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PubMed1998

Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry.

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-epsilon-N-fluorescei n (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83-87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054) and the photochemical cross-linking data.

Affinity Labels↗
PubMed1984

Specific lipid components in membrane receptors of 12-O-tetradecanoylphorbol-13-acetate as revealed by direct photoaffinity labelling.

The use of [20-3H]-12-O-tetradecanoylphorbol-13-acetate (3H-TPA) as a direct photoaffinity probe of receptors was investigated. When membrane receptors in a particulate fraction from mouse brain were loaded with the high-affinity agonist 3H-TPA, irradiation of such preparations with ultraviolet (UV) light resulted in specific irreversible binding of the label to the membrane lipids phosphatidylethanolamine (PE) and phosphatidylserine (PS); evidence for labelling of proteins was lacking. The labelled lipids were tentatively identified by co-chromatography; they corresponded to selected reference lipids obtained by photoaffinity labelling. When a variety of natural and synthetic reference lipids was irradiated in the presence of 3H-TPA, photoadducts were obtained primarily from natural lipids containing unsaturated acyl chains in position 2. The synthetic 1,2-dipalmitoyl derivatives of phosphatidylcholine (PC), PE and phosphatidyl glycerol were refractory. When the photoadducts from PC and PE were treated with phospholipases A2 and C, the chromatographic pattern of cleavage products obtained was in accordance with that of phospholipids carrying the 3H-TPA label on the acyl moiety in the 2-position. Since previous evidence had suggested that a lipid-protein complex was the phorbol ester receptor, it may, therefore, be concluded that the receptor(s) contain PE and PS in specific interaction with protein(s). Accordingly, PE- and PS-dependent enzymes (or other proteins) are considered prime candidates for phorbol ester receptors.

Affinity Labels↗
PubMed1990

Photoaffinity labeling of GDP-fucose:nLcOse4Cer alpha 1----3-fucosyltransferase from human small cell lung carcinoma NCI-H69 cells with the GDP-fucose analog GDP-hexanolaminyl-4-azidosalicylic acid.

An iodinatable photoactive analog of GDP-fucose, GDP-hexanolaminyl-4-azidosalicylic acid, has been prepared and applied to studies of the previously described alpha 1----3-fucosyltransferase from NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627). The NCI-H69 cell alpha 1----3-fucosyltransferase was obtained from a 0.2% Triton X-100-solubilized enzyme fraction after affinity purification on a GDP-hexanolamine-Sepharose column and gel filtration through a fast protein liquid chromatography Superose 12 column. Increasing concentrations of the photoaffinity reagent were found to result in loss of up to 35% of the original enzyme activity at under 100 microM final concentrations. The inactivation was photolysis dependent and could be prevented by the addition of GDP-fucose prior to photolysis. The photoprobe behaved as a competitive inhibitor with respect to GDP-fucose with a Ki of 23 microM, identical to that of GDP. Photoincorporation of 125I-labeled GDP-hexanolaminyl-4-azidosalicylic acid into the enzyme fraction labeled a slow migrating protein band in a native polyacrylamide gel which corresponded to enzyme activity. Inclusion of GDP-fucose prevented photolabeling of this band. Sodium dodecyl sulfate gel electrophoresis of the photolabeled, GDP-fucose-protected band yielded a 125I-labeled protein band that migrated at Mr 45,000, most probably corresponding to an alpha 1----3-fucosyltransferase protein subunit. These studies suggest photoaffinity labeling using nucleotide affinity ligands linked to photoactivatable, heterobifunctional cross-linking reagents may be generally applicable to photoaffinity labeling glycosyltransferase enzyme proteins.

Affinity Labels↗
PubMed1992

Electron microscopic localization of photoaffinity-labelled delta opioid receptors in the neostriatum of the rat.

