skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

Abstract

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from (..gamma..-/sup 32/P)2-azidoATP and (..cap alpha..-/sup 32/P)8-azidoAPT by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p/sub 3/A/sub 4/(/sup 32/P)pCp from RNase L with affinity equivalent to p/sub 3/A/sub 3/. The 8-azido photoprobe also activates RNase L to hydrolyze poly(U)(/sup 32/P)pCp 50% at 7 /times/ 10/sup /minus/9/ M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p/sub 3/A/sub 3/ activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10/sup /minus/9/ M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The (..gamma..-/sup 32/P)2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the (..cap alpha..-/sup 32/P)8-azido adenylate trimermore » 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p/sub 3/A/sub 3/ allosteric binding site was obtained.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Temple Univ. of School of Medicine, Philadelphia, PA (USA)
OSTI Identifier:
6208678
Alternate Identifier(s):
OSTI ID: 6208678
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 27:24
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AZIDO COMPOUNDS; BIOSYNTHESIS; RNA-ASE; STEREOCHEMISTRY; ATP; INTERFERON; LABELLING; MICE; MOLECULAR STRUCTURE; PHOSPHORUS 32; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; DAYS LIVING RADIOISOTOPES; ENZYMES; ESTERASES; GROWTH FACTORS; HYDROLASES; ISOTOPES; LIGHT NUCLEI; LYMPHOKINES; MAMMALS; MITOGENS; NUCLEI; NUCLEOTIDES; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PHOSPHODIESTERASES; PHOSPHORUS ISOTOPES; PROTEINS; RADIOISOTOPES; RODENTS; SYNTHESIS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Suhadolnik, R.J., Kariko, K., Sobol, R.W. Jr., Li, S.W., Reichenbach, N.L., and Haley, B.E. Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins. United States: N. p., 1988. Web.
Suhadolnik, R.J., Kariko, K., Sobol, R.W. Jr., Li, S.W., Reichenbach, N.L., & Haley, B.E. Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins. United States.
Suhadolnik, R.J., Kariko, K., Sobol, R.W. Jr., Li, S.W., Reichenbach, N.L., and Haley, B.E. Tue . "Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins". United States. doi:.
@article{osti_6208678,
title = {Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins},
author = {Suhadolnik, R.J. and Kariko, K. and Sobol, R.W. Jr. and Li, S.W. and Reichenbach, N.L. and Haley, B.E.},
abstractNote = {The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from (..gamma..-/sup 32/P)2-azidoATP and (..cap alpha..-/sup 32/P)8-azidoAPT by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p/sub 3/A/sub 4/(/sup 32/P)pCp from RNase L with affinity equivalent to p/sub 3/A/sub 3/. The 8-azido photoprobe also activates RNase L to hydrolyze poly(U)(/sup 32/P)pCp 50% at 7 /times/ 10/sup /minus/9/ M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p/sub 3/A/sub 3/ activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10/sup /minus/9/ M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The (..gamma..-/sup 32/P)2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the (..cap alpha..-/sup 32/P)8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p/sub 3/A/sub 3/ allosteric binding site was obtained.},
doi = {},
journal = {Biochemistry; (United States)},
number = ,
volume = 27:24,
place = {United States},
year = {Tue Nov 29 00:00:00 EST 1988},
month = {Tue Nov 29 00:00:00 EST 1988}
}
  • The photoaffinity probes (..gamma..-/sup 32/P)2-azidoATP (2-N/sub 3/ATP) and (..cap alpha..-/sup 32/P)8-azido-ATP (8-N/sub 3/ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N/sub 3/APT and 8-N/sub 3/ATP are substrates for 2-5A synthetase. In this study the authors show that 2- and 8-N/sub 3/ATP are competitive inhibitors of the enzymatic conversion of ATP to 2-5A. Ultraviolet irradiation results in the photoinsertion of 2-N/sub 3/ATP and 8-N/sub 3/ATP into the enzyme. The covalent photoinsertion of (..cap alpha..-/sup 32/P)8-N/sub 3/ATP into the 2-5A synthetase is proportional to the inactivation of the enzyme as UV irradiation is increased. Photolabeling ofmore » 2-5A synthetase is saturated at 1.5 mM 2-N/sub 3/ATP and 2.0 mM 8-N/sub 3/ATP. Computer analysis of the curvilinear Scatchard plots of the 2-5A synthetase suggest the presence of high-affinity and low-affinity binding sites that may correspond to the acceptor and the 2'-adenylation sites of the enzyme. The competition of nucleotides for the covalent photoinsertion of 8-N/sub 3/ATP into the binding site(s) of the synthetase was determined. Photoinsertion of 8-N/sub 3/ATP into 2-5A synthetase increases with the addition of poly(rI)/times/poly(rC). Without the addition of poly(rI)/times/poly(rC) to the synthetase, the (..cap alpha..-/sup 32/P)8-N/sub 3/ATP is photoinserted into the enzyme; however, in the absence of dsRNA and in the absence of UV irradiation, the synthetase cannot convert ATP to 2-5A. The finding suggest that the formation of the enzyme/substrate complex can occur in the absence of dsRNA but dsRNA is essential to activate the 2-5A synthetase to form the productive complex needed for synthesis of 2-5A from ATP.« less
