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PubMed1990

Direct photoaffinity labeling of gizzard myosin with [3H]uridine diphosphate places Glu185 of the heavy chain at the active site.

The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with [3H]UDP. [3H] UDP was stably trapped at the active site by addition of vanadate (Vi) and Co2+. The extraordinary stability of the myosin.Co2+.[3H]UDP.Vi complex (t1/2 greater than 5 days at 0 degrees C) allowed it to be purified free of extraneous [3H]UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped [3H]UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results (Maruta, H., and Korn, E. (1981) J. Biol. Chem. 256, 499-502) using [3H]UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a "zero-length" cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by [3H]UTP photolabeling in Acanthamoeba myosin II (Atkinson, M. A., Robinson, E. A., Appella, E., and Korn, E. D. (1986) J. Biol. Chem. 261, 1844-1848).

Affinity Labels↗
PubMed1990

Flunitrazepam photoaffinity labeling of the GABA(A) receptor reduces inhibition of [3H]Ro15-4513 binding by GABA.

The benzodiazepine drugs modulate gamma-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA(A) receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [3H]Ro15-4513, [3H]flumazenil (Ro15-1788), and [3H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [3H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]Ro15-4513. The increase in [3H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Kd, with no change in Bmax. Following photoaffinity labeling, negative modulation of [3H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [3H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [3H]Ro15-4513.

Affinity Labels↗
PubMed1989

Direct photoaffinity labeling of leukotriene binding sites.

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.

Animals↗
PubMed1984

Photoaffinity-labeled hapten-binding T-cell receptor on a suppressor T-cell hybridoma.

A T-cell hydridoma, 7C3-13-Ag6, which produces a (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific suppressor T-cell factor associated with an I-J determinant, was utilized to study the hapten-binding receptor of T-cells. This hybridoma had been shown to express NP-binding receptor molecules on the cell surface with heteroclitic fine specificity for a cross-reactive hapten, (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP). The stoichiometric analysis of the hapten binding by 7C3-13-Ag6 cells was performed by the measurement of direct binding of highly radioactive haptens to the cell surface. The affinity constant (Ka) of the receptor for N125IP-epsilon-aminocaproic acid (N125IP-cap), as calculated from a Hill plot, was 5.75 X 10(7) M-1 [Hill coefficient (a) = 0.86; expression of receptor sites per cell = approximately 1 X 10(3) on average]. The receptor molecule was specifically affinity labeled with photoreactive nitroaryl azide derivatives of N125IP (510-570 Ci/mmole). The specificity of photoaffinity labeling was demonstrated both by competitive inhibition of labeling with NIP- or NP-cap and by differential photoaffinity labeling based on the reversibility of hapten-receptor interaction. The gel electrophoretic analysis of the photoaffinity-labeled molecule indicated that the hapten-binding receptor of 7C3-13-Ag6 has a mol. wt of 28,000 +/- 3000 and an isoelectric point of 5.6-5.7. No immunoglobulin determinants were detected on the molecule. A comparative immunoprecipitation analysis of the membrane lysate of 7C3-13-Ag6 with monoclonal anti-I-J reagents identified a separate I-J molecule of 25,000 +/- 1000 mol. wt that is distinct from the photoaffinity-labeled hapten-binding molecule.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5.

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.

Affinity Labels↗
PubMed1981

Isolation and characterization of a photoaffinity-labeled peptide from the catalytic site of prenyltransferase.

Previously we presented evidence for the selective modification of the catalytic site of prenyltransferase by photoaffinity labeling with o-azidophenylethyl pyrophosphate [Brems, D. N., & Rilling, H. C. (1979) Biochemistry 18, 860]. In the present work, we report the isolation and characterization of a CNBr fragment of 30 amino acid residues from the photoaffinity-labeled enzyme. This CNBr fragment contains over 809% of the total label attached to prenyltransferase as a result of photoaffinity labeling. Several lines of evidence indicate that a number of residues in this CNBr fragment have been modified. First, Edman degradation of this labeled peptide demonstrates that at least 16 of the 30 amino acids have been modified by the photoaffinity reagent. The two most extensively modified amino acids are a specific arginine and alanine. Second, two-dimensional chromatography of Pronase digestions of the labeled CNBr fragment indicates that at least 11 different products resulted from photoaffinity labeling. Third, peptide maps of a trypsin digest of this CNBr fragment show that the attached affinity label is distributed among at least three of the resulting products of tryptic hydrolysis. Finally, comparison of amino acid analysis of this CNBr fragment with that of its counterpart isolated from native enzyme is consistent with the modification of a number of amino acids rather than a few y the photoaffinity labeling process.

