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PubMed1989

Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate.

In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1986

Purification of a benzo[a]pyrene binding protein by affinity chromatography and photoaffinity labeling.

Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29,000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30,000 was estimated by gel filtration chromatography of [3H]B[a]P- and [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 +/- 0.66 nM and a maximum number of binding sites of 2.07 +/- 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29,000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1-azidopyrene.

Affinity Labels↗
PubMed1986

Photoaffinity labelling of androgen receptors from human foreskin and cultured genital fibroblasts with tritiated methyltrienolone [3H]R1881.

Androgen receptors were partially purified by affinity chromatography of cytosols prepared from either normal foreskins or normal cultured fibroblasts. Partially purified receptors were covalently labelled with [3H]R1881 (tritiated methyltrienolone) by ultraviolet photoactivation. Gel electrophoresis of cytosols from both sources showed two peaks of specific binding, Mr approximately 40 K and approximately 85 K, under denaturing conditions. Photoaffinity labelling of the human androgen receptor may thus provide a useful tool in further studies on disorders of androgenization.

Affinity Labels↗
PubMed1988

Photoaffinity labeling of the acetylcholine binding sites on the nicotinic receptor by an aryldiazonium derivative.

p-(Dimethylamino)benzenediazonium fluoroborate (DDF) behaves, in the dark, as a reversible competitive antagonist of the electrical response of Electrophorus electricus electroplaque to acetylcholine and of the acetylcholine-gated single-channel currents recorded in the C2 mouse cell line. This chemically stable but highly photoreactive compound binds irreversibly to the acetylcholine receptor when irradiated by visible light. In vivo, it irreversibly blocks the postsynaptic response of E. electricus electroplaque to agonists. In vitro, it reduces the alpha-bungarotoxin-binding capacity of acetylcholine receptor rich membrane fragments prepared from Torpedo marmorata electric organ. Once reversibly bound to the T. marmorata acetylcholine receptor, this ligand can be selectively photodecomposed by an energy-transfer reaction involving a tryptophan residue(s) of the protein. By use of reagent concentrations that are below the dissociation constant at equilibrium, up to 60% of the agonist-binding sites are covalently labeled. Under these conditions the alpha subunit of the acetylcholine receptor is preferentially labeled, and this labeling is partially prevented by agonists or competitive antagonists. This protective effect is substantially increased by prior incubation with phencyclidine, a compound known to prevent the binding of DDF at the level of the high-affinity site for noncompetitive blockers [Kotzyba-Hibert, F., Langenbuch-Cachat, J., Jaganathen, J., Goeldner, M. P., & Hirth, C. G. (1985) FEBS Lett. 182, 297-301]. The incorporation of about one molecule of label in an agonist/competitive antagonist protectable manner per alpha-bungarotoxin-binding site suffices to fully block alpha-bungarotoxin binding to the membrane-bound receptor. Thus, DDF behaves as a monovalent photoaffinity label of the acetylcholine-binding site.

Acetylcholine↗
PubMed1980

Photoaffinity labelling of arginine kinase and creatine kinase with a gamma-P-substituted arylazido analogue of ATP.