  • A photoaffinity analog of colchicine, 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine, was synthesized by reacting deacetylcolchicine or (TH)deacetylcochicine with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Homogeneity of the photoaffinity analog was established by thin-layer chromatography and high-pressure liquid chromatography. The structure of the photoaffinity analog was determined by H and TC NMR, infrared and ultraviolet-visible spectroscopies, and elemental analysis. Binding of 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine to bovine renal tubulin was measured by competition with (TH)colchicine. The value of the apparent Ki for the photoaffinity analog was 0.28 microM in the concentration range of 0.8-1.2 microM of the analog. A value of 0.50 microM for the apparent Kd was measured by the direct bindingmore » of the tritiated photoaffinity analog to tubulin. The analog is slightly more potent an inhibitor of microtubule formation than colchicine. The photoaffinity analog reacted with renal tubulin upon irradiation with a mercury lamp equipped with a 420-nm cutoff filter. Spectral and radiochemical analyses of the tubulin after photolysis and dialysis have demonstrated a stoichiometric incorporation of the photoaffinity analog in the alpha-subunit of the tubulin. Covalent labeling of tubulin with the photoaffinity analog decreases the extent of (TH)colchicine binding by more than 90%.« less
  • To produce light sensitive DNA for the study of protein-DNA interactions, 5-azido-2'-deoxyuridine-5'-triphosphate (5-N/sub 3/dUTP), was synthesized to serve as a substrate for the enzymatic incorporation of a photoactive nucleotide analog into DNA. 5-N-dUTP was synthesized from dUMP in five steps with the key reaction being the nitration of dUMP to 5-NO/sub 2/dUMP in 98% yield using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. The triphosphate form of the nucleotide (5-N/sub 3/dUTP), formed through a chemical coupling of pyrophosphate to 5-N/sub 3/dUMP, was found to support DNA synthesis by E. coli DNA polymerase I at a rate indistinguishable to thatmore » supported by dTTP. Using UMP as the starting compound 5-N/sub 3/UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare (..gamma../sup 32/P) 5-N/sub 3/dUTP for use as an active site directed photoaffinity labeling reagent, a simple method of preparing (..gamma../sup 32/P) labeled pyrimidine nucleotides was developed. (..gamma../sup 32/P) 5-N/sub 3/dUTP is an effective photoaffinity labeling reagent for DNA polymerase I which binds to the active site of the enzyme with a two fold higher affinity than dTTP.« less
  • Vulnerability of 25-hydroxy-(26,27-{sup 3}H)vitamin D{sub 3} 3{beta}-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D{sub 3} (25-OH-D{sub 3}) toward standard conditions of carboxymethylationin promoted the authors to synthesize 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D{sub 3}, and 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE), the radiolabeled counterpart of 25-ANE competes for the 25-OH-D{sub 3} binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with {sup 3}H-25-ANE, covalently labeled the protein. However, very little covalent labeling wasmore » observed in the absence of UV light or in the presence of a large excess of 25-OH-D{sub 3}. These results provide strong evidence for the covalent labeling of the 25-OH-D{sub 3} binding site in rDPB by {sup 3}H-25-ANE.« less
  • Photoreactive analogs of 2-5A have been enzymatically synthesized from 8-azidoATP by 2-5A synthetase from lysed rabbit reticulocytes (LRR) in 1% average yield, but not from 100,000-fold purified 2-5A synthetase. The structure of the 2,5-8-N3p3A3 was established by enzymatic hydrolyses and HPLC. The ability of the 2,5-8-N3p3A3 to bind to and activate RNase L was compared to authentic p3A3 in radiobinding, core-cellulose and rRNA cleavage assays using RNase L in L929 cell extracts. The 2,5-8-N3p3A3 can bind to activate RNase L as well as does p3A3. Furthermore, ( -TSP)2,5-8-