Affinity Labels↗
PubMed1986

Solubilization and separation of the human erythrocyte D-glucose transporter covalently and noncovalently photoaffinity-labeled with [3H]cytochalasin B.

The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [3H]cytochalasin B and solubilized with n-octyl beta-D-glucopyranoside (octyl glucoside). [3H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [3H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [3H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. With preparative NaDod-SO4/polyacrylamide gel electrophoresis, the radioactivity of peak I could be released, but that of peak II remained with the D-glucose transporter. These results indicate that [3H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of conformational changes of the protein as a result of covalent binding with [3H]cytochalasin B by photoaffinity labeling.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of felodipine-binding proteins in vascular smooth muscle.

[3H]-Felodipine and high-intensity ultraviolet irradiation were used in the photoaffinity labeling of soluble proteins prepared from porcine mesenteric vascular smooth muscle. Irradiation of the soluble proteins in the presence of [3H]-felodipine resulted in the labeling of a protein with an apparent molecular weight of 62 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Labeling of the protein did not occur without ultraviolet irradiation. An [3H]-azido analog of felodipine was found to show less specificity than felodipine in its protein labeling when irradiated, since proteins with apparent molecular weights of 44, 29, and 14, as well as 62 kDa, were labeled. The photoaffinity labeling of the proteins were inhibited by excess of unlabeled felodipine.

Affinity Labels↗
PubMed1984

Photoaffinity labeling of acetylcholine receptor in millisecond time scale.

Photoaffinity labeling of acetylcholine receptors can be performed with a time resolution allowing to discriminate reaction sites within the receptor protein in its different functional states. This is achieved by a combination of a stopped-flow apparatus with a high energy pulse laser. The photoaffinity label used is the lipophilic cation [3H]TPMP+ which has been shown to be a non-competitive antagonist and a specific ion channel blocker. AChR in its resting (channel closed) and active (channel open) state incorporates the label mainly into the alpha-polypeptide chain of the receptor. Only several hundred milliseconds after mixing AChR with agonist labeling of delta-chains becomes significant.

Affinity Labels↗
PubMed1980

Structure-function characterization for ethidium photoaffinity labels as mutagens in Salmonella.

The development of photoaffinity probes to characterize the binding process and subsequent biological activity of a drug has recently been emphasized by the synthesis of two ethidium azide analogs. The initial finding showed that one of the azido analogs, the 8-azido-3-amino derivative, was at least 40-fold more mutagenic and toxic in Salmonella tester strain TA1538 than the other analog, the 3.8-diazido derivative. These observations suggested the need to examine the structural requirements of ethidium photoaffinity labels for frameshift mutagenic activity in Salmonella. Thus, the isomer of the monoazide, the 3-azido-8-amino derivative, and two deaminated monoazide derivatives were synthesized and all of the ethidium analogs were screened in two Salmonella frameshift tester strains, TA1537 and TA1538, and in their excision-repair positive isogenic strains. The results presented in this paper demonstrate that two substituents are needed to produce significant mutagenicity and toxicity by the compound. One substituent, usually the amino group, is required for mutagenic activity, perhaps by orienting the phenanthridinium ring into its mutagenic configuration. The other substituent, the azido group, is required for covalent attachment, a requisite for mutagenic activity. Thus, photoaffinity labeling has provided a means of comparing structure with mutagenic activity for ethidium compounds.

Affinity Labels↗
PubMed1990

The sites in the I-Ak histocompatibility molecule photoaffinity labeled by an immunogenic lysozyme peptide.

