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PubMed1985

Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate.

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1986

Identification of polypeptides of the phencyclidine receptor of rat hippocampus by photoaffinity labeling with [3H]azidophencyclidine.

Polypeptide components of the phencyclidine (PCP) receptor present in rat hippocampus were identified with the photolabile derivative of phencyclidine [3H]azidophencyclidine ( [3H]AZ-PCP). The labeled affinity probe was shown to reversibly bind to specific sites in the dark. The number of receptor sites bound is equal to those labeled by [3H]PCP, and their pharmacology and stereospecificity are identical with those of the PCP/sigma-opiate receptors. The dissociation constant of [3H]AZ-PCP from these receptors is 0.25 +/- 0.08 microM. Photolysis of hippocampus membranes preequilibrated with [3H]AZ-PCP, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the existence of five major labeled bands of which a Mr 90 000 band and a Mr 33 000 band were heavily labeled. Inhibition experiments, in which membranes were incubated with [3H]AZ-PCP in the presence of various PCP analogues and opiates, indicate that labeling of both the Mr 90 000 band and the Mr 33 000 band is sensitive to relatively low concentrations (10 microM) of potent PCP/sigma receptor ligands, while similar concentrations of levoxadrol, naloxone, morphine, D-Ala-D-Leu-enkephalin, atropine, propranolol, and serotonin were all ineffective. Stereoselective inhibition of labeling of the Mr 90 000 band and of the Mr 33 000 band was also observed by the use of dexoxadrol and levoxadrol. The Mr 33 000 band was not as sensitive as the Mr 90 000 band to inhibition by the selective PCP receptor ligands N-[1-(2-thienyl)cyclohexyl]piperidine and PCP.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1986

Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K+ channel subunits and target size analysis.

Two photoreactive apamin derivatives were prepared with an aryl azide [[(azidonitrophenyl)amino]acetate (ANPAA)] group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca2+-activated K+ channel. 125I-[alpha-ANPAA-Cys1] apamin labeled a single Mr 86 000 chain in cultured neurons whereas two bands corresponding to Mr 86 000 and 59,000 were detected in synaptic membrane preparations, suggesting that the Mr 59,000 polypeptide may be a degradation product. 125I-[epsilon-ANPAA-Lys4]apamin however incorporated uniquely into two smaller components with Mr 33,000 and 22,000 in both cultured neurons and synaptic membranes. Randomly modified 125I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K+ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in 125I-apamin receptor capacity, yielding a target size for the functional binding unit of Mr 84,000-115,000, which could be constituted by the Mr 86,000 subunit alone or by the Mr 86,000 subunit in conjuction with one of the two smaller subunits.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine.

A photoactive, radioactive analogue of vinblastine, N-(p-azido[3,5-3H]benzoyl)-N'-(beta-amino-ethyl)vindesine ([ 3H]NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 microM NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other's binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 microM for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 microM for NABV and vinblastine, respectively. Mixtures of tubulin and [3H]NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the alpha- and beta-subunits of tubulin with increasing concentrations of [3H]NABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an alpha:beta ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of [3H]NABV/mol of alpha-tubulin and beta-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3.

3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of the thymidine triphosphate binding domain in Escherichia coli DNA polymerase I: identification of histidine-881 as the site of cross-linking.

Using the technique of ultraviolet-mediated cross-linking of substrate deoxynucleoside triphosphates (dNTPs) to their acceptor site [Abraham, K. I., & Modak, M. J. (1984) Biochemistry 23, 1176-1182], we have labeled the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) with [alpha-32P]dTTP. Covalent cross-linking of [alpha-32P]dTTP to the Klenow fragment is shown to be at the substrate binding site by the following criteria: (a) the cross-linking reaction requires dTTP in its metal chelate form; (b) dTTP is readily competed out by other dNTPs as well as by substrate binding site directed reagents; (c) labeling with dTTP occurs at a single site as judged by peptide mapping. Under optimal conditions, a modification of approximately 20% of the enzyme was achieved. Following tryptic digestion of the [alpha-32P]dTTP-labeled Klenow fragment, reverse-phase high-performance liquid chromatography demonstrated that 80% of the radioactivity was contained within a single peptide. The amino acid composition and sequence of this peptide identified it as the peptide spanning amino acid residues 876-890 in the primary sequence of E. coli Pol I. Chymotrypsin and Staphylococcus aureus V8 protease digestion of the labeled tryptic peptide in each case yielded a single smaller fragment that was radioactive. Amino acid analysis and sequencing of these smaller peptides further narrowed the dTTP cross-linking site to within the region spanning residues 876-883. We concluded that histidine-881 is the primary attachment site for dTTP in E. coli DNA Pol I, since during amino acid sequencing analysis of all three radioactive peptides loss of the histidine residue at the expected cycle is observed.

