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PubMed1996

Synthesis and characterization of a carbene-generating biotinylated N-acetylglucosamine for photoaffinity labeling of beta-(1-->4)-galactosyltransferase.

A photoreactive N-acetylglucosamine derivative, N-[2-[2-[2-(2-biotinylaminoethoxy)-ethoxy]ethoxy]-4-[3-(trifluo rom ethyl)-3-H-diazirin-3-yl]benzoyl]-N4-[2-(acetylamino)-deoxy-beta-D -glucopyranosyl]-L-aspartamide (BDGA), was synthesized as a carbene-generating biotinylated probe for UDP-galactose:N-acetylglucosamine beta-(1-->4)-galactosyltransferase (GalT). The photoaffinity labeling experiments of bovine GalT with BDGA under various condition were examined based on the quantitative chemiluminescent detection of the biotinyl residue which was photochemically introduced into in GalT protein. A progressive decrease in the yield of specific photolabeling was observed upon lowering the incubation temperature from 37 degrees C to 20 degrees C or 4 degree C. The amount of photoincorporation was also decreased when UMP was not included in the incubation mixture. Using a crude protein mixture of recombinant human GalT, a band corresponding to the glutathione S-transferase fusion GalT protein was also specifically visualized. Furthermore, combine use of BDGA photolabeling with an immobilized avidin was found to be effective for the selective retrieval of photolabeled GalT from a reaction mixture containing a large amount of unlabeled GalT protein. The results obtained clearly demonstrate that the covalent biotinylation using the carbene-generating photoaffinity reagent BDGA would be useful for the analysis of acceptor substrate binding sites within the GalT protein.

Acetylglucosamine↗
PubMed2000

Halothane binding to a G protein coupled receptor in retinal membranes by photoaffinity labeling.

General anesthetics have been reported to alter the functions of G protein coupled receptor (GPCR) signaling systems. To determine whether these effects might be mediated by direct binding interactions with the GPCR or its associated G protein, we studied the binding character of halothane on mammalian rhodopsin, structurally the best understood GPCR, by using direct photoaffinity labeling with [(14)C]halothane. In the bleached bovine rod disk membranes (RDM), opsin and membrane lipids were dominantly photolabeled with [(14)C]halothane, but none of the three G protein subunits were labeled. In opsin itself, halothane labeling was inhibited by unlabeled halothane with an IC(50) of 0.9 mM and a Hill coefficient of -0.8. The stoichiometry was 1.1:1.0 (halothane:opsin molar ratio). The IC(50) values of isoflurane and 1-chloro-1,2, 2-trifluorocyclobutane were 5.0 and 15 mM, respectively. Ethanol had no effect on opsin labeling by halothane. A nonimmobilizer, 1, 2-dichlorohexafluorocyclobutane, inhibited halothane labeling by 50% at 0.05 mM. The present results demonstrate that halothane binds specifically and selectively to GPCRs in the RDM. The absence of halothane binding to any of the G protein subunits strongly suggests that the functional effects of halothane on GPCR signaling systems are mediated by direct interactions with receptor proteins.

Affinity Labels↗
PubMed1989

Interaction of p-aminobenzoic acid with erythrocyte membrane: photoaffinity labeling of the binding sites.

p-Aminobenzoic acid (PABA) was found to prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right-side-out membrane vesicles revealed a similar number of binding sites and Kd values for both normal and sickle cell membranes. A [14C]Azide analog of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface. The azide was found to covalently incorporate into the membrane components upon irradiation; 52-35% of the label was associated with the proteins and the remaining with the lipids. Electrophoretic analysis of photolabeled membranes revealed that the azide interacts mainly with Band 3 protein in the case of intact erythrocytes and right-side-out sealed vesicles; however, if unsealed ghosts are used, other membrane proteins besides Band 3 are photolabeled. PABA was found to inhibit both high and low affinity calcium-binding sites situated on either surface of the membrane apparently in a non-competitive manner. However, calcium binding stimulated by magnesium and ATP was only slightly affected. Calcium transport into inside-out vesicles was inhibited by PABA, but it did not affect the calcium ATPase activity.

