site, which is accessible from the outside surface of the plasma membrane, and the phosphorylation site, which is accessible from the inside surface, are both on the larger polypeptide subunit of (Na(+) + K(+)) adenosinetriphosphatase, this polypeptide has sequences exposed to both sides of the membrane.

Adenosine Triphosphatases↗
PubMed1983

Photoaffinity labeling of the chick progesterone receptor proteins. Similar hormone binding domains detected after removal of proteolytic interference.

Chick progesterone receptor subunits A and B have been photoaffinity-labeled using [3H]R5020 ([17 alpha-methyl-3H]17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) by a modification of the procedures previously reported by our laboratory (Dure, L. S., IV, Schrader, W. T., and O'Malley, B. W. (1980) Nature (Lond.) 283, 784-786). [3H]R5020 binds to the same receptor sites as authentic progesterone, and has an apparent Kdiss of 2.0 nM. Use of a CuSO4 filter raised the coupling efficiency to 5% and labeled exclusively the receptor proteins. Smaller labeled macromolecules were found to be proteolytic fragments of receptors. The protease(s) could not be inhibited by any of the commonly used protease inhibitors. However, the proteolytic activity was completely removed by passage of crude receptor preparations through phosphocellulose columns. Receptor preparations, photoaffinity-labeled after this procedure, showed exclusively one radioactive band at Mr = 79,000 (subunit A) or Mr = 108,000 (subunit B) with no detectable side-reaction products. Labeled receptors A and B were digested with Staphylococcus aureus V8 protease to yield smaller [3H]R5020-protein fragments derived from both. Molecular weight estimates (Mr = 9,500) and apparent isoelectric points indicate similarities of these regions of both A and B. The photoaffinity protocol described here thus provides a method for study of the hormone-binding domain of progesterone receptors and of receptor proteolysis in crude extracts.

Affinity Labels↗
PubMed1982

Photoaffinity labeling of insulin receptors in viable cultured human lymphocytes. Demonstration of receptor shedding and degradation.

A photosensitive derivative of radiolabeled insulin, SANAH-125I-insulin, was prepared by reacting N-succinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate (SANAH) with 125I-insulin. Cultured IM-9 cells were incubated with SANAH-125I-insulin at 16 degrees C in the dark. They were then washed, photolyzed, solubilized, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Under disulfide reducing conditions, a single specific band of Mr 125,000 was obtained. The characteristics of the labeling of this band with SANAH-125I-insulin (specificity, time course, concentration effect) were the same as that of 125I-insulin interaction with the IM-9 cells and the labeling process did not affect cell viability. The solubilized photolabeled insulin receptor fraction was enriched by first adsorbing to agarose-bound wheat germ agglutinin and the material eluted with N-acetyl-D-glucosamine was then analyzed by SDS-PAGE and autoradiography. Under nonreducing conditions, a major receptor band of Mr 320 K and a minor band of 280 K were obtained. Upon disulfide bond reduction with increasing concentrations of dithiothreitol, a major band of Mr 125 K and two minor bands of Mr 210 K and 94 K were seen. When cells photolabeled at 16 degrees C were further incubated at 37 degrees C, there was a time-dependent loss of intact receptors into the incubation buffer. In contrast, no similar shedding of labeled receptors was observed from isolated rat adipocytes. Following shedding, the labeled IM-9 insulin receptors rapidly disappeared from the incubation buffer (half-time approximately 1.5 h). These results demonstrate the feasibility of photoaffinity labeling, characterizing, and following the fate of insulin receptor in viable cells. Thus receptor photoaffinity labeling should provide a suitable approach for studies of the biologic fate of insulin receptors in cells that are targets for insulin action.

Autoradiography↗
PubMed1986

Photoaffinity labeling of the K562 cell membrane D-glucose transporter with cytochalasin B.

D-glucose carrier protein in K562 cell membrane was studied by photoaffinity labeling with cytochalasin B. The saturable cytochalasin B binding in purified K562 cell membranes was 90 pmol/mg and 200 pmol/mg protein in the presence of D-glucose and D-sorbitol, respectively. More than half of the total cytochalasin B binding could be depressed by D-glucose. The results of SDS-PAGE analysis of K562 cell membranes after photoaffinity labeling at 0.1 microM cytochalasin B showed that the main peak of covalently bound [3H]-cytochalasin B was in the Mr range of 46-65 KDa. The label found in the peak was reduced by more than 50% in the presence of 0.5 M D-glucose, the inhibition similar being to that obtained in the binding experiment. This polypeptide has a slightly higher molecular weight than that of the human erythrocyte cell membrane.

Affinity Labels↗
PubMed1995

Identification of a 23 kDa protein from maize photoaffinity-labelled with 5-azido-[7-3H]indol-3-ylacetic acid.

