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PubMed1991

Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP: identification of the viral RNA polymerase.

Rotavirus single-shelled particles have several enzymatic activities that are involved with the synthesis of capped mRNAs both in vivo and in vitro. Because single-shelled particles must be structurally intact to carry out transcription, it has proven to be difficult to identify the protein within such particles that possesses associated RNA polymerase activity. One approach for characterizing the function of the individual proteins within single-shelled particles is to use nucleotide analogs to specifically label those proteins, such as the viral RNA polymerase, that have affinity for nucleotides. In this study, 8-azido-ATP (azido-ATP), a photoreactable nucleotide analog, was used to identify the viral RNA polymerase on the basis of the ability of the analog to inhibit transcription activity associated with rotavirus particles on exposure to UV light. When single-shelled particles were treated with UV light in the presence of [alpha-32P]azido-ATP, the structural protein VP1 became radiolabeled because of cross-linking of the nucleotide analog, and there was a corresponding decrease in the ability of the particles to synthesize mRNA. In parallel experiments in which single-shelled particles were not exposed to UV light, VP1 was not radiolabeled and the particles successfully used azido-ATP as a substrate for the synthesis of viral mRNAs. Taken together, these results are consistent only with the conclusion that VP1 is the rotavirus RNA-dependent RNA polymerase.

Adenosine Triphosphate↗
PubMed1996

UV photoaffinity labeling of Tn3 transposase--DNA complexes: identification of DNA binding domains.

The prokaryotic transposon Tn3 requires the transposase protein, as well as the cis-acting terminal inverted repeats (IRs), for transposition. The first step in the transposition process requires transposase binding to the IRs, as well as target site selection for element insertion. The primary aim of this study is to define the relationship between the structure of Tn3 transposase and its DNA binding functions. We have defined, by UV cross-linking, two broad regions of transposase that interact with DNA: a 70-kDa N-terminal domain and a 30-kDa C-terminal domain. The 70-kDa N-terminal domain encompasses the IR sequence specific binding domain, as well as a nonspecific DNA binding domain that has been previously described. We have also defined, by UV cross-linking, a region in the nonspecific DNA binding domain centered at amino acids 376 and 381 that is in contact with DNA. We have used site-directed mutagenesis of amino acids 376 and 381 to help delineate the function of this region of the transposase protein. Mutations in this region reduce transposition frequency to 30-40% of the wild type. These mutations reduce nonspecific DNA binding three- to four-fold but do not appear to affect specific binding to the IR. Transposition immunity is unaffected by mutations in the nonspecific DNA binding domain. This suggests that this region may be involved in target site selection.

Affinity Labels↗
PubMed1993

Timed photoaffinity labeling and characterization of bile acid binding and transport proteins in rat ileum.

Rat ileal enterocytes were radiolabeled by flash photolysis with a photolabile derivative of taurocholate (7,7-azo-[3H]TC) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal labeling of the bile acid binding proteins (BABPs) was achieved between 15 and 90 s. When enterocytes were pulsed with 7,7-azo-[3H]TC for 2 min, and then 0.5 mM TC was added to chase the radiolabel, the radioactivity in the BABPs was displaced by 50% after 2 min. The 99-kDa brush-border membrane (BBM) protein had the highest initial labeling rate, followed by 43-kDa actin, 35- and 14-kDa cytosolic proteins, 54-kDa basolateral membrane (BLM) protein, 59-kDa BLM-associated protein, and 20-kDa microsomal protein. When a mixed microsomal and cytosolic fraction was photolabeled with 7,7-azo-[3H]TC and then separated, the 20-kDa microsomal protein was labeled. However, if the microsomal fraction alone was photolabeled, the 20-kDa protein was not labeled, suggesting this protein required a cytosolic cofactor for labeling. Using Triton X-114 phase separation and EDTA extraction, the BABPs were separated into amphiphilic integral membrane proteins (99- and 54-kDa proteins) and hydrophilic proteins (14-, 35-, 43-, and 59-kDa proteins). From these data, a model is proposed for transcellular bile acid transport in rat ileal enterocytes.

Affinity Labels↗
PubMed1984

Photoaffinity labeling of adenosine transporter in cardiac membranes with nitrobenzylthioinosine.

