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PubMed1995

Photoaffinity labeling of HIV reverse transcriptase: utilization of 2',3'-dideoxyuridylate analog bearing aryl(trifluoromethyl)diazirine moiety as a chain terminator.

In order to develop a photoaffinity labeling reagent for HIV-1 reverse transcriptase, we utilized 2',3'-dideoxy-E-5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl] styryl]UTP (TDS-ddUTP) for formation of a photolabile primer. This compound was incorporated into the 3'-terminus of the primer by reverse transcriptase activity, and thus the generated photo-reactive primer was able to bind to the enzyme molecule by photo-irradiation.

Affinity Labels↗
PubMed1993

Identification of the fragment photoaffinity-labeled with azidodansyl-rhizoxin as Met-363-Lys-379 on beta-tubulin.

The rhizoxin (RZX)-binding site on porcine brain tubulin was investigated by photoaffinity labeling with the 5-azido-1-naphthalene sulfonyl (azidodansyl) derivative of RZX, nor-rhizoxin-22-al-5'-azidonaphthalene-1'-sulfonylhydrazo ne (azidodansylrhizoxin: Adan-RZX). Upon ultraviolet irradiation, Adan-RZX generates a highly reactive nitrene, which irreversibly binds to an amino acid residue(s) near the RZX-binding site. The label was found to be on beta-tubulin. Enzymatic digestion of the labeled tubulin generated only one major fluorescent peak on C18 reverse phase HPLC analysis. The labeled site(s) was mapped by using various combinations of highly specific peptidases in succession. That is, the labeled fragment generated by the first peptidase was purified by HPLC and exposed to a second peptidase; if the retention time in HPLC changed after the second digestion, the fragment generated in the first digestion must have contained the recognition site(s) of the second enzyme. From the results of these successive digestions and the known polypeptide sequences, we could identify the labeled fragment as Met-363-Lys-379 of beta-tubulin. This peptidase combination technique should be widely applicable.

Affinity Labels↗
PubMed1993

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA.

Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.

Acyl Coenzyme A↗
PubMed1995

Determination of the photoaffinity-labeled site on the ligand-binding domain of retinoic acid receptor alpha.

The ligand-binding domain of human retinoic acid receptor alpha (hRAR alpha) was photoaffinity-labeled with a fluorescent retinoid, ADAM-3, by the use of a recombinant fused protein constructed from a maltose-binding protein and the E/F-domain of hRAR alpha (MBP-RAR alpha/E), which was expressed in E. coli. The labeled site was identified as Arg-589 (this corresponds to amino acid residue 385 of hRAR alpha) or a residue in its vicinity.

Affinity Labels↗
PubMed1992

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane.

The mechanism of steroid uptake by the cell remains controversial. [3H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (Kd = 33 +/- 4 nM, Bmax = 32 +/- 2 pmol/mg). [3H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 +/- 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.

Affinity Labels↗
PubMed1986

Difference in molecular size of receptors for alpha-rat atrial natriuretic polypeptide among the kidney, aorta, and adrenal gland as identified by direct UV-photoaffinity labeling.

In order to identify the molecular size of receptors for alpha-rat atrial natriuretic polypeptide (alpha-rANP), we utilized the direct UV irradiation method for photoaffinity labeling with the biologically active [125I] alpha-rANP. In the preparation of isolated glomerulus and the inner medullary collecting duct (IMCT)-rich fraction, the autoradiograms of the electrophoresed sodium dodecyl sulfate (SDS)-polyacrylamide gels showed a single radioactive band which is displaceable with unlabeled alpha-rANP. The dose-dependent displacement fit very well with a binding-inhibition curve representing the binding affinity of 6.5 X 10(-10) M. The molecular size of the ligand-receptor complex was about 65,000 daltons for both glomerulus and IMCT-rich fraction. In contrast, in homogenate of the aorta and adrenal gland, the ligand-receptor complex was 140,000 daltons.

Adrenal Glands↗
PubMed1988

Photoaffinity labeling of peripheral-type benzodiazepine receptors in rat kidney mitochondria with [3H]PK 14105.