The class II histocompatibilty molecule I-Ak was photoaffinity labeled by NH2- and COOH-terminal photoreactive conjugates of an immunogenic hen egg white lysozyme (HEL) peptide. The labeled alpha and beta chains were digested with protease from Staphylococcus aureus strain V-8 (protease V-8) and/or trypsin, and the proteolytic fragments were separated by high performance liquid chromatography (HPLC) (peptide mapping). Reproducible peptide maps containing a major labeled component were obtained from the three conjugates reported here whose photoreactive group was attached via short spacers of limited flexibility. The COOH-terminal conjugate N-acetyl HEL-(49-61)-iodo-4-azidosalicyloyl thioester (compound 1) labeled hydrophilic tryptic digest fragments on both chains of I-Ak. The labeled digest fragments were homogeneous in reverse-phase and anion-exchange HPLC, indicating that the photoaffinity labeling was site-specific. Conversely, the NH2-terminal conjugate iodo-4-azidosalicyloyl HEL-(46-61) (compound 2: IASA-(46-61)) labeled exceptionally hydrophobic sequences on both chains of I-Ak. The labeling was also site-specific because reverse-phase HPLC of primary digests with protease V-8 and secondary digests with trypsin showed single major labeled components. The labeling of I-Ak by IASA-(46-61) was fully inhibitible by HEL-(46-61). In contrast, IASA attached to the smallest immunogenic peptide 52-61 (compound 3) labeled a distinctly different hydrophilic tryptic fragment. The site of the I-Ak molecule that was photoaffinity labeled by IASA-(46-61) (compound 2) was determined. IASA-(46-61) labeled selectively at Pro-118 of a primary alpha chain fragment most likely encompassing residues 115-134. It labeled Thr-121 of a primary beta chain fragment most likely encompassing residues 109-138. We also obtained evidence that IASA-(46-61) occupied the antigen-specific site; the conjugate stimulated a T-cell hybridoma that recognizes the sequence 52-61 and also competed for the binding of this smaller peptide to I-Ak. Thus, peptides that bind to the allele-specific binding site and are long enough to extend beyond it can interact with a hydrophobic area of class II molecules. This area is formed by sequences of the first halves of the second domain of both alpha and beta chains.

Affinity Labels↗
PubMed1988

Identification of the monkey lens glucose transporter by photoaffinity labelling with cytochalasin B.

Polypeptide constituents of the lens glucose transporter have been identified by photoaffinity labelling with cytochalasin B. The urea-insoluble fraction of monkey lens was irradiated at 280 nm for 30 min in the presence of 5 X 10(-7) M 3H-cytochalasin B. After extensive washing, the membranes were solubilized and their polypeptide composition determined by SDS-PAGE. Radioactivity was extracted from gel slices to determine the position of photoincorporated label. 3H-cytochalasin B was irreversibly incorporated into a broad molecular weight region from Mr greater than 94,000 to 43,000 with the peak of activity occurring at Mr 53,000. Photoincorporation was inhibited by D-glucose (500 mM) and phloretin (1 X 10(-5] but was unaffected by L-glucose (500 mM), cytochalasin E (1 X 10(-5) and phloridzin (1 X 10(-5) M). Cortex and nucleus membrane preparations contained the same range of labelled polypeptides after photoaffinity labelling but nuclear membranes contained approximately twice the activity of cortical membranes indicating an enrichment of glucose transporters in the nucleus. Treatment of labelled membranes with endoglycosidase F converted the broad band of labelling to a sharp band of Mr 45,000. The lens glucose transporter is therefore a glycoprotein and the broadness of the photaffinity labelled peak is due to heterogeneous N-linked glycosylation of a core polypeptide. From these studies it appears that the monkey lens glucose transporter closely resembles that of the human erythrocyte.

Affinity Labels↗
PubMed1985

Mapping labeled sites in Escherichia coli ribosomal RNA: distribution of methyl groups and identification of a photoaffinity-labeled RNA region putatively at the peptidyltransferase center.