The kinetic and molecular properties of the adenosine transporter in guinea pig cardiac membranes were studied using nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport. [3H]-NBMPR bound tightly but reversibly to guinea pig cardiac membranes (apparent dissociation constant 0.24 +/- 0.07 nM; maximum binding capacity 1.24 +/- 0.45 pmol of NBMPR bound/mg protein). Reversible high-affinity [3H]NBMPR binding was inhibited in an apparent competitive manner by adenosine (apparent inhibition constant 0.14 mM). L-N-phenylisopropyladenosine (L-PIA) had no effect on NBMPR binding. Exposure of cardiac membranes in the presence of [3H]-NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent molecular weight 66,000-50,000. Covalent attachment of [3H]NBMPR under equilibrium binding conditions was inhibited by adenosine, nitrobenzylthioguanosine , and dipyridamole but was unaffected by the adenosine receptor agonist L-PIA. These data suggest that the photolabeled molecular weight protein (apparent mol wt 66,000-50,000) is involved in adenosine permeation by guinea pig cardiac membranes.

Adenosine↗
PubMed1986

Characterization of the human lymphocyte beta-adrenergic receptor by photoaffinity labeling. Alterations with desensitization.

Desensitization of the leukocyte beta-receptor system has been associated with a functional uncoupling of the components of the beta-receptor complex. In order to determine whether desensitization and uncoupling of the leukocyte beta-receptor is associated with any structural alterations in the beta-receptor, we studied labeling of lymphocytes using the photoactive beta-adrenergic antagonist p-azido-m-[125I]iodobenzylcarazolol. Labeled peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using autoradiographic techniques. In broken cell preparations, specific labeling was demonstrated in two major peptide bands: mol wt approximately equal to 68,000 and mol wt approximately equal to 55,000. Inhibition of photolabeling was stereospecific and demonstrated an order of potency for agonists consistent with labeling of a beta 2-receptor. Preincubation of cells with the beta-agonist, isoproterenol, resulted in a reduction in beta-adrenergic-mediated adenylate cyclase activity to 60% of control, but no change in total binding sites as determined by [125I]iodocyanopindolol binding. In photolabeling studies, desensitization was associated with a reduction in proportional labeling of the 55,000 mol wt band as compared to the 68,000 mol wt band to 58 +/- 3% of control and a reduction in mobility of the upper band. These studies suggest that structural alterations in the human lymphocyte beta-receptors occur with desensitization, analogous to changes in several other beta-receptor model systems. Also, since the techniques described can identify alterations in human beta-receptor structure, these methods may be exploited to determine whether structural alterations in lymphocyte beta-receptors may occur in human disease states.

Adenylyl Cyclases↗
PubMed1981

Photoaffinity labeling of luteinizing hormone releasing hormone receptor of rat pituitary membrane preparations.

Specific LHRH receptor proteins of plasma membrane preparation from pituitary glands of the rat were identified using an 125I-labeled photoreactive LHRH derivative, [D-Lys6-N epsilon-azidobenzoyl]LHRH. This analog retained high binding affinity (apparent Kd value of 0.1 nM) to a single class of receptors. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated the specific photolabeling of two proteins, a major band with an apparent molecular weight of 60,000 daltons and a minor band of 48,000 daltons. The latter is probably a degradation product of the receptor.

Affinity Labels↗
PubMed1982

Some physicochemical characteristics of photoaffinity-labeled rabbit testosterone-binding globulin.

Photolabeled testosterone-binding globulin (TeBG), obtained from the plasma of sexually immature male rabbits, was produced using 17 beta-hydroxy-[1,2-3H]4,6-androstadien-3-one. Photolabeled TeBG could not be distinguished from unlabeled TeBG by gel filtration chromatography, electrophoresis on nondenaturing gels, or sucrose gradient ultracentrifugation. Rabbit TeBG had a Stokes radius of approximately 45 A and a sedimentation coefficient of approximately 4.6S. Based on these parameters, its native molecular weight was calculated to be approximately 78,000. The frictional ratio of rabbit TeBG was found to be approximately 1.6. When photolabeled TeBG was examined on polyacrylamide gels containing sodium dodecyl sulfate, a single androgen-specific peak of approximately 40,000 daltons was obtained. When photolabeled TeBG was treated with the cross-linking reagent disuccinimidyl suberate before electrophoresis on sodium dodecyl sulfate gels, an additional androgen-specific peak of radioactivity corresponding to approximately 100,000 daltons was obtained. We interpret this peak to represent oligomers of the TeBG subunits. The 40,000-dalton subunit was obtained regardless of whether the proteins were treated with 2-mercaptoethanol, indicating that the monomers are not linked by disulfide bonds.