4-Aminobenzoic Acid↗
PubMed1991

Efficient and selective photoaffinity labeling of the estrogen receptor using two nonsteroidal ligands that embody aryl azide or tetrafluoroaryl azide photoreactive functions.

3-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-6-hydroxy-2-(4- hydroxyphenyl)benzo[b]thiophene 1 (tetrafluoroaryl azide, TFAA) and its protio analogue 3-(4-azidobenzoyl)-6- hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene 2 (protioaryl azide, PAA), photoaffinity labeling (PAL) reagents for the estrogen receptor (ER), have been prepared in high specific activity tritium-labeled form (19 Ci/mmol) and shown to undergo selective and efficient photocovalent attachment to ER from rat uterus. Both azides 1 and 2 demonstrate high binding affinity for ER as determined by both a competitive binding assay (relative binding affinities: estradiol = 100; TFAA = 9.3; PAA = 66) and a direct binding assay (Kd: estradiol = 0.24 nM; TFAA = 2.64 nM; PAA = 0.37 nM). When unlabeled TFAA and PAA are irradiated at greater than 315 nm, they demonstrate site-specific photoinactivation of ER that reaches 43% and 55%, respectively, by 30 min. Specific photocovalent attachment to ER can be effected by irradiation of the tritium-labeled azides; the covalent attachment efficiency is good (1 = 20-30%, 2 = ca. 25%) and the selectivity of ER labeling is high. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of a major component at Mr 60,000 and a minor species at Mr 46,000, the same two species that are labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. The ER-specific antibodies H222Sp gamma and D547Sp gamma show a clean precipitation of only these two species. In the MCF-7 human breast cancer cell line, PAA is a full estrogen agonist in terms of stimulation of cell proliferation and induction of progesterone receptor. These two azides provide the first system in which the photocovalent attachment efficiency of an aryl azide can be compared to its tetrafluorosubstituted aryl azide analogue in a complex biological receptor system. Azides 1 and 2 are the most efficient and selective PAL reagents prepared to date for ER, and they should be useful in further studies of the hormone-binding domain of this protein.

Affinity Labels↗
PubMed1995

Photoaffinity labeling of a cell surface polyamine binding protein.

Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N4 position via an alkyl linkage, and the second is a norspermine conjugate with 4-azidosalicylic acid at the N4 position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4 degrees C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N4-spermidine conjugate by SDS-polyacrylamide gel electrophoresis revealed two heavily labeled proteins at approximately 118 and approximately 50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N1-norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (approximately 100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.

Affinity Labels↗
PubMed1992

Modification of cytochrome P450 1A2 enzymes by the mechanism-based inactivator 2-ethynylnaphthalene and the photoaffinity label 4-azidobiphenyl.

2-Ethynylnaphthalene (2EN) had previously been demonstrated to be a mechanism-based inactivator of rat cytochrome P450 (P450) 1A2 [Hammons, G.J., Alworth, W.L., Hopkins, N.E., Guengerich, F. P., & Kadlubar, F. F. (1989) Chem. Res. Toxicol. 2, 367-374]. In this work 2EN was also demonstrated to be a useful inactivator of rabbit P450 1A2 (k(inactivation) 0.094 min-1, K(i) 11 microM) but it did not inactivate human P450 1A2, although the sequences of the three proteins are approximately 80% identical. Rat and rabbit P450 1A2 were modified by incubation with NADPH-P450 reductase, NADPH, and [3H]2EN to levels of 0.35 and 0.47 nmol of adduct (nmol of P450)-1, respectively. In each case only a single tryptic peptide was labeled; recovery of labeled peptides was low under the acidic HPLC conditions. The rabbit P450 1A2 peptide FQELMAAVGR (positions 175-184) and the rat P450 1A2 peptide L(S)QQYGDVLQIR (positions 67-78) were identified. 4-Azidobiphenyl (4-N3BP) was developed as a photoaffinity label for P-450 1A2 proteins because of its similarity to 4-aminobiphenyl, a known substrate for the enzymes. 4-N3BP was shown to be photolyzed with 350-nm light and radioactive label could be incorporated into rat P450 1A2. Labeling of the protein was found to be saturable with increasing concentrations of 4-N3BP and up to 0.59 nmol of label could be incorporated (nmol P450 1A2)-1. The substrate 4-aminobiphenyl and the competitive inhibitor 7,8-benzoflavone blocked photolabeling of P450 1A2 with 4-N3BP, and 4-N3BP inhibited N-hydroxylation of 4-aminobiphenyl by P450 1A2 in the usual enzyme assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1988

Direct photoaffinity labeling of ribonucleotide reductase from Escherichia coli using dTTP: characterization of the photoproducts.

