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PubMed1978

A photoaffinity labelling study of the messenger RNA-binding region of Escherichia coli ribosomes.

A photoaffinity labelling study of the messenger RNA-binding region of E. coli ribosomes has been made, using oligoadenylic acids as mRNA analogs. The oligonucleotides, of chain length 6 to 8 and thus several nucleotides longer than oligonucleotides previously employed for this purpose, carried a radioactive photolabile aromatic azide reagent bound covalently to the 3'-terminal ribose moiety. The synthesis of the reagent, p-azidobenzoyl-(3H)-glycylhydrazide, is described. The derivatized oligonucleotides were shown to be functional messengers. They stimulated the binding of the cognate aminoacyl-tRNA, lysyl-tRNA: their binding was reciprocally stimulated by lysyl-tRNA; and they competed with underivatized oligoadenylates for ribosomal binding sites. When the 70 S ribosomal binding complex was irradiated, the photolabile reagent reacted covalently with both RNA and proteins of the 30 S subunit and with tRNA, but not with the 50 S subunit. The 16 S RNA appeared to be labelled at more than one site. Of the proteins, S3 and S5 reacted with the reagent with high specificity; and the possibility was not eliminated that S4 may have been labelled to a minor degree. Functional studies in other laboratories have implicated S3 and S5 in the decoding process, but these proteins were not labelled by any of the previously reported mRNA affinity labelling analogs. The results reported here therefore indicate that S3 and S5 not only affect the decoding process, but are located in the mRNA-binding region of the ribosome, presumably to the 3' side of the decoding site.

Affinity Labels↗
PubMed1991

Photoaffinity labeling of leukotriene binding sites in hepatocytes and hepatoma cells.

The method of direct photoaffinity labeling in the frozen state using the leukotrienes as suitable photolabile compounds may serve to identify and characterize polypeptides which interact with these eicosanoids during their hepatobiliary transport and metabolism. Furthermore, it will be a helpful technique to evaluate changes in the cell-specific protein pattern during neoplastic dedifferentiation of hepatocytes.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of extracellular and intracellular androgen binding proteins.

A method is described for the photoaffinity labeling of human and rabbit serum sex hormone binding globulin (SHBG) in ammonium sulphate precipitates utilizing 3H-delta 6-testosterone as affinity label. The precipitation step diminished albumin contamination and at the same time concentrated the binding globulin. Photolysis was conveniently carried out with an electronic flash. Unbound and non-covalently bound steroids were adsorbed by prolonged dextran-coated charcoal treatment. Sephadex G-200 gel filtration column chromatography showed a single peak of covalently bound radioactivity with the elution volume of SHBG. With some modifications, the method was applied to the affinity labeling of the prostate androgen receptor utilizing 3H-methyltrienolone (R 1881) as affinity label. The receptor was also precipitated from prostate cytosol with ammonium sulphate. After labeling, photolysis and heating at 50 degrees C, non covalently bound 3H-R 1881 was removed by dextran coated charcoal treatment. Sephadex G-25 microcolumn chromatography after heating showed a peak of radioactivity eluting with macromolecules if photolysis had been carried out, while it disappeared in the absence of previous photolysis. However, the photolabeled receptor had a sedimentation coefficient different from the non-irradiated receptor, suggesting that photolysis induced a change in the configuration of the complex.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of peripheral-type benzodiazepine-binding sites.

The use of a novel photoaffinity label for the peripheral-type benzodiazepine-binding site is described. This compound, PK 14105, has high affinity (4 nM) and selectivity for cardiac benzodiazepine-binding sites. Under ultraviolet light, PK 14105 couples covalently to an 18,000-Da membrane protein which apparently corresponds to the (or a part of the) cardiac benzodiazepine-binding site. Since covalent attachment of PK 14105 totally precludes the binding of other ligands to this binding site, it is suggested that, during ultraviolet irradiation, this compound inserts covalently into the binding domain of the peripheral-type benzodiazepine-binding site.