Affinity Labels↗
PubMed1983

Irreversible stimulation of hydroosmotic response in toad bladder by photoaffinity labeling with [Phe2,Phe-(p-N3)3]Vasopressin.

The photoreactive analogs of vasopressin, [Phe2,Phe-(p-N3)3]AVP (3a) and [Phe-(p-N3)2]AVP (2a), and the chemically reactive analogs of vasopressin, [Phe2,Phe-(p-NHCOCH2Br)3]AVP (3b) and [Phe-(p-NHCOCH2Br)2]AVP (2c), have been tested in the toad bladder for irreversible stimulation or inhibition of the water permeability response. Analog 3a was found to be an agonist with an ED50 of 4.5 X 10(-7) M and to induce a maximal osmotic water flow across bladders equivalent to 74% of that observed with the parent hormone (AVP). Photolysis of this analog in Ringer's fluid resulted in a decrease in its biological activity, with a half-time of 7 min. However, UV irradiation of the analog in the presence of toad bladders triggered an irreversible increase in the permeability of the bladders to water. Under optimal conditions of irradiation, water permeability remained at about 60% of maximum for more than 3 h after washout of analog 3a. The addition of AVP raised the permeability of these bladders to 100%. Analog 3a did not cause irreversible stimulation without photolysis, nor did this analog induce its characteristic effect when added to the mucosal solution. Compound 2a was found to be a potent antagonist of AVP. This inhibitory action of 2a was readily reversed in both the presence and absence of UV irradiation. Compound 3b was also found to be a reversible inhibitor of AVP. Compound 2c was found to be inactive as agonist or antagonist. These studies suggest that analog 3a binds covalently at or near the toad bladder hydroosmotic receptors, resulting in a persistent increase in permeability to water of the bladder wall.

Affinity Labels↗
PubMed1991

Photoaffinity labeling of rat type I iodothyronine deiodinase.

The photoreactive compound p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PAL) was coupled to [125I]rT3, T4, or T3 and incubated with liver and kidney microsomes of hypo-, hyper-, or euthyroid rats to identify the type I iodothyronine deiodinase. Various substrates or inhibitors of the enzyme, including rT3, T4, T3, 6-n-propylthiouracil (PTU), and iopanoic acid, were used as competitors to establish the specificity of protein labeling. The PAL derivatization enhanced the behavior of T4 and T3 as substrates for the type I enzyme. No specific labeling of microsomal proteins was observed with either rT3 or T4-PAL, presumably due to deiodination of the labeled compound. In contrast, T3-PAL labeled a 27-kDa band, the presence of which paralleled thyroid status. The labeling of only this protein was blocked by either substrates or enzyme inhibitors in a dose-dependent fashion, with a rank order of potency predicted by the activity of such compounds in type I enzyme assays. The specific nature of these competitions provides further evidence that this 27-kDa protein, identified in previous studies using N-bromoacetyl [125I]T3 or -T4, contains the active site of the rat type I deiodinase. This is in agreement with the mol wt of the rat type I deiodinase deduced from the recently identified cDNA coding for this protein.

Affinity Labels↗
PubMed1990

Photoaffinity labeling of the electroplax sodium channel with tetrodotoxin derivatives. II. Comparison of the photoreactivity of different photoactivable groups in the tetrodotoxin binding site.

Four photoactivable tetrodotoxin (TTX) derivatives and the corresponding tritiated compounds were synthesized and purified. The photoactivable groups introduced were a nitro-azidophenyl (NAP) group and a trifluoromethyl diazirinobenzoyl (TDB) group, as nitrene and carbene precursors, respectively, as well as two isomers of the fluoro-nitrophenoxy group, a new photoreactive group selective for nucleophiles. The binding affinities of the four derivatives to the sodium channel were similar to that of TTX, but the values of specific photoincorporation were unexpectedly low (up to 2.5%), suggesting that the photoactivable groups are likely to be oriented unfavorably for labeling reactions in the TTX binding site. Two of the labeled sodium channel proteins were purified and digested. Peptides labeled with TTXenNAP lost most of the label during high performance liquid chromatography in 0.1% trifluoroacetic acid-acetonitrile, while peptides labeled with TTXenTDB retained the labels during the procedures. The TTXenTDB-labeled peptides were eluted in four major fractions with 80% recovery of the amount applied on a reverse-phased C4 column. However, further purification is necessary to identify the labeled sites of the peptides.

Animals↗