A 23 kDa protein (p23) was identified in microsomal extracts from maize coleoptiles by photoaffinity labelling with 5-azido-[7-3H]indol-3-ylacetic acid ([3H]N3IAA). Labelling of p23 was blocked by unlabelled IAA, N3IAA, indol-3-ylbutyric acid and indol-3-yl-lactate. In addition, labelling was efficiently decreased by tryptophan, as well as by the scavenger p-aminobenzoic acid. Labelling was, however, not affected by synthetic auxins such as 1-naphthylacetic acid or 2,4-dichlorophenoxyacetic acid. Competition data suggest that the label was probably bound via the indole ring, and hence labelling was not specific for auxins. The 23 kDa protein was solubilized from crude microsomes by extraction with Triton X-100 and purified to homogeneity by ion-exchange, size-exclusion and reversed-phase chromatography. After electroblotting, the amino acid sequences of the p23 N-terminus as well as the several tryptic peptides were obtained. Database comparisons revealed sequence identity with a maize manganese superoxide dismutase. We conclude that photoaffinity labelling of p23 was pseudo-affinity, and therefore the binding site for IAA is not specific.

4-Aminobenzoic Acid↗
PubMed2001

Application of photoaffinity labeling with [(3)H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid--binding proteins I and II.

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [(3)H]atRA or [(3)H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [(3)H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA beta-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-beta-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [(3)H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins.

Alitretinoin↗
PubMed1987

Phylogenetic comparison of the photoaffinity-labeled benzodiazepine receptor subunits.

The late evolutionary appearance of the benzodiazepine receptor (BZR) allows an experimental approach for evaluation of the qualitative development of its subunits. Photoaffinity labeling of brain membranes with [3H]flunitrazepam followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography offers a suitable method for tracing the qualitative evolution of the BZR. A systematic comparison of the subunit patterns in fishes, amphibians, reptiles, birds, and mammals revealed that the subunit of 53K is phylogenetically the oldest photoaffinity labeled subunit; whereas it is the only band present in the lungfish and most amphibians, additional bands are apparent in higher tetrapods. In fishes, the evolution of the BZR subunits leads to the loss of the 53K subunit. KD values are discussed in relation to specific subunit patterns. Possible explanations for the observed variation of the subunits are discussed, with special emphasis placed on the possible evolution by gene duplication and subsequent divergence.

Amphibians↗
PubMed1998

ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity.

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.

Adenosine Triphosphatases↗
PubMed1976

2-diazo-3,3,3-trifluoropropionyl chloride: reagent for photoaffinity labeling.

2-Diazo-3,3,3-trifluoropropionyl chloride has been synthesized from trifluorodiazoethane and phosgene. Its derivatives are acid stable, can be used to label enzymes, and undergo photolysis with substantially less rearrangement than do derivatives of other known diazoacyl reagents designed for photoaffinity labeling. In particular, the diazotrifluoropropionyl thioester of methyl N-acetylcysteine undergoes photolysis in methanol with about 40% insertion into the - OH bond of the solvent; by contrast, photolysis of other diazoacyl thioesters gives substantially quantitative Wolff rearrangement. The trifluoro compounds hold promise for the photoaffinity labeling of thiols.

Affinity Labels↗
PubMed2003

Localization and photoaffinity labelling of the levetiracetam binding site in rat brain and certain cell lines.

Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA, a novel antiepileptic drug, has been shown to bind to a specific binding site located in the brain (Eur. J. Pharmacol. 286 (1995) 137). To identify the protein constituent of the levetiracetam binding site in situ, we synthesized the photoaffinity label [3H]ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide), a levetiracetam analog with higher affinity for the levetiracetam binding site. This radioligand was used to map the levetiracetam binding site within the brain and to study its cellular and subcellular distribution. Autoradiography experiments using [3H]ucb 30889 in rat brain revealed a unique distribution profile that did not match that of classical receptors known to be involved in the generation of epileptic seizures. There was a high level of binding in the dentate gyrus, the superior colliculus, several thalamic nuclei, the molecular layer of the cerebellum and to a lesser extent in the cerebral cortex, the striatum and the hypothalamus. The levetiracetam binding site was restricted to neuronal cell types, undifferentiated PC12 cells and was highly enriched in synaptic vesicles. [3H]ucb 30889 was also used in photoaffinity labelling studies and shown to bind covalently to a membrane protein with a molecular weight of approximately 90 kDa.

Animals↗
PubMed1994

Rat liver and kidney contain high densities of sigma 1 and sigma 2 receptors: characterization by ligand binding and photoaffinity labeling.