Affinity Labels↗
PubMed1984

Photoaffinity labeling of human chorionic gonadotropin-binding sites in rat ovarian plasma membranes.

A photoaffinity derivative of human chorionic gonadotropin (hCG-N-hydroxysuccinimidyl-4-azidobenzoate (hCG-HSAB) ) was used to identify components of the lutropin (LH)/hCG receptor in plasma membranes prepared from pregnant mare serum-pretreated rat ovaries. In intact plasma membranes, the Kd for hCG (1 X 10(-10) M) was the same with both 125I-hCG and 125I-hCG-HSAB, and the binding capacity for the photoaffinity label (1 pmol/mg of membrane protein) was the same as for native hCG. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions of 125I-hCG-HSAB cross-linked to plasma membranes revealed newly formed complexes of Mr = 106,000, 85,000, and 80,000 (representing membrane protein components of Mr = 86,000, 65,000, and 60,000, respectively). However, only the Mr = 106,000 complex displayed an affinity for hCG (Kd of 1 X 10(-10) M) consistent with the physiological LH/hCG receptor. The other binding sites were nonsaturable. Half-maximal saturation by 125I-hCG-HSAB of the Mr = 106,000 complex (1-2 X 10(-9) M) was similar to the Kd of the specific binding of 125I-hCG-HSAB to intact plasma membranes (8 X 10(-10) M). The present data suggest that among the different components of the LH/hCG receptor, only one component (of Mr = 86,000) possesses the affinity and binding capacity characteristics of the LH/hCG receptor as determined using radiolabeled, underivatized hCG.

Affinity Labels↗
PubMed1993

Identification of a 27-kDa high affinity phenylalkylamine-binding polypeptide as the sigma 1 binding site by photoaffinity labeling and ligand-directed antibodies.

The verapamil-like arylazide (-)-[3H]azidopamil specifically photoaffinity labeled two low molecular mass polypeptides, with apparent molecular masses of 22 and 27 kDa, in the endoplasmic reticulum of guinea pig liver, kidney, adrenal gland, and lung. It was recently shown that the 22-kDa polypeptide binds the anti-ischemic phenylalkylamine (-)-[3H]emopamil and other anti-ischemic drugs with high affinity. We now provide evidence that the photolabeling of the 27-kDa polypeptide is blocked by nanomolar concentrations of sigma ligands [order of potency, haloperidol > pentazocine > 1,3-ditolylguanidine > dextromethorphan > (+)-SKF10,047]. The apparent affinities of these and other drugs closely corresponded to those for 1,3-[3H]ditolylguanidine-labeled sigma binding sites. Based on its high affinity for the (+)-enantiomer [but not the (-)-enantiomer] of SKF10,047 (Ki = 51 nM), pentazocine (Ki = 3 nM), and dextromethorphan (Ki = 30 nM), the (-)-[3H]azidopamil-labeled site on the 27-kDa polypeptide was classified as being of the sigma 1 subtype. Using antiphenylalkylamine antibodies, we developed a novel immunological detection method that allows the rapid and sensitive staining of the photolabeled 27-kDa polypeptide after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the phenylalkylamines emopamil and azidopamil represent a novel class of sigma ligands, highly suitable for the further structural characterization of polypeptides carrying sigma 1 binding sites.

Affinity Labels↗
PubMed1999

Photoaffinity labeling of brain glutamate dehydrogenase isoproteins with an azido-ADP.

The ADP binding site within two types of bovine brain glutamate dehydrogenase isoproteins (GDH I and GDH II) was identified using photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-diphosphate (8N3ADP). 8N3ADP, without photolysis, mimicked the activatory properties of ADP on GDH I and GDH II activities, although maximal activity with 8N3ADP was about 75% of maximal ADP-stimulated activity. Saturation of photoinsertion with [alpha-32P]8N3ADP occurred at around 40 approximately 50 microM photoprobe with apparent Kd values near 25 and 40 microM for GDH I and GDH II, respectively. Photoinsertion of [alpha-32P]8N3ADP was decreased best by ADP in comparison with other nucleotides. With the combination of immobilized aluminum affinity chromatography and reversed-phase high performance liquid chromatography, photolabel-containing peptides generated by tryptic digestion were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as in the region containing the sequence, EMSWIADTYASTIGHYDIN. Photolabeling of the peptide was prevented over 90% by the presence of 1 mM ADP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as ADP. These results demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide with the residues Glu179-Asn197 is within the ADP binding domain of the brain GDH isoproteins.