Amino Acid Sequence↗
PubMed1989

Photoaffinity labeling by [3H]-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity.

N5-Methyl-N5-isobutylamiloride (MIA) is one of a series of 5-N-substituted amiloride analogues which exhibit high affinity and specificity for inhibition of Na+/H+ antiport. Amiloride-sensitive [3H]MIA binding to renal brush border membranes exhibited a Kd of 250 nM and a Bmax of 8.6 pmol/mg of protein. Specific binding was optimal at pH 7.5 and inhibited in the presence of Na+ and Li+. Inhibition by amiloride exhibited biphasic kinetics. After resolution of solubilized membranes by high-pressure liquid chromatography, MIA binding activity cofractionated together with Na+/H+ antiport activity, measured after reconstitution in asolectin vesicles, into a major and a minor peak. When fractions containing the major peak of Na+/H+ antiport activity were incubated with [3H]MIA and then photolyzed with a mercury arc lamp, covalent incorporation of label into polypeptides of apparent molecular mass 81 and 107 kDa was observed. These photolabeled bands were also observed in intact brush border membranes in addition to labeled polypeptides of apparent molecular mass 60 and 46 kDa, respectively. Labeling was inhibited by amiloride, reduced in the presence of Na+, and not observed in the absence of photolysis. These data point to the 81- and 107-kDa polypeptides as candidates for identification as components of a Na+/H+ antiport system in renal brush border membranes.

Affinity Labels↗
PubMed1990

Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine.

A photoactivatable analogue of phosphatidylserine, 125I-labeled 4-azidosalicylic acid-phosphatidylserine (125I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin (a crude soybean lipid extract) vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the alpha subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR alpha subunit that incorporated 125I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the alpha subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the alpha subunit incorporated little or no detectable amount of probe.

Affinity Labels↗
PubMed1990

Photoaffinity labeling of bacteriorhodopsin.

14C-Labeled optically pure 3S- and 3R-(diazoacetoxy)-all-trans-retinals were incorporated separately into bacterioopsin to reconstitute functional bacteriorhodopsin (bR) analogues, 3S- and 3R-diazo-bRs. UV irradiation at 254 nm generated highly reactive carbenes, which cross-linked the radiolabeled retinals to amino acid residues in the vicinity of the beta-ionine ring. The 3S- and 3R-diazo analogues were found to cross-link, respectively, to cyanogen bromide fragments CN 7/CN9 and CN 8/CN 9. More specifically, Thr121 and Gly122 in fragment CN 7 were found to be cross-linked to the 3S-diazo analogue. The identification of cross-linked residues and fragments favors assignments of the seven helices A-G-F-E-D-C-B or B-C-D-E-F-G-A to helices 1-2-3-4-5-6-7 in the two-dimensional electron density map (Henderson et al., 1975, 1986; Mogi et al., 1987). The present results show that the chromophore chain is oriented with the ionone ring inclined toward the outside of the membrane (the 9-methyl group also faces the extracellular side of the membrane).

Affinity Labels↗
PubMed1981

Photoaffinity labeling of Crotalus atrox phospholipase A2 by a substrate analogue.