Subunit B1 of Escherichia coli ribonucleotide reductase contains one type of allosteric binding site that controls the substrate specificity of the enzyme. This site binds the allosteric effector dTTP as well as other nucleoside triphosphates. Cross-linking of dTTP to protein B1 by direct photoaffinity labeling, as well as the isolation and sequence determination of the labeled tryptic peptide, has recently been reported [Eriksson, S., Sjöberg, B.-M., Jörnwall, H., & Carlquist, M. (1986) J. Biol. Chem. 261, 1878-1882]. In this study, we have further purified the dTTP-labeled peptide and characterized it using UV spectroscopy. Two types of dTTP-cross-linked peptide were found: one having an absorbance maximum at 261 nm typical for a dTTP spectrum, i.e., containing an intact 5,6 double bond, and one minor form with low absorbance at 261 nm. In both cases, the same amino acid composition was found, corresponding to the peptide Ser291-X-Ser-Gln-Gly-Gly-Val-Arg299 in the B1 sequence with X being Cys-292 cross-linked to dTTP. Isotope labeling experiments revealed that one proton in the 5-methyl group of thymine was lost during photoincorporation. Therefore, the cross-linking occurs via the 5-methyl group to Cys-292 in a majority of incorporated dTTPs, but a second, possibly 5,6-saturated form of incorporated nucleotide was also detected. The reasons for the high stereospecificity of the reaction and the possible structure of the allosteric site of protein B1 are discussed.

Affinity Labels↗
PubMed1996

Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase.

Farnesyl pyrophosphate (FPP) is involved in a large number of cellular processes including the prenylation of transforming mutants of Ras proteins implicated in cancer. Photoactive analogs could provide useful information about enzyme active sites that bind farnesyl pyrophosphate; however, the availability of such compounds is extremely limited. Molecules that incorporate benzophenone moieties are attractive photoaffinity labeling reagents because of their useful photochemical properties. Here, the syntheses of two compounds, 3a and 3b, containing para- and meta-substituted benzoylbenzoates are described. Compounds 3a and 3b are competitive inhibitors (with respect to FPP) of yeast protein farnesyltransferase (PFTase) with K(i) values of 910 and 380 nM, respectively. Both compounds inactivate PFTase upon photolysis, resulting in as much as 44% inactivation of enzyme activity. Photolysis of PFTase in the presence of [(32)P]3a or of [(32)P]3b results in preferential labeling of the beta subunit, suggesting that this subunit is involved in prenyl group recognition. These compounds should be valuable tools for studying enzymes that utilize FPP as a substrate.

Journal Article↗
PubMed2003

Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain.

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.

Affinity Labels↗
PubMed1988

Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil.

Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells. In this study two photoactive radioactive analogs of verapamil, N-(p-azido[3,5-3H]benzoyl)aminomethyl verapamil and N-(p-azido[3-125I]salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistant DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine. The results show that a specifically labeled 150- to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for P-glycoprotein. Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive analogs as well as with verapamil. Photoaffinity labeling was also inhibited by 50 microM concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine. Bay K8644, a calcium channel agonist, also inhibited P-glycoprotein photolabeling. Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 microM compared to that by verapamil at 8 microM. Photolabeling was also partially inhibited by two of the drugs to which these cells are cross-resistant, doxorubicin and actinomycin D, at 100 microM, but not by colchicine. These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1983

Identification of the insulin receptor in plasma membranes of Ehrlich ascites carcinoma cells by photoaffinity labeling.