Adenosine↗
PubMed1994

Photoaffinity labeling with progesterone-11 alpha-hemisuccinate- (2-[125I]iodohistamine) identifies four protein bands in mouse brain membranes.

The radiolabeled progesterone (PG) analogue progesterone-11 alpha-hemisuccinate-(2-[125I]iodohistamine) was used to label PG binding proteins in brain membranes from mouse cerebellum. Photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified specific PG binding protein bands 1-4 of 64-29 kDa. Bands 1 and 4 were well resolved on the gel and easily quantified. Preincubation with PG inhibited photolabeling in a dose-dependent manner. The labeling was specific with respect to steroid structure. For band 1, the extent of inhibition of labeling by PG and 3 alpha, 5 alpha-pregnanolone (3 alpha) was pronounced. Other steroids such as testosterone (Tes), estradiol (Est), and corticosterone (Cor) were less effective, whereas pregnenolone sulfate (PS) and cholesterol (Cho) were ineffective. With respect to band 4, Est was the most effective; PG, 3 alpha, and Tes were intermediate; and PS, Cho, and Cor were ineffective. The results describe specific membrane proteins that bind PG (band 1) and Est (band 4).

Affinity Labels↗
PubMed1989

3-Diazirine-derivatives of bile salts for photoaffinity labeling.

New carbene-generating photolabile bile salt derivatives, 3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oic acid and (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oyl)-2- aminoethanesulfonic acid were synthesized with high specific radioactivity. These 3-diazirine-derivatives could be activated to the corresponding carbenes by irradiation with ultraviolet light at 350 nm with a half-life time of 2 min. The 3-diazirine derivatives behaved in enterohepatic circulation like the natural bile salts. The uptake of [3H]taurocholate into isolated hepatocytes was competitively inhibited by (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2- aminoethanesulfonic acid indicating that the 3,3-azo-derivative of taurocholate shares the hepatic transport systems for natural bile salts. It was demonstrated that the radioactively labeled 3-diazirine bile salt derivatives are useful probes for photoaffinity labeling of bile salt binding proteins especially in intact cells and tissues.

Affinity Labels↗
PubMed1999

Characterization of sheep brain ryanodine receptor ATP binding site by photoaffinity labeling.

Two high Mr protein bands (440 and 420 kDa) in sheep brain microsomal membranes were labeled with the photoaffinity ATP analog, O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP). The 420 kDa band is labeled by [alpha-32P]-Bz2ATP with about 1000-fold higher affinity than the 440 kDa band. The heavily labeled 420 kDa band is identified as dynein heavy chain based on its partial amino acid sequence, and cross-reactivity with anti-dynein antibodies. The 440 kDa protein is immunologically identified as the type-2 RyR. Bz2ATP binding is obtained in the absence of divalent cations. Bz2ATP and ATP increased the binding of ryanodine to its receptor up to 3-fold, and increased the binding affinity up to 6-fold. Other nucleotides stimulate ryanodine binding with decreasing effectiveness: Bz2ATP > ATP > ADP > AMP > AMP-PNP > GTP > cAMP. With respect to nucleotide specificity, this binding site is similar to the skeletal muscle RyR (type 1). However, the brain RyR may have additional one or more sites with lower affinity with inhibitory effect on ryanodine binding. These results suggest that the major RyR isoform in sheep brain corresponds to the type-2 isoform, and that modulation of ryanodine binding by ATP involves its binding to the RyR protein. The association of dynein with brain microsomal membranes may reflect a linkage of RyR to the cytoskeleton.