We have developed a method for the rapid localization of sites of ribosomal RNA labeling to limited regions (approximately 200 bases). The method is based on the formation and polyacrylamide gel electrophoretic separation of hybrids between restriction fragments of rrnB DNA and isotopically labeled rRNA and the subsequent determination of radioactivity across the gel. Using [3H]adenine-labeled rRNA as a control sample, we optimized experimental conditions with respect to a number of variables, including rRNA:DNA stoichiometric ratio, temperature of the annealing step, and levels of nucleases. An important result is that different rRNA X DNA hybrid fragments are obtained in different yields. The method was then applied to analyses of C3H3-labeled rRNA, giving results in good accord with known and proposed sites of rRNA methylation, and of rRNA that has been photoaffinity-labeled with 5-azido-2-nitrobenzoyl-[3H]Phe-tRNAPhe, a probe directed toward the peptidyltransferase center. The latter study showed a single major site of RNA labeling, falling within bases 2445-2668 of 23S rRNA. The extent of labeling was shown to be dependent on light-induced formation of a reactive intermediate and to be decreased in the absence of poly(uridylic acid) or in the presence of puromycin. The location of this major site of labeling is consistent with recent results obtained with an analogous tRNA photoaffinity label [Barta, A., Steiner, G., Brosius, J., Noller, H. F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611] and with related genetic and biochemical studies of antibiotic interaction with ribosomes suggesting that the peptidyltransferase center falls within region V (bases 2043-2625) of 23S rRNA.

Acyltransferases↗
PubMed1988

The oligosaccharide component of alpha 1-adrenergic receptors from BC3H1 and DDT1 muscle cells. Studies with glycosidases and photoaffinity labelling of intact cells.

In this study, we clarify the structural aspects of the oligosaccharides associated with the alpha 1-adrenergic receptor in two muscle cell lines. Photoaffinity labelling of intact BC3H1 or DDT1 muscle cells with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline ([125I]azidoprazosin) followed by SDS/polyacrylamide-gel electrophoresis (PAGE) and autoradiography revealed specifically labelled proteins of molecular mass = 87,000 and 81,000, respectively. Treatment of photoaffinity-labelled receptors in DDT1 cells with 33 u. of endoglycosidase F/ml for 24 h resulted in the loss of the 81 kDa receptor and the appearance of a 52.5 kDa protein. When lower concentrations of glycosidase or shorter incubation times were used, the 81 kDa receptor was converted to a 66 kDa protein. Treatment of the photoaffinity-labelled BC3H1 receptor with endoglycosidase F resulted in the appearance of a 50.5 kDa protein. Neither alpha-mannosidase nor endoglycosidase H had an effect on the photoaffinity labelling patterns of the receptor from the two cell types. alpha 1-Adrenergic receptors, solubilized from membranes prepared from BC3H1 and DDT1 cells, bound to wheat germ agglutinin-Sepharose and were displaced by N-acetylglucosamine. Taken together, these results indicate that alpha 1-adrenergic receptors in BC3H1 and DDT1 cells contain complex, but not high, mannose oligosaccharide chains; differences in the composition or number of chains partially accounts for the different molecular mass of the receptor in the two cell lines. The results further indicate that the oligosaccharide chains contribute substantially to the apparent molecular mass of alpha 1-adrenergic receptors, as detected by SDS/PAGE, and that the protein backbone of these receptors is likely to be approximately 50 kDa.

Acetylglucosaminidase↗
PubMed1985

Regional heterogeneity of rat brain phencyclidine (PCP) receptors revealed by photoaffinity labeling with [3H] azido phencyclidine.

Photoaffinity labeling of rat brain phencyclidine (PCP) receptors with [3H] azido phencyclidine ([3H]AZ-PCP) reveals the existence of five polypeptides which are specifically labeled by the affinity probe (Mr's 90,000, 62,000, 49,000, 40,000 and 33,000). These labeled components are unevenly distributed in rat brain. In the frontal cortex, thalamus and olfactory bulb, the major bands labeled are the Mr's 90 K and 62 K polypeptides; in the cerebellum most of the labeling is in the 90 K and 33 K bands; and in the hippocampus all but the Mr 40 K band are heavily labeled. Together with dexoxadrol/[3H]PCP competition binding data, which indicated the existence of high and low affinity dexoxadrol/PCP binding sites, these results suggest regional heterogeneity of PCP receptors. The regional distribution of the high affinity dexoxadrol binding sites correlates best with that of the Mr 90 K polypeptide.