A photolabile analogue of phosphatidylethanolamine (photolabile PE analogue), 1,2-di-O-hexylglycero-3-(ethyl diazomalonamidoethyl phosphate), was synthesized in nonisotopic and 14C-radiolabeled form and in both the L configuration (that of the naturally occurring phospholipids) and the racemic form. When the unlabeled racemic compound was photolyzed in the presence of phospholipase A2 of Crotalus atrox, extensive enzyme inactivation was observed. The rate of inactivation was stimulated by Ca2+ and by formation of micelles of the photolabile compound. The dihexyl ether analogue of phosphatidylethanolamine protected the enzyme from inactivation. Phospholipase A2 gave rise to a covalently labeled polypeptide when irradiated in the presence of either L or racemic 14C-labeled photolabile PE analogue. The racemic compound labeled both the N-terminal region (residues 1--15) and the central region (residues 43--97) of the polypeptide while the L compound labeled only the N-terminal region. The lone methionine at position 10 of the C. atrox phospholipase A2 permitted degradation by cyanogen bromide, which showed that labeling by the L compound was restricted to the first ten amino acid residues at the N-terminal end. Phospholipase A2 has an absolute specificity for L-phospholipids, and D-phospholipids are competitive inhibitors. The results of these studies underscore the importance of the head-group region of the phospholipid in phospholipase--substrate interactions and suggest that the two optical isomers of the substrate may be rather differently oriented on the enzyme surface.

Affinity Labels↗
PubMed1981

Photoaffinity labeling of mitochondrial adenosine triphosphatase by an azido derivative of the natural adenosine triphosphate inhibitor.

The natural mitochondrial ATPase inhibitor (IF1) was modified with a radioactivity labeled heterobifunctional and photosensitive reagent, methyl 4-azido(14C)benzimidate ((14C)MABI). Titration experiments of IF1 by (14C)MABI and tryptic maps of (14C)MABI-IF1 indicated that specific lysine residues in IF1 are preferentially labeled by (14C)MABI. Under appropriate conditions of labeling (1 to 2 lysine residues modified per IF1), MABI-IF1 exhibited the same inhibitory potency as native IF1 on the hydrolytic activity of the coupling factor 1 of mitochondrial ATPase (F1). The same conditions were required for inhibition of F1 by MABI-IF1 and IF1 (slightly acidic pH and presence of ATP and MgCl2). In photolabeling experiments, (14C)MABI-IF1 was used to investigate the localization of IF1 binding sites on F1. Upon photoirradiation, MABI-IF1 bound selectively to the beta subunit of soluble or membrane-bound F1. Adenylyl imidodiphosphate and quercetin, two compounds which partially mimic the inhibitory effect of IF1 on ATPase activity of F1, markedly prevented the binding of (14C)MABI-IF1 to F1; on the other hand, aurovertin, a specific ligand of the beta subunit of F1, did not affect the interaction between (14C)MABI-IF1 and F1. In the absence of light, (14C)MABI-IF1 was used as a reversible radiolabeled ligand with respect to membrane bound F1 to investigate F1-IF1 interactions to inside-out submitochondrial particles as a function of the energy state of the particles. Oxidation of NADH by submitochondrial particles resulted in a decrease of bound (14C)MABI-IF1; the effect was counteracted by antimycin. The data suggested that added (14C)MABI-IF1 is capable of exchanging with IF1 bound to F1 in submitochondrial particles and that the rate and extent of (14C)MABI-IF1 release are triggered by the proton-motive force developed by the particles.

Adenosine Triphosphatases↗
PubMed1981

Hydrodynamic characterization of the photoaffinity-labeled insulin receptor solubilized in Triton X-100.

The insulin receptor in isolated rat liver plasma membranes was covalently labeled with the photoreactive insulin analogue NB-29-[(4-azido-2-nitrophenyl)acetyl]insulin and solubilized with the nondenaturing detergent Triton X-100. The resulting protein-detergent complex was characterized by gel filtration on Sepharose 6B, sedimentation rate determination in linear sucrose gradients, and equilibrium isopycnic centrifugation in NaBr and CsCl. The labeled insulin receptor was found in two forms. The Strokes radii and s20,w's of the two receptor-detergent complexes (R1 and R2) were (mean +/- SEM) 7.08 +/- 0.04 and 3.62 +/- 0.05 nm and 10.45 +/- 0.04 and 6.54 +/- 0.15 S, respectively. The two forms appeared to have the same buoyant density, 1.285 +/- 0.002 g cm-3. The dissociation of R2 from R1, or its reaggregation, either with itself or with other unlabeled proteins, to give R1 proceeded without chemical modification. Mild reduction of disulfide bonds (1 mM 1,4-dithiothreitol) increased the dissociation of R2 from R1. These results indicate that the solubilized receptor binds significant amounts of detergents, that the insulin binding component of the receptor binds to other receptor components by hydrophobic interactions, and that one or more components of the insulin receptor contain intrachain disulfide bonds.