The peripheral-type benzodiazepine receptor in rat kidney has been identified by photoaffinity labeling. PK 14105, a derivative of the selective peripheral-type ligand PK 11195, was used to covalently label peripheral-type benzodiazepine receptors. In the absence of UV light PK 14105 demonstrated reversible, high affinity (KD = 4.8 nM) binding to rat kidney mitochondrial membranes. Inhibition of the reversible binding of [3H]PK 14105 by various benzodiazepine and other ligands demonstrated that this ligand bound with all the characteristics expected of a ligand interacting specifically with peripheral-type benzodiazepine receptors. A similar order of relative potencies was obtained for inhibition of photolabeling, indicating that reversible binding and photolabeling occurred at the same class of binding sites. Examination of photolabeled binding sites from kidney, heart, brain and adrenal membranes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the probe is photoincorporated into a single peptide of Mr = 18,500. The results indicate that [3H]PK 14105 identifies the ligand binding domain of the peripheral-type benzodiazepine receptor, which is a peptide with Mr = 18,500, that is of similar size in kidney, heart, brain and adrenals.

Affinity Labels↗
PubMed1982

Photoaffinity labelling of the rat liver nuclear thyroid hormone receptor with [125I]triiodothyronine.

[125I]Triiodothyronine (T3) was used as a photoreactive probe for the thyroid hormone nuclear receptor in photoaffinity labelling experiments. Autoradiograms of photolysis products electrophoresed on either one or two-dimensional gels showed that [125I]T3 covalently, but nonspecifically, labelled many proteins in the partially purified receptor preparations used. However, one of these proteins with an estimated molecular weight of 47,000 and an isoelectric point of approximately 6.2 +/- 0.5 pH units appears to be the thyroid hormone receptor, since, in contrast to the other proteins, its photoinduced labelling was blocked by concentrations of T3 and thyroxine (T4) similar to those that inhibit binding of [125I]T3 by the receptor in equilibrium binding assays. In addition, the isoelectric point of the photolabelled protein agrees with that determined in separate equilibrium isoelectric focusing studies. These results indicate that [125I]T3 can serve as a photoreactive probe for the thyroid hormone nuclear receptor, and they suggest that this receptor is a single polypeptide chain of molecular weight 47,000 with an isoelectric point of 6.2 +/- 0.5 pH units.

Affinity Labels↗
PubMed1996

High affinity thyroid hormone-binding protein in human kidney: kinetic characterization and identification by photoaffinity labeling.

The binding of thyroid hormones and its regulation of NADPH and NADP+ were studied in human kidney cytosol, and a 38-kDa polypeptide (p38) was identified by photoaffinity labeling of cytosol with underivatized [125I]T3, SDS-PAGE, and autoradiography. The cytosolic thyroid hormone binding and p38 photolabeling were strongly activated by NADPH (maximum at 10(-7) M), whereas other nucleotides were less effective or ineffective. NADP+ did not activate T3 binding and p38 photolabeling, provided it was protected from conversion to NADPH by the addition of an exogenous oxidizing enzymatic system (oxidized glutathione plus glutathione reductase). Furthermore, NADP+ inhibited NADPH activation (half-maximum inhibitory effect at approximately 2 x 10(-5)M), and oxidation of NADPH to NADP+ induced dissociation of bound T3. The equilibrium dissociation constant (Kd) of the NADPH-activated cytosolic T3-binding sites was 0.3 nM, similar to the Kd of the nuclear T3 receptors. The kidney contained 200 times more cytosolic NADPH-activated thyroid hormone-binding sites than nuclear T3 receptors. Nonradioactive iodothyronines competed with [125I]T3 for both NADPH-activated binding and p38 photolabeling, with the following order of decreasing affinity: D-isomer of T3 > T3 > T4 > triiodothyroacetic acid > 3'-isopropyl-3,5-diiodothyronine > rT3. NADPH-activated T3 binding and photolabeled p38 were also detected in human heart and liver cytosols, but not in pancreas, cultured fibroblast and erythrocyte cytosols, or plasma. Rat kidney cytosol contained a 35-kDa photolabeled polypeptide homolog to human p38. The native molecular mass of the human photolabeled protein was 50 kDa, whereas that of the rat protein was 60 kDa, as determined by nondenaturing polyacrylamide gel electrophoresis. Two-dimensional PAGE of photolabeled p38 indicated an isoelectric point of 5.3. These findings describe the molecular properties of a NADPH/NADP+-regulated thyroid hormone-binding protein not previously identified in human and rat kidney cytosol.

Affinity Labels↗
PubMed1995

[3H]propyl-6-azido-beta-carboline-3-carboxylate: a new photoaffinity label for the GABAA-benzodiazepine receptor.