Binding of 125I-insulin to Ehrlich ascites carcinoma cells revealed the presence of high affinity binding sites characterized by a dissociation constant of 6.1 . 10(-9) M and a number of sites per cell of 2.6 . 10(3). These values are in line with respective data for other cell types reported in the literature. The receptor was identified by photoaffinity labeling of highly purified plasma membranes with 4-azido-benzoyl-125I-insulin as a probe. The only receptor protein detectable has a molecular mass of 120-122 kD. This finding is discussed in terms of metabolic and cell proliferation effects of insulin.

Affinity Labels↗
PubMed1984

Synthesis and characterisation of an iodinated bile-salt derivative for photoaffinity labelling.

The synthesis and characterisation of a novel iodinated bile salt derivative, 125I-labelled 3 beta-azidocholylhistamine, is described. The derivative is handled by rat liver in a similar manner to taurocholate and binding to bovine serum albumin, a well-characterised bile acid-binding protein, is demonstrated. The suitability of the derivative for photoaffinity labelling is assessed.

Affinity Labels↗
PubMed1987

Identification of identical binding polypeptides for cephalosporins and dipeptides in intestinal brush-border membrane vesicles by photoaffinity labeling.

The uptake of a photolabile derivative of the orally effective cephalosporin cephalexin, N-(4-azidobenzoyl)cephalexin, was investigated in brush-border membrane vesicles. The compound was taken up into the intravesicular space and inhibited the active uptake of cephalexin in a concentration-dependent manner. Therefore, this probe interacts with the transport system shared by alpha-aminocephalosporins and dipeptides. Photoaffinity labeling of brush-border membrane vesicles from rat small intestine with N-(4-azido[3,5-3H]benzoyl) derivatives of the cephalosporin cephalexin and the dipeptide glycyl-L-proline resulted in the covalent incorporation of radioactivity into membrane polypeptides with apparent molecular weights of 127,000, 100,000, 94,000 and 86,000, the polypeptide of molecular weight 127,000 being predominantly labeled. The specificity of labeling was demonstrated by a decrease in the labeling of the polypeptide of apparent molecular weight 127,000 in the presence of beta-lactam antibiotics and dipeptides, whereas glucose, taurocholate or amino acids had no effect on the labeling pattern. These data demonstrate an interaction of cephalosporins and dipeptides with a common membrane protein of molecular weight 127,000, which could be a component of the intestinal transport system(s) responsible for the uptake of orally effective cephalosporins and dipeptides.

Affinity Labels↗
PubMed1981

Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes.

2-Azido-4-ethylamino-6-isopropylamino-s-triazine (azido-atrazine) inhibits photosynthetic electron transport at a site identical to that affected by atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine). The latter is a well-characterized inhibitor of photosystem II reactions. Azido-atrazine was used as a photoaffinity label to identify the herbicide receptor protein; UV irradiation of chloroplast thylakoids in the presence of azido[(14)C]atrazine resulted in the covalent attachment of radioactive inhibitor to thylakoid membranes isolated from pea seedlings and from a triazine-susceptible biotype of the weed Amaranthus hybridus. No covalent binding of azido-atrazine was observed for thylakoid membranes isolated from a naturally occurring triazine-resistant biotype of A. hybridus. Analysis of thylakoid polypeptides from both the susceptible and resistant A. hybridus biotypes by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by fluorography to locate (14)C label, demonstrated specific association of the azido[(14)C]atrazine with polypeptides of the 34- to 32-kilodalton size class in susceptible but not in resistant membranes.

Journal Article↗
PubMed1991

Reversal of multidrug resistance by calcium channel blocker SR33557 without photoaffinity labeling of P-glycoprotein.