The distribution of delta opioid receptors, selectively labelled in vitro with the photoaffinity probe monoiodo azido-DTLET ([D-Thr2,pN3Phe4, Leu5]enkephaly-Thr6), was analyzed by light and electron microscopic radioautography in sections from rat neostriatum. Preliminary experiments indicated that up to 65% of specific 125I-azido-DTLET binding to rat striatal sections was still detectable following prefixation of the brain with 0.5% glutaraldehyde. These experiments also showed that up to 20-30% of the specifically bound radioactivity was covalently linked following ultraviolet irradiation and was thereby retained in tissue during subsequent postfixation and dehydration steps. Accordingly, the topographic distribution of the covalently attached azido-DTLET molecules was similar to that seen in fresh frozen sections and characteristic of that previously described for delta sites. Light and electron microscopic examination of the label in prefixed, striatal sections irradiated with ultraviolet light revealed that a significant proportion of specifically bound 125I-azido-DTLET molecules was intraneuronal. Specifically, 16% of the labelled binding sites were found in dendrites, 12% in perikarya and 4% in axon terminals. These results suggest that an important proportion of delta opioid binding sites labelled in the neostriatum correspond to receptors that are undergoing synthesis, transport and/or recycling. They also imply that a major fraction of delta sites are associated with intrastriatal neurons, as opposed to afferent axons. Approximately 44% of the labelled binding sites were associated with neuronal plasma membranes. Although most of these were found at the level of axodendritic (20%) and dendrodendritic (7%) appositions, comparison of the labelling incidence of these two compartments with their frequency of occurrence in tissue suggested that delta sites are fairly widely dispersed along neuronal plasma membranes. Only a small proportion (smaller than that of mu or kappa sites labelled in the same region) was associated with synaptic specializations. These results support the concept that delta receptors correspond to molecular entities that are distinct from mu and kappa sites and suggest that delta ligands act primarily nonjunctionally on the plasma membrane of striatal neurons.

Affinity Labels↗
PubMed1985

Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan.

The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm). The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value [S]0.5 = 6.3 microM). Photolysis of the quinonoid form of the alpha 2 beta 2 complex results in time-dependent inactivation of the beta 2 subunit but not of the alpha subunit. The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis. The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation. The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation. The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the alpha 2 beta 2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation. The modified alpha 2 beta 2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the alpha subunit. 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-[14C]serine by the alpha 2 beta 2 complex of tryptophan synthase on a preparative scale and has been isolated. Incorporation of 14C label from 6-azido-L-[14C]tryptophan is stoichiometric with inactivation. Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.

Affinity Labels↗
PubMed1991

Identification by photoaffinity labeling of fatty acid-binding protein as a potential warfarin receptor in rat liver.

Two different photoaffinity analogs of 4-hydroxy coumarin, 3-(p-azidobenzyl)-4-hydroxycoumarin (AzBHC) and 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin (AzISAHC), are being used in the identification of warfarin-binding proteins present in mammalian tissue (Myszka, D. G., and Swenson, R. P. (1990) Biochem. Biophys. Res. Commun. 172, 415-422; Myszka, D. G., and Swenson, R. P. (1991) J. Biol. Chem. 266, 4789-4797). In this study, [14C]AzBHC, but not [125I]AzISAHC, was observed to specifically label a 15,000-dalton protein present in both the microsomal and cytosolic fractions of rat liver. Pretreatment of the crude protein samples with warfarin or dicoumarol completely protected the 15-kDa protein from modification by [14C]AzBHC, indicating that this photoaffinity reagent is specifically labeling a coumarin-binding protein. 4-Hydroxycoumarin itself and AzISAHC were unable to block the incorporation of this photoaffinity probe. The 15-kDa protein was isolated by two-dimensional electrophoresis and subjected to amino-terminal sequence analysis. The first 20 amino acid residues analyzed were found to be identical with the amino-terminal sequence of rat liver fatty acid-binding protein (L-FABP) (Gordon J. I., Alpers, D. H., Ockner, R. K., and Strauss, A. W. (1983) J. Biol. Chem. 258, 3356-3363). Photoaffinity labeling and protection experiments carried out on purified preparations of L-FABP paralleled the labeling results obtained in the microsomes and cytosol, confirming that L-FABP is capable of specifically binding AzBHC, warfarin, and dicoumarol. Oleic acid, an established ligand for L-FABP, can compete with the binding of the photoaffinity probe; however, it was less effective in protecting the protein than warfarin. The specificity of labeling of crude liver fractions by warfarin photoaffinity analogs reported here as well as the high concentration of FABP in liver tissue together suggest that this protein may represent a major hepatic receptor responsible for the uptake and/or transport of various oral 4-hydroxycoumarin-based anticoagulant drugs.