Affinity Labels↗
PubMed1989

Azido derivatives of dicarboxylic acids for photoaffinity labeling of mitochondrial carriers.

New photoaffinity probes, N-(4-azidosalicylic)-aminosuccinic acid, 3-(4-azidophenylazo)-4-hydroxyphenylmalonic acid, (4-azido-2-nitroanilino)-N-succinic acid, 4-azidophenacylthiosuccinic acid and 4-azidophenylsuccinic acid, were synthesized and characterized chemically. They differ in the distance between dicarboxylic and azido groups, hydrophobicity and acidic moiety. These between dicarboxylic and azido groups, hydrophobicity and acidic moiety. These reagents can be applied for photoaffinity labeling of mitochondrial anion carriers and enzymes interacting with dicarboxylic acids. Inhibition and labeling of the dicarboxylate carrier is presented.

Affinity Labels↗
PubMed1985

Comparative characterization of thyroid hormone receptors and binding proteins in rat liver nucleus, plasma membrane, and cytosol by photoaffinity labeling with L-thyroxine.

Photoaffinity labeling with underivatized thyroxine (T4) was used to identify and compare the T4 binding proteins in rat liver cytosol, nuclear extract, and purified plasma membrane. When these subcellular fractions were incubated with a tracer concentration of [125I]T4, irradiated with light above 300 nm, and individually analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactivity profiles revealed the presence of T4 binding proteins of molecular masses of 70, 52, 43, 37, 30, and 26 kilodaltons (kDa) in cytosol, of 96, 56, 45, and 35 kDa in nuclear extract, and of 70, 44, and 30 kDa in plasma membrane. Competition experiments performed in the presence of a 1000-fold excess of unlabeled T4 demonstrated that these binding proteins display different hormone binding activities. The similar electrophoretic mobilities of some binding proteins present in the different subcellular fractions, i.e., the 70-, 43-45-, and 30-kDa proteins, suggested that these proteins might be identical. However, double-labeling experiments in which plasma membrane, nuclear extract, and cytosol were photolabeled with either [125I] or [131I]T4 and mixed, two at a time, in all possible combinations showed that from one cellular fraction to another, the radioactivity peaks corresponding to the approximately 70-, 43-45-, and 30-kDa proteins were not superimposed. Their relative positions on the gel differed by one or two slices, which indicated differences in molecular mass of 1.9-3.6 kDa. Moreover, enzymatic digestion with Staphylococcus aureus V8 protease of these three proteins, prepared from each subcellular fraction, yielded dissimilar peptide patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1980

Photoaffinity labeling of corticotropin receptors.

A photoaffinity label for corticotropin (ACTH) receptors was prepared by selective chemical modification of the single tryptophan residue in the hormone by reaction with 2-nitro-5-azidophenylsulfenyl chloride. The photoreactive derivative, [(2-nitro-5-azidophenylsulfenyl)-Trp9]ACTH (2,5-NAPS-ACTH), stimulated corticosterone synthesis to 60% of the maximal rate induced by ACTH in isolated rat adrenocortical cells. 2.5-NAPS-ACTH caused only a marginal stimulation of cyclic AMP production compared to the unmodified hormone. Stimulation of corticosterone production and cyclic AMP accumulation induced by ACTH were both inhibited in a competitive manner by 2,5-NAPS-ACTH. Photolysis of adrenocortical cells in the presence of 2,5-NAPS-ACTH resulted in a 40% inactivation of ACTH receptors mediating steroidogenesis, as shown by the decrease in response to subsequent stimulation with ACTH. No loss of function was observed when photolysis was conducted in the presence of the photoresistant analog [(2,4-dinitrophenylsulfenyl)-Trp9]ACTH. Covalent attachment of the hormone to the receptors was also demonstrated by photolyzing adrenocortical cells in the presence of tritiated 2,5-NAPS-ACTH of high specific radioactivity (90 Ci/mmol) and analyzing the cell proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A protein with an approximate molecular weight of 100,000 was specifically labeled by this procedure. The unique labeling of an adrenocortical cell protein and the concomitant loss of ACTH responsiveness suggest that physiologically relevant receptors are photolabeled by this method.

Adrenal Cortex↗