The altered pharmacology of drugs in multidrug-resistant cells (decreased accumulation and retention) appears to be mediated by a high molecular weight integral membrane protein, called P-glycogprotein (P-gp). Agents known to reverse this pleiotropic drug resistance (chemosensitizers) have been shown to interact with P-gp; and as such, the inhibition of photoaffinity labeling by P-gp probes (such as [3H]azidopine) has been proposed as a basis for mass screening of chemosensitizers. In this study, we provide direct evidence that a novel calcium channel blocker (SR33557), which was 4.5 times more potent in sensitizing P388/ADR cells to doxorubicin as compared to verapamil (while inducing a similar increase in uptake and decrease in efflux of [14C]doxorubicin, did not compete for the [3H]azidopine-binding site on P-gp, whereas verapamil did. Moreover, SR33557, which is inherently photoactivable, did not photolabel P-gp, but a 65-kDa protein did appear to be an acceptor; and this binding was displaced by diltiazem and nifedipine, but not by verapamil. Finally, the implication for the participation of a sphingomyelin/sphingosine cycle (as a potential lipid second messenger system) in the chemosensitization of P388/ADR cells was investigated. 30 microM SR33557 induced a 72% inhibition in acid lysosomal sphingomyelinase activity, a 5-fold increase in sphingosine levels, and a 75% inhibition in intracellular protein kinase C activity. Although no direct link is established between these observations and P-gp activity, further studies on a possible sphingosine-mediated regulation of P-gp may yield information on the involvement of this second messenger system in the action of SR33557.

Animals↗
PubMed1988

Photoaffinity labeling of pyruvate kinase from rabbit muscle.

Several studies have been performed on the structure of muscle pyruvate kinase. X-ray diffraction has provided a three-dimensional picture of the active site, and chemical modification studies have revealed essential amino acid residues for substrate binding or catalysis. We have shown that 8-azido-ADP (N3 ADP) behaves as a photoaffinity label for the enzyme. This reagent upon irradiation produces inactivation of the enzyme, and the activity loss is protected by nucleotides. The partially modified enzyme shows the same Km for ADP as the native one suggesting an "all or none" inactivation effect. The incorporation of 1 mole of 14C-N3 ADP per subunit correlates with complete inactivation. A radioactive peptide was isolated from the enzyme labeled with 14C-N3 ADP. The partial sequence of this peptide showed that it corresponds to the same peptide isolated from rabbit muscle pyruvate kinase labeled with dialdehyde-ADP and with trinitrobenzenesulfonate. This peptide is identical to a region in the cat and chicken muscle enzymes, and also a high degree of homology is found in a region of the rat liver and yeast enzymes. These studies show that N3 ADP binds to the same site as dialdehyde-ADP in rabbit muscle pyruvate kinase, and this site seems to be the nucleotide binding site.

Adenosine Triphosphatases↗
PubMed1992

Photoaffinity labeling of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) on intact platelets with 8-azido-[gamma-32P]ATP.

The fibrinogen receptor GPIIb-IIIa plays a crucial role in platelet aggregation. Here we show that the adenine nucleotide, 8-azido-ATP, inhibits ADP-induced conformational change of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3). Photoaffinity labeling of intact platelets with 8-azido-[gamma-32P]ATP exclusively modifies two plasma-membrane glycoproteins which are identical with both subunits of GPIIb-IIIa. The presence of adenine-nucleotide-binding sites on GPIIb-IIIa implies that the platelet fibrinogen receptor is directly regulated by extracellular adenine nucleotides.

Adenosine Diphosphate↗
PubMed1987

Photoaffinity labeling of opioid receptor of rat brain membranes with 125I(D-Ala2, p-N3-Phe4-Met5)enkephalin.

A photoreactive (D-Ala2, p-N3-Phe4-Met5)enkephalin derivative was prepared, iodinated with carrier-free 125I, and then purified by high-performance liquid chromatography. The purified radioactive photoprobe was monoiodinated at the amino terminal tyrosine residue. This radioactive photoprobe was used to photoaffinity label membranes prepared from the rat brain (minus cerebellum) and the spinal cord. The photolabeled membranes were analyzed by sodium dodecyl sulfate gel electrophoresis. A 46,000-Da protein was specifically photolabeled in these membrane preparations. The photolabeling of this protein was inhibited by peptides related to enkephalin but not by unrelated substance P or gastrin tetrapeptide. A concentration-dependent inhibition of the photolabeling of the 46,000-Da protein was observed in the presence of competing ligands specific for the mu-, delta-, and kappa-opioid receptors. These data demonstrate that the radioactive photoprobe labels the mu-, delta-, and kappa-opioid receptors. Although there is no evidence available to show that the 46,000-Da protein is identical in all the cases, our data strongly suggest that it is a binding protein common to all of the opioid receptor subtypes.

Affinity Labels↗