[3H]Propyl-6-azido-beta-carboline-3-carboxylate ([3H]ACCP) exhibited a high affinity for GABAA receptors affinity purified from the brains of adult rats, and binding of this compound could be inhibited by several ligands of the benzodiazepine binding site of GABAA receptors. On irradiation with UV light, [3H]ACCP, similarly to [3H]flunitrazepam, irreversibly labeled a protein with an apparent molecular weight of 51 kDa in affinity-purified GABAA receptors, and this labeling could be inhibited in the presence of diazepam. These data indicate that [3H]ACCP can be used as a photoaffinity label for GABAA receptors.

Affinity Labels↗
PubMed1993

Direct photoaffinity labeling of rat liver carbamoyl phosphate synthetase I with ATP.

The adenine subsites of the ATP sites of rat liver carbamoyl phosphate synthetase I have been localized by direct photoaffinity labeling with ATP. The synthetase is known to utilize two molecules of ATP, apparently in mechanistically discrete steps and at separate ATP sites. UV irradiation of the synthetase in the presence of [alpha-32P]ATP resulted in the incorporation of label. Peptide analysis of the ATP-photolabeled synthetase demonstrated that the labeling was extremely selective. To localize the sites of ATP photoincorporation to discrete regions of the synthetase which appear to be structural domains, the enzyme was photolabeled with [alpha-32P]ATP and subjected to limited proteolytic digestion. Consideration of these data indicated that the internal domains B and C were preferentially labeled and that there was lesser, but significant, labeling of the N-terminal domain A. Omission of the required allosteric activator N-acetylglutamate from the photolabeling mixture resulted in an approximately 60% decrease in label incorporation and an accompanying decrease in the extent of label incorporation in domain B. Consideration of these N-acetylglutamate effects, together with previous findings on the effects of the allosteric activator, confirmed the following functional identification of the ATP sites: domain B participates in binding the molecule of ATP involved in bicarbonate activation, whereas domain C participates in binding the molecule of ATP involved in carbamate phosphorylation.

Adenosine Triphosphate↗
PubMed1985

Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyl ADP.

3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF1). As with the CF1-ATPase (Bar-Zvi, D. and Shavit, N. (1984) Biochim. Biophys. Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-ATPase. BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF1-ATPase, it has no effect on the Vmax. In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound. Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase. Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP/mol TF1. Photoinactivation of TF1 by BzADP is prevented if excess of either ADP or ATP is present during irradiation. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz[3H]ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the beta subunit.

Adenosine Diphosphate↗
PubMed1977

Photoaffinity labeling of acyl-coenzyme A:glycine N-acyltransferase with p-azidobenzoyl-coenzyme A.

A photolabile reagent, p-azidobenzoyl-CoA, has been synthesized and tested as a photoaffinity label for acyl-CoA:glycine N-acyltransferase (EC 2.3.1.13) from beef liver. p-Azidobenzoyl-CoA is an active-site-directed reagent for this N-acyltransferase, since it is an alternate substrate (Km = 26 micronM, when [glycine] = 100 mM). Ultraviolet irradiation of a mixture of p-azidobenzoyl-CoA and the N-acyltransferase produces irreversible inhibition. Benzoyl-CoA protects the enzyme from inhibition by photoactivated p-azidobenzoyl-CoA. Acyl-CoA:glycine N-acyltransferase is composed of a single polypeptide with a molecular weight of about 35 000. Photolabeling experiments show that there is one active site per molecule of enzyme.

Acyltransferases↗
PubMed1983

Gonadotropin releasing-hormone receptors: photoaffinity labeling with an antagonist.

A photoaffinity antagonist of gonadotropin releasing hormone (GnRH), D pGlu-D-Phe-D-Trp-Ser-D-Lys6(N epsilon-azidobenzoyl)-Leu-Arg-Pro-Gly-NH2 (photoaffinity antagonist) was prepared by reacting [D-pGlu1, D-Phe2, D-Trp3, D-Lys6]GnRH with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. The analog appeared homogeneous when analyzed by thin-layer chromatography and its photoreactivity was demonstrated by spectral changes when exposed to light. The photoaffinity antagonist retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in vitro in whole pituitaries. Pituitary membrane preparations were incubated with the radioactive photoaffinity GnRH antagonist and irradiated with light. Sodium dodecyl sulfate gel electrophoresis after solubilization and reduction showed the specific labeling of a single specific protein with an apparent molecular weight of 60,000 daltons. These results indicate that GnRH agonists and antagonists bind to the same receptor.

Affinity Labels↗
PubMed1975

Photoaffinity-labeled Auxins: Synthesis and Biological Activity.