Affinity Labels↗
PubMed1982

Subunit structure of insulin receptor of rat adipocytes as demonstrated by photoaffinity labeling.

Isolated rat adipocytes were incubated in the dark with either one or two radioiodinated photoreactive insulin derivatives, N epsilon B29-(azidobenzoyl) insulin (B29-MABI) and N alpha B1-(azidobenzoyl) insulin (B1-MABI), and were then exposed to light. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and radioautography of the crude plasma membrane fraction after reduction showed that B29-MABI labeled specifically three proteins of Mr 130 000, 90 000, and 40 000 whereas B1-MABI labeled specifically two proteins of Mr 130 000 and 40 000. B1-MABI also variably labeled some bands of intermediate Mr between 130 000 and 90 000. In contrast, the labeling of the 40-kilodalton protein was not observed in our previous studies in which photolabeling was carried our on isolated plasma membrane preparations [Yip, C. C., Yeung, C. W. T. & Moule, M. L. (1980) Biochemistry 19, 70-76; Yeung, C. W. T., Moule, M. L., & Yip, C. C. (1980) Biochemistry 19, 2196-2203]. Without reduction, an Mr 300 000 band and a larger band which barely entered a 5-15% gradient gel were specifically labeled by both photoreactive insulins. Reduction of these two high molecular weight bands gave rise to the 130-, 90-, and 40-kilodalton bands. The labeling of these proteins was affected neither by the time or temperature of incubation nor by the addition of methylamine, chloroquine, bacitracin, phenylmethanesulfonyl fluoride, p-(chloromercuri) benzenesulfonic acid, Trasylol, N-ethylmaleimide, or benzamidine. The labeling of these proteins by the photoreactive insulin derivatives was inhibited by first incubating the adipocytes with a human autoimmune serum to insulin receptor. We therefore conclude that these proteins are subunits of the insulin receptor in intact adipocytes.

Adipose Tissue↗
PubMed1980

Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations.

The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N epsilon B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis, and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these two derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high molecular weight when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with [125I]iodine. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity, and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse, and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that two proteins were specifically labeled. The molecular weights of the two radioactive bands were estimated to be about 130 000 and 90 000 in all three species of animals.

Affinity Labels↗
PubMed1977

Estrogen photoaffinity labels. 1. Chemical and radiochemical synthesis of hexestrol diazoketone and azide derivatives; photochemical studies in solution.

Two photosensitive estrogen derivatives, hexestrol diazoketopropyl ether (5) and hexestrol azide (8a), have been synthesized in radiolabeled form, and their photochemical behavior in solution has been studied. The radiolabeled compounds were prepared in good yields according to improved synthetic procedures; they are stable and were obtained with specific activities in the range of 50-100 Ci per mmol and radiochemical purities in excess of 95%. A simpler model system, phenyl diazoketoprophyl ether, was used to study the photochemical behavior of the diazoketopropyl ether group. Direct irradiation of this compound at 254 nm in methanol led to 33% insertion product (methoxyketone) and 67% Wolff rearrangement product (ester). Irradiation of [3H]hexestrol diazoketopropyl ether (5) in methanol gives mainly nonpolar photoproducts (presumed to be the methoxy ketone and ester); however, irradiation in aqueous medium leads to large amounts of free hexestrol (52%). Photolysis of hexestrol azide (8a) in either methanol or water gives the corresponding amine in low yield as the only identifiable photoproduct.

Affinity Labels↗