Adenosine Triphosphate↗
PubMed2006

A new photoactive building block for investigation of DNA backbone interactions: photoaffinity labeling of human DNA polymerase beta.

The cross-linking of target proteins or nucleic acids to light-activatable ligands is an important tool for elucidating molecular interactions. Through the use of photoaffinity-labeling reagents, several new insights into nucleic acid interactions have been obtained, for example in DNA replication and repair. In most known photoprobes, the applied light-sensitive functionalities are placed directly at the nucleobase or are attached via linkers to either the nucleobase or the phosphate backbone. Here we describe the first photoprobe that bears a light-sensitive aryl(trifluoromethyl)diazirine at the sugar moiety of a DNA oligonucleotide. We devised a route for the synthesis of the modified nucleoside and its incorporation into an oligonucleotide. The photoactive species was proven to be stable under the conditions employed in routine automated DNA synthesis. The modified oligonucleotide was shown by subsequent photolabeling studies of human DNA polymerase beta to form a covalent complex to the enzyme upon irradiation with near-UV light.

DNA↗
PubMed2003

Photoaffinity labeling of the N-methyltransferase domains of cyclosporin synthetase.

The multifunctional polypeptide cyclosporin synthetase (CySyn) remains one of the most complex nonribosomal peptide synthetase described. In this study we used a highly specific photoaffinity labeling procedure with the natural cofactor S-adenosyl-L-methionine (AdoMet), 14C-isotopically labeled at the Sdelta methyl group to probe the concerted AdoMet-binding interaction of the N-methyltransferase (N-MTase) centers of CySyn. The binding stoichiometry for the enzyme-AdoMet complex was determined to be 1:7, which is in agreement with inferences made from analysis of the complementary DNA sequence of the simA gene encoding the CySyn polypeptide. The photolabeling of the AdoMet-binding sites displayed homotropic negative cooperativity, characterized by a curvilinear Scatchard plot with upward concavity. Although, the process of N-methyl transfer is not a critical event for peptide elongation, the destabilizing homotropic interactions between N-MTase centers that were observed may represent a mechanism whereby the enzyme preserves the proficiency of the substrate-channeling process of cyclosporin peptide assembly over a broad range of cofactor concentrations. Furthermore, we demonstrated the utility of the photolabeling procedure for tracking the enzyme during purification.

Kinetics↗
PubMed1999

Photoaffinity labeling with the activator IMP and site-directed mutagenesis of histidine 995 of carbamoyl phosphate synthetase from Escherichia coli demonstrate that the binding site for IMP overlaps with that for the inhibitor UMP.

Photoaffinity labeling with IMP was used to attach covalently this activator to its binding site of Escherichia coli carbamoyl phosphate synthetase. We now identify histidine 995 of the large enzyme subunit as the amino acid that is cross-linked with IMP. The identification was carried out by comparative peptide mapping in two chromatographic systems of peptides differentially labeled with [3H]IMP and with the labeled inhibitor [14C]UMP, followed by automated Edman degradation and radiosequence analysis. Site-directed substitution of His995 by alanine confirmed His995 to be the only amino acid in the protein forming a covalent adduct with IMP. The His995Ala mutant protein was soluble and active and exhibited normal kinetics for the activator ornithine and for the substrates in the presence of ornithine. However, the mutation selectively induced changes in the activation by IMP and the inhibition by UMP, and it abolished the photolabeling of the enzyme by IMP without affecting the photolabeling by the inhibitor UMP. Since UMP is cross-linked to Lys993 [Cervera, J., et al. (1996) Biochemistry 35, 7247-7255] only two residues upstream of the site of IMP labeling, the results provide structural evidence for earlier proposals which suggested that UMP and IMP bind in a single or overlapping site. The two residues are within the region previously proposed as the binding fold for the nucleotide effectors. In the crystal structure of the enzyme, Lys993 and His995 are exposed and line a crevice where a Pi molecule was found [Thoden, J. B., et al. (1997) Biochemistry 36, 6305-6316]. UMP and IMP appear to bind in this crevice, possibly toward the C-side of the beta-sheet in a Rossman fold. Their binding in this site is consistent with the selectivity of adduct formation of UMP with Lys993 and of IMP with His995. It is also consistent with the nonessentiality of His995 for the binding, since the interactions with other residues that line the crevice must contribute a large part of the binding energy. The lack of an effect of the mutation on the activation by ornithine is consistent with the binding of this activator in a separate site in the protein.