1. An ATP analogue with a photoactivated azide group attached to the gamma-phosphate via an amide bond, ATP gamma-p-azidoanilide, appeared to have potential use as a photoaffinity label for the nucleotide-binding regions of ATP: guanidine phosphotransferases. Upon photolysis in the presence of lobster muscle arginine kinase and rabbit muscle creatine kinase, the analogue is converted to a potent inhibito of these two kinases. This photo-dependent inhibition is specific as it cannot be induced by azidoaniline, a mixture of azidoaniline and ATP or by ATP gamma-p-aminoanilide. Preirradiated under suitable conditions, the photoanalogue still shows a transitory inhibitory effect which, however, slowly vanishes with time (t0.5 = 3 h). 2. The photoinhibition is significantly decreased by the presence of ATP or ADP but is completely prevented by the addition of a mixture of nucleotide and guanidine substrates. Differential spectroscopy and affinity chromatography on Sepharose-ATP demonstrated the inability of photoinactivated arginine kinase and creatine kinase to recognize their nucleotide substrates. 3. Experiments with [14C]ATP gamma-p-azidoanilide indicated that photolysis is associated with an irreversible and stoichiometric binding of the ATP analogue to the enzymes. Autoradiographs made with the peptide maps corresponding to the tryptic digests of each 14C-labelled photomodified enzyme showed an unexpected highly specific labelling of the proteins. 4. Thiiol titrations of the kinases which have been subjected to various photolysis conditions led to the conclusion that the arylnitrene moiety of the photoanalogue is covalently attached to the single reactive cysteinyl side chain present in the active-site region of the two homologous kinases. This amino acid residue appears, therefore, to be located near the phosphate chain binding subsite occupied by the ATP analogue and probably also by the natural nucleotide substrates.

Adenosine Triphosphate↗
PubMed1979

Analogues of [3H] chloramphenicol for photoaffinity labeling.

The synthesis of [3H]chloramphenicol and its erythro-diastereoisomer with specific activities of 1.25 Ci/mmol, and the further transformation of the [3H]chloramphenicol to a series of azido and diazo-substituted derivatives are described. The antibiotic activity of the compounds was considered insufficient for their use as photoaffinity labels.

Affinity Labels↗
PubMed1984

Specific photoaffinity labeling of the cAMP surface receptor in Dictyostelium discoideum.

The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.

Azides↗
PubMed1982

Identification of the fibrinogen receptor on human platelets by photoaffinity labeling.

Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. In order to identify the platelet fibrinogen receptor, we modified fibrinogen with the photoreactive, heterobifunctional cross-linking reagent methyl 4-azidobenzoimidate (MABI). MABI-fibrinogen was fully clottable and able to support platelet aggregation. To photoaffinity label the fibrinogen receptor, gel-filtered human platelets were incubated at 37 degrees C in the dark with 200 micrograms/ml of MABI-fibrinogen, 10 microM ADP, and 0.5 mM calcium. Irradiation of these platelets with ultraviolet light resulted in the incorporation of MABI-fibrinogen into the platelet surface. Incorporation could be prevented by excess native fibrinogen suggesting that MABI-fibrinogen had interacted with the fibrinogen receptor before photolysis. Examination of the irradiated platelets by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the photoactivated MABI-fibrinogen had been incorporated into a 105,000 molecular weight membrane polypeptide that also contained the PlA1 antigen. Thus, this polypeptide has the characteristics of the membrane glycoprotein IIIa. Previous studies have shown that thrombasthenic platelets lack this glycoprotein and fail to bind fibrinogen after stimulation by ADP. Consequently, our data suggest that glycoprotein IIIa constitutes at least one component of the platelet fibrinogen receptor.

Affinity Labels↗
PubMed2005

Insights into interactions between the alpha-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling.

Fish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT(8-32) containing a single photolabile p-benzoyl-l-phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa(19)]sCT(8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys(134)-Lys(141) (within the amino terminus of the receptor, adjacent to transmembrane 1) (Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met(49) as the cross-linking site for [Bpa(8)]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa(8)]human CT (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa(8) analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core.

Amino Acid Sequence↗
PubMed1979

Photoaffinity labeling of the catalytic site of prenyltransferase.

Three photoreactive substrate analogues, o-azidophenethyl pyrophosphate, p-azidophenethyl pyrophosphate, and 3-azido-1-butyl pyrophosphate, have been synthesized as site-directed probes to label the catalytic site of prenyltransferase. Due to the relatively poor affinity of p-azidophenethyl pyrophosphate and 3-azido-1-butyl pyrophosphate for the enzyme, only o-azidophenethyl pyrophosphate (aryl azide) was utilized for photoaffinity labeling. This aryl azide has a UV absorption maximum at 250 nm. In the absence of activating light, binding studies demonstrate that the o-aryl azide competes for binding with both the natural substrates, isopentenyl pyrophosphate and geranyl pyrophosphate. More than 90% enzymatic activity is lost when enzyme is irradiated in the presence of the aryl azide as compared to irradiation in the absence of the azide, and the protein loses its capacity for substrate binding in direct proportion to photolabeling. A stoichiometry of 2 mol of affinity label covalently bound per mol of enzyme dimer was established with [1-3H]-o-azidophenethyl pyrophosphate. Since there are two catalytic sites per enzyme dimer, the o-aryl azide appears specifically to label the enzyme at its catalytic sites. Additional evidence that the reagent was specific for the catalytic site came from the observation that farnesyl pyrophosphate afforded complete protection against photoinactivation, while isopentenyl pyrophosphate provided partial protection. Gel isoelectric focusing verified this stoichiometry and indicated that the labeled enzyme has a more acidic isoelectric point than the native enzyme.

Affinity Labels↗
PubMed1990

Properties of the nucleic acid photoaffinity labeling agent 3-azidoamsacrine.

This paper reports the study of the photochemical, physical, and biological properties of 3-azidoamsacrine. The binding of 3-azidoamsacrine to DNA was studied with UV spectroscopy. The UV spectral behavior is quite similar to that of the parent amsacrine and argues that 3-azidoamsacrine is a good photoaffinity labeling agent for amsacrine. The biological properties (cytotoxicity and mutagenicity) of 3-azidoamsacrine in the mammalian mutagenesis V79 and L5178Y assay systems were measured. Light-activated 3-azidoamsacrine is toxic, but not mutagenic, to V79 cells. 3-Azidoamsacrine with and without light activation, as well as amsacrine, are toxic and mutagenic to L5178Y cells. To probe the interactions of 3-azidoamsacrine with DNA, studies of the photoreactivity of this compound were conducted. 3-Azidoamsacrine was photolyzed in the presence of the plasmid pBR322, and the effect of the photoadducts on restriction endonuclease cleavage was investigated. Amsacrine and 3-azidoamsacrine, without light activation, did not block any of the restriction endonucleases. Light-activated 3-azidoamsacrine blocked cleavage by the restriction endonucleases AluI, HinfI, NciI, NaeI, DraI, Sau96I, HpaII, and HaeIII. Photolysis experiments with mononucleosides, blocked mononucleosides, dinucleotides, and DNA all indicated that 3-azidoamsacrine formed adducts with G and A. The structures of these adducts are discussed based upon mass spectral data. Thus, it appears that 3-azidoamsacrine covalently attaches to DNA and that this covalent binding results in the production of toxic and, in some cases, mutagenic lesions in mammalian cells and the inhibition of restriction endonuclease cleavage of DNA.

Affinity Labels↗
PubMed1990

Photoaffinity labelling of the beta-methoxyacrylate binding site in bovine heart mitochondrial cytochrome bc1 complex.

A carbene-generating 14C-labelled beta-methoxyacrylate derivative ((E)-methyl-3-methoxy-2-[4-(3-trifluoromethyl-3-diazirinyl) benzoyloxyphenyl] propanoate, uniformly labelled with 14C in the benzene ring of the benzoyl group) has been used to locate the proteins involved in binding this class of inhibitors to bovine heart mitochondrial ubihydroquinone:cytochrome c oxidoreductase. The beta-methoxyacrylate photoaffinity label was shown to be a competent inhibitor of electron transport through the protein complex. Under illumination through a narrow bandpass filter, allowing specific photolysis of the diazirine group, the compound bound to cytochrome b and weakly to an 8 kDa polypeptide. Apart from some binding to a cytochrome b aggregate, other proteins were left unlabelled. The binding could be prevented in the presence of excess amounts of unlabelled beta-methoxyacrylate, myxothiazol or stigmatellin but not by antimycin A or HQNO. At high concentrations DBMIB partially competed for the binding site. The binding site for this class of inhibitors is therefore the 'o-site'. Our results indicate that this site is comprised of residues from cytochrome b and possibly the 8 kDa polypeptide and that the site may be close to the Reiske iron-sulphur protein.

Acrylates↗
PubMed1987

Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine-sensitive calcium channels.

Partially purified fractions of dihydropyridine and phenylalkylamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalkylamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner.

Affinity Labels↗
PubMed1988

Anions induce conformational changes and influence the activity and photoaffinity-labelling by 8-azido-ATP of isolated cytochrome c oxidase.

The biphasic effect of anions on the activity of isolated bovine heart cytochrome c oxidase is paralleled by changes in the visible oxidized spectra, indicating the different conformational changes in the enzyme induced by bromide, chloride, sulphate, phosphate, ADP and ATP. Photoaffinity-labelling of most subunits of the isolated enzyme by low concentrations of 8-azido-[gamma-32P]ATP is strongly increased by ATP, ADP and unlabelled 8-azido-ATP in an unspecific manner. With the reconstituted enzyme less subunits are labelled and this labelling is only little affected by nucleotides. The data suggest a highly dynamic structure for isolated bovine heart cytochrome c oxidase.

Adenosine Triphosphate↗
PubMed1990

A 75-kDa polypeptide, located primarily at the plasma membrane of carrot cell-suspension cultures, is photoaffinity labeled by the calcium channel blocker LU 49888.

Calcium channel blockers of the phenylalkylamine family bind specifically to membranes and inhibit calcium uptake in carrot protoplast. LU 49888, an azido derivative of phenylalkylamine, behaves as its unmodified homolog in terms of affinity and specificity and therefore allows us to probe the receptor by photoaffinity labeling. Upon UV irradiation, a 75-kDa peptide was specifically labeled. Incubation of microsomes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent, led to the solubilization of the LU 49888-binding protein. Electrophoretic analysis under denaturing conditions and gel filtration of the solubilized "receptor-ligand" complex show a 75-kDa peptide mainly located at the plasma membrane. Consequently the LU 49888-binding protein in plants differs significantly from its animal counterpart by its size and may be a primary target for external signal molecules.

Journal Article↗
PubMed1990

Photoaffinity labelling of isopenicillin N synthetase.

The alpha-aminoadipoyl group of the natural substrate of isopenicillin N synthetase (IPNS), L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), has been replaced by a diazirinyl-containing group, which can be photoactivated. This has allowed investigation of the substrate binding site of IPNS by photoaffinity labelling. Laser flash photolysis of this analogue, [3H]DCV, in the presence of IPNS leads to the incorporation of radioactivity into the enzyme. Tryptic digestion of this labelled enzyme, followed by separation and sequencing of the resultant fragments, identified two labelled regions of the protein. These are the fragments Asp-40 to Arg-78 and Thr-237 to Gly-256.

Affinity Labels↗
PubMed1990

Equivalent molecular mass of cytosolic and nuclear forms of Ah receptor from Hepa-1 cells determined by photoaffinity labeling with 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin.

The structure of the Ah receptor previously has been extensively characterized by reversible binding of the high affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. We report the use of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as a photoaffinity ligand for Ah receptor from the mouse hepatoma cell line Hepa-1c1c9. Both cytosolic and nuclear forms of Ah receptor could be specifically photoaffinity-labeled, which allowed determination of molecular mass for the two forms under denaturing conditions. After analysis by fluorography of polyacrylamide gels run in the presence of sodium dodecyl sulfate, molecular mass for the cytosolic form of Ah receptor was estimated at 92,000 +/- 4,300 and that for the nuclear form was estimated at 93,500 +/- 3,400. Receptor in mixture of cytosol and nuclear extract (each labeled separately with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin) migrated as a single band. These results are consistent with the presence of a common ligand-binding subunit of identical molecular mass in both cytosolic and nuclear complexes.

Affinity Labels↗