Affinity Labels↗
PubMed1992

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.

3T3 Cells↗
PubMed1995

Photoaffinity labeling of human placental S-adenosylhomocysteine hydrolase with [2-3H]8-azido-adenosine.

The potential photoaffinity probe 8-azido-adenosine (8-N3-Ado) was shown to serve as a substrate for the 3'-oxidative activity of human S-adenosylhomocysteine (AdoHcy) hydrolase (Aiyar, V. N., and Hershfield, M. S. (1985) Biochem. J. 232, 643-650). In this study, we have determined the equilibrium binding properties of 8-N3-Ado with AdoHcy hydrolase (NAD+ form) and identified the specific amino acid residues that are covalently modified. After irradiation of the reaction mixture of [2-3H]8-N3-Ado and AdoHcy hydrolase (NAD+ form) and followed by tryptic digestion, peptides specifically photolabeled by [2-3H]3'-keto-8-N3-Ado were effectively separated from peptides nonspecifically labeled with [2-3H]8-N3-Ado using boronate affinity chromatography. After purification by reverse phase high performance liquid chromatography, two photolabeled peptides were isolated and identified as Val175-Lys186 and Val319-Arg327, in which Ala177 and Ile321 were associated with radioactivity. The specificity of the photoaffinity labeling with [2-3H]3'-keto-8-N3-Ado was demonstrated by the observation that these photolabeled peptides were not isolated when [2-3H]8-N3-Ado was incubated with apo AdoHcy hydrolase and irradiated. The two photolabeled peptides are assumed to be parts of the adenine-binding domain for substrates. They are both within well conserved regions of AdoHcy hydrolases. The peptide Val175-Lys186 is located very close to Cys195 and Glu197. Ser198, both of which were indicated to be located in the active site of the enzyme by chemical modification and limited proteolysis methods. The peptide Val319-Arg327 is adjacent to Leu330, which is proposed by a computer graphics model to interact with the C-6-NH2 group of Ado.

Adenosine↗
PubMed1997

Photoaffinity labeling of the ATP transporter of rat liver microsomes.

A photoreactive azido derivative of ATP, 3 (2')-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimidyl-4-azidobenzoate (NHS-AB) to photolabel the ATP transporter of rat liver microsomes. AB-ATP specifically inhibited the transport of ATP into microsomes, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe for the identification of the ATP transporter in rat liver microsomes. An SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of microsomes photolabeled with [gamma-32P]AB-ATP indicates the presence of four major protein bands with apparent molecular sizes of 97, 56, 53, and 47 kDa. Among these labeled proteins, the 56 kDa protein was completely protected from the photoaffinity labeling by 30 microM ATP but not by the same amount of GTP, which is consistent with the specific labeling of the ATP binding site of the ATP transporter. The specific labeling of the only 56 kDa protein among them was sensitive to the anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) but not sensitive to the mitochondrial ADP/ATP carrier inhibitor, atractyloside (ATR). Moreover, the 56 kDa protein was uniquely photolabeled with [gamma-32P]AB-ATP in the highly purified rough endoplasmic reticulum (RER) vesicles. These results strongly suggested that the 56 kDa protein represents the ATP transporter of rat liver endoplasmic reticulum (ER).

ATP-Binding Cassette Transporters↗
PubMed1991

Binding proteins for a peptide hormone in the shrimp, Sicyonia ingentis: evidence from photoaffinity labeling with red pigment concentrating hormone analogs.

Two photoaffinity analogs of the crustacean erythrophore (red pigment) concentrating hormone (RPCH) have been synthesized and shown to cause pigment concentration in the shrimp Sicyonia ingentis. These two modified oligopeptides have azidosalicylamide groups which allow introduction of an 125I label and enable photochemically induced covalent attachment to a specific binding site. Incubation of [125I]-ASA-Glu1-CC-2 with the 100,000g membrane pellet and cytosol fraction from epidermis, eyestalks, muscle, and central nervous system (CNS), followed by irradiation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography results in covalent modification of certain protein bands in the membranes of selected tissues. Two such proteins were observed in neural tissues and showed competitive displacement by excess RPCH, indicative of specific high-affinity binding. This is the first report of peptide hormone-binding proteins in an invertebrate and provides further evidence of a role for RPCH as a neurotransmitter in the CNS.