Alanine↗
PubMed1998

Syntheses of photoactive analogues of adenosine diphosphate (hydroxymethyl)pyrrolidinediol and photoaffinity labeling of poly(ADP-ribose) glycohydrolase.

Two isomeric azidoadenosyl analogues of adenosine diphosphate (hydroxymethyl)pyrrolidinediol [ADP-HPD; Slama, J. T., et al. (1995) J. Med. Chem. 38, 389-393] were synthesized as photoaffinity labels for poly(ADP-ribose) glycohydrolase. 8-Azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N3-ADP-HPD) inhibited the enzyme activity by 50% at ca. 1 microM, a concentration 80-fold lower than that where the isomeric 2-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol did. [alpha-32P]-8-N3-ADP-HPD was therefore synthesized and used to photoderivatize poly(ADP-ribose) glycohydrolase. Irradiation of recombinant poly(ADP-ribose) glycohydrolase and low concentrations of [alpha-32P]-8-N3-ADP-HPD with short-wave UV light resulted in the covalent incorporation of the photoprobe into the protein, as demonstrated by gel electrophoresis followed by autoradiography or acid precipitation of the protein followed by scintillation counting. No photoincorporation occurred in the absence of UV light. The photoincorporation saturated at low concentrations of the photoprobe and photoprotection was observed in the presence of low concentrations of ADP-HPD, an indication of the specificity of the photoinsertion reaction. These results demonstrate that [alpha-32P]-8-N3-ADP-HPD can be used to specifically covalently photoderivatize the enzyme to characterize the polypetides that constitute the ADP-HPD binding site of poly(ADP-ribose) glycohydrolase. The photoincorporation reaction was further used to determine the ability of ADP-ribose polymers of varying size to compete with [alpha-32P]-8-N3-ADP-HPD for binding to the enzyme. Photoincorporation of [alpha-32P]-8-N3-ADP-HPD was inhibited by 80% in the presence of low concentrations of short, unbranched ADP-ribose oligomers (5-15 ADP-ribose units in length). No similar photoprotection was afforded by the addition of a high-molecular weight highly branched polymer. These results indicate that the photolabel shares a binding site with the short, linear polymer, but not with the long, highly branched polymer.

Adenosine Diphosphate↗
PubMed2001

Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair.

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.

Animals↗
PubMed2006

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling.

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.

Amino Acid Sequence↗
PubMed1992

Binding site mapping of a photoaffinity-labeled juvenile hormone binding protein.

The juvenile hormone binding protein (JHBP) of larval Manduca sexta was labeled by a photoaffinity analog of JH II and purified by preparative IEF and ion-exchange HPLC. The purified [3H]EHDA-labeled JHBP was selectively cleaved by CNBr and by endoproteinases Lys-C and Glu-C. The radioactive peptides were separated by tricine SDS-PAGE and sequenced after blotting to a PVDF membrane. The sequence revealed that Ala184-Asn226 contained a primary binding site of [3H]EHDA. Furthermore, peptide mapping indicated that Asp1-Glu34 also contained a second covalent attachment site of [3H]EHDA. Labeling of the N-terminal region increased when the photolysis was performed at lower temperature. Since Ala184-Asn226 is predicted to be a hydrophobic beta-sheet region, it may participate in the recognition of lipophilic backbone of JH. Five out of six cysteines are located in these two regions, consistent with a model in which the two binding regions connected by disulfide bridges provide a two-sided binding pocket for juvenile hormone.

Affinity Labels↗