Rat liver and kidney were investigated for the presence of sigma (sigma) receptor subtypes by radioligand binding with three highly selective sigma probes and by photoaffinity labeling using [3H]azido-di-o-tolylguanidine ([3H]azido-DTG). [3H](+)-Pentazocine, a highly selective sigma 1 probe, bound to sites in liver membranes with Kd = 7.5 nM and Bmax3 = 2929 fmol/mg protein. [3H](+)-Pentazocine binding sites in kidney had Kd = 23.3 nM and Bmax = 229 fmol/mg protein. [3H]1,3-Di-o-tolylguanidine ([3H]DTG) and [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3H](+)-3-PPP) label both sigma 1 and sigma 2 receptors. Parameters for [3H]DTG in the liver were Kd = 17.9 nM and Bmax = 11,895 fmol/mg protein. Similar parameters were observed for [3H](+)-3-PPP, Kd = 51.9 nM and Bmax = 11,070 fmol/mg protein. [3H]DTG bound to rat kidney with Kd = 45.8 nM and Bmax = 1190 fmol/mg protein. The observation that either [3H]DTG or [3H](+)-3-PPP and [3H](+)-3-PPP labeled a higher number of sites relative to [3H](+)-pentazocine suggested that liver and kidney contain both subtypes of sigma receptor. This was confirmed by competition studies vs. [3H](+)-pentazocine and [3H]DTG (in the presence of dextrallorphan to mask sigma 1 sites). In both tissues, [3H](+)-pentazocine labeled sites with high affinity for haloperidol and enantioselectivity for (+)-benzomorphans over (-)-benzomorphans. [3H]DTG + dextrallorphan labeled sites in both tissues which also had high affinity for haloperidol, but which had the characteristic sigma 2 property of low affinity for (+)-benzomorphans and enantioselectivity for (-)-benzomorphans over the corresponding (+)-isomer. Similar results were obtained with [3H](+)-3-PPP + dextrallorphan. Several novel aryl diamines, such as 1S,2R-cis-N-[2-(3,4-dichlorophenylethyl]-N-methyl-2- (1-pyrrolidinyl)cyclohexylamine (BD737) and N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine (BD1008), bound to both sites with high affinity. Photoaffinity labeling with 10 nM [3H]azido-DTG resulted in specific labeling of polypeptides of 25 kDa and 21.5 kDa. Dextrallorphan (100 nM or 500 nM) completely blocked labeling of the 25 kDa polypeptide, but had no effect on labeling of the lower molecular weight protein. (+)-10,11-Dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10- imine((+)-MK-801) had no effect on labeling of either polypeptide. These data are consistent with the notion that the 25 kDa and 21.5 kDa proteins represent sigma 1 and sigma 2 receptors, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1988

Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog.

Multidrug-resistant human KB carcinoma cells express a 170,000-dalton membrane glycoprotein (P-glycoprotein) that can be photoaffinity labeled with the vinblastine analog N-(p-azido-[3-125I]salicyl]-N'-(beta-aminoethyl)vindesine. Several agents that suppress the multidrug-resistant phenotype, including N-solanesyl-N,N'-bis(3,4-dimethylbenzyl)ethylenediamine, cepharanthine, quinidine, and reserpine, were found to inhibit photolabeling of P-glycoprotein at doses comparable to those that reverse multidrug resistance. However, the phenothiazines chlorpromazine and trifluoperazine, which also effectively reverse multidrug resistance, were poor inhibitors of the photoaffinity labeling of P-glycoprotein. Chloroquine, propranolol, or atropine, which only partially reversed the drug resistance, also did not inhibit photolabeling. Naphthalene sulfonamide calmodulin inhibitors, W7 and W5, as well as many other drugs that did not circumvent multidrug resistance, did not inhibit photolabeling. These studies suggest that most, but not all, agents that phenotypically suppress multidrug resistance also inhibit drug binding to a site on P-glycoprotein with which a photoaffinity analog of vinblastine interacts.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Direct photoaffinity labeling of the putative sulfonylurea receptor in rat beta-cell tumor membranes by [3H]glibenclamide.

The oral antidiabetic sulfonylurea [3H]glibenclamide specifically binds to plasma membranes from a rat beta-cell tumor indicating a receptor for sulfonylureas in these membranes. Irradiation of [3H]glibenclamide at 254 or 300 nm in the presence of albumin resulted in covalent labeling of the albumin molecule. Direct photoaffinity labeling of beta-cell membranes with [3H]glibenclamide resulted in the covalent modification of two membrane polypeptides with apparent molecular masses 140 and 33 kDa. The extent of labeling of the 140 kDa polypeptide was specifically decreased by sulfonylureas. This suggests that a membrane polypeptide of 140 kDa is a component of the sulfonylurea receptor in the beta-cell membrane.

ATP-Binding Cassette Transporters↗