Amino Acid Sequence↗
PubMed1990

Identification of the UDP-glucose-binding polypeptide of callose synthase from Beta vulgaris L. by photoaffinity labeling with 5-azido-UDP-glucose.

The photoaffinity probe 5-azidouridine 5'-[beta-32P]diphosphate glucose (5N3[32P]UDP-Glc) was used to identify a 57-kDa polypeptide as a strong candidate for the UDP-Glc-binding polypeptide of UDP-glucose: (1,3)-beta-glucan (callose) synthase from red beet (Beta vulgaris L.) storage tissue. Unlabeled 5N3UDP-Glc was a competitive inhibitor of callose synthase with a Ki of 310 microM. Callose synthase was purified from plasma membranes by a two-step solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, followed by product entrapment, and photoincorporation of radioactivity from 5N3[32P]UDP-Glc was used to identify UDP-Glc-binding polypeptides that copurified with callose synthase activity. Photoinsertion into the 57-kDa band was closely correlated with all catalytic properties examined. Photolabeling of the 57-kDa polypeptide was enriched upon purification of callose synthase by product entrapment, was abolished with increasing levels of unlabeled UDP-Glc, was dependent upon the presence of divalent cations, and the pH dependence of photolabeling correlated with the pH activity profile of callose synthase. In addition, photolabeling of the 57-kDa band did not occur after phospholipase treatment, which destroys enzyme activity. The extent of labeling of this polypeptide thus correlates closely with the activity of callose synthase under a wide variety of conditions. These results imply that the polypeptide at 57 kDa represents the substrate-binding and cation-regulated component of the callose synthase complex of higher plants.

Affinity Labels↗
PubMed2000

Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin.

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe(10),Tyr(14)]nociceptin ([Bpa(10),Tyr(14)]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa(10),Tyr(14)]noc binds the receptor with high affinity (K(i) approximately 0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC(50) approximately 0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa(10),Tyr(14)]noc results in the irreversible labeling of a glycoprotein of approximately 65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (approximately 6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of approximately 3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu(295) --> Asp mutant receptor also yields a single labeled fragment of approximately 6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296-302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa(10)]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile(300) within the experimentally identified photoreactive region.

Animals↗
PubMed1986

Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

Adenosine↗
PubMed1988

delta 9-[16 alpha-125I]iodo-19-nortestosterone: a gamma-emitting photoaffinity label for the progesterone receptor.

We have synthesized 16 alpha-iodo-4,9-estradien-17 beta-ol-3-one [delta 9-16 alpha-iodo-19-nortestosterone (delta 9-INT)] labeled with 125I (delta 9-[16 alpha-125I]INT) to provide a new gamma-emitting photoaffinity ligand for the progesterone receptor that has many advantages over the currently available [3H]R5020. We have characterized the interaction of delta 9-[16 alpha-125I]INT with the rabbit uterine progesterone receptor and have demonstrated the usefulness of this compound for studies of receptor structure. The binding of 2 nM [3H]progesterone to receptor in rabbit uterine cytosol was specifically competed for by 19-nortestosterone, 16 alpha-iodo-19-nortestosterone, and delta 9-INT. Scatchard analysis demonstrated that delta 9-[16 alpha-125I]INT and [3H]progesterone estimated the same number of binding sites in rabbit uterine cytosol, with a Kd for delta 9-[16 alpha-125I]INT of about 2.7 nM. The binding of delta 9-[16 alpha-125I]INT was inhibited by both progesterone and R5020, whereas testosterone, estradiol, and 5 alpha-dihydrotestosterone were ineffective. In cytosol, delta 9-[16 alpha-125I]INT covalently labeled the same mol wt receptor forms as [3H]R5020. Although the efficiency of cross-linking was similar for [3H]R5020 (3%) and delta 9-[16 alpha-125I]INT (4%), the radioactivity was 10-fold greater due to the higher specific activity of delta 9-[16 alpha-125I]INT and the lack of sample quench. The use of delta 9-[16 alpha-125I]INT greatly increases the sensitivity and efficiency of the photoaffinity labeling technique; it will provide a valuable tool for further studies of the progesterone receptor, allowing the detection of receptor in dilute cytosol after gel electrophoresis under denaturing conditions.

Animals↗