Two light-sensitive analogs of 2,4-dichlorophenoxyacetic acid, namely 4-azido-2-chlorophenoxyacetic acid and 3-azido-5-chlorophenoxyacetic acid, have been synthesized for use as auxin photoaffinity labels. The preparation and biological activity of the compounds are described. Both contain the photolabile azido group; the 2,4-substituted compound shows auxin activity and the 3,5-substituted compound does not. These photoaffinity analogs of 2,4-dichlorophenoxyacetic acid may be useful in the identification of the auxin receptor molecules in plant cells and eventually of the receptor sites within these molecules.

Journal Article↗
PubMed1976

Photoaffinity labeling of the beta-adrenergic receptors and receptor reserve for isoprenaline.

The beta-adrenergic receptors in the taenia isolated from the guinea pig caecum were irreversibly inactivated by photoaffinity labeling with 1-isoprenaline and 2-(2-hydroxy-3-isopropylaminopropoxy)iodobenzene. The photoinactivation was eliminated in the presence of protectors of the receptors. Photoinactivation caused a considerable parallel shift in the concentration-action curve of 1-isoprenaline. The results possibly point to a certain receptor reserve for 1-isoprenaline.

Affinity Labels↗
PubMed1990

[3H]DU41165: a high affinity ligand and novel photoaffinity labeling reagent for the progesterone receptor.

17 alpha-Acetoxy-6-fluoro-16-methylene-(9 beta, 10 alpha)pregna-4,6-dien- 3,20-dione (DU41165), a retroprogestin (9 beta, 10 alpha) embodying a fluorine-substituted dienone system, has been prepared in high specific activity tritium-labeled form (4 Ci/mmol) and shown to be a high affinity ligand for the progesterone receptor (PgR) and a highly selective photoaffinity labeling reagent for PgR. The radiosynthesis involved conversion of DU41231 (the 17 alpha-hydroxy analog of DU41165) to DU41165 by treatment with tritium-labeled acetic anhydride. The binding affinity of DU41165 for PgR was determined by both a competitive binding assay and a direct binding assay (Scatchard analysis) to be 1.6-2.2-times higher than that of the high affinity synthetic progestin promegestone (R5020). In unlabeled form, DU41165 demonstrates photoinactivation of PgR to the extent of 60% at 60 min. In radiolabeled form [3H]DU41165 demonstrates specific covalent attachment with an efficiency of 5-7%. SDS-polyacrylamide gel electrophoresis of photoattached [3H]DU41165 confirms that there is covalent labeling of both the B subunit (Mr = 118,000), and the A subunit (Mr = 88,000) of PgR in a molar ratio of approximately 1:3.

Affinity Labels↗
PubMed1975

Photoaffinity labeling of the ribosomal A site with S-(p-azidophenacyl)valyl-tRNA.

S-(p-Azidophenacyl)valyl-tRNA, an analog of valyl-tRNA which has a photoaffinity label attached to its 4-thiouridine residue, was bound to the ribosomal A site at 10 mM Mg2+. Binding was stimulated 25-fold by the presence of elongation factor EFTu. Photoactivation of the p-azidophenacyl group by irradiation resulted in covalent linking of 6% of the noncovalently bound tRNA to the ribosomes. Covalent linking was dependent on the simultaneous presence of ribosomes, poly(U2,G),EFTu.GTP, required irradiation, and did not occur when S-(phenacyl)valyl-tRNA, a nonphotolyzable analog, replaced S-(p-azidophenacyl)valyl-tRNA. The attached tRNA was distributed approximately equally between both the 30S and 50S subunits. At the 30S subunit, 30% of the tRNA was bound to protein while 70% was linked to 16S RNA. At the 50S subunit, however, negligible binding to the 23S RNA was observed. More than 90% of the tRNA was attached to low molecular weight material according to sodium dodecyl sulfate-sucrose gradient analysis, and more than 87% of this fraction consisted of tRNA-protein complexes as assayed by phenol solubility and electrophoretic mobility before and after protease treatment. These results, in conjunction with our previous report (I. Schwartz and J Ofengand (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 3951) which showed that covalent linking of this same tRNA derivative at the ribosomal P site resulted in attachment solely to the 16S RNA, demonstrate that 16S, but not 23S or 5S rRNA, is an important component of the tRNA binding site in the region of the 4-thiouridine residue and furthermore show that ribosomal A and P sites are topologically distinct.

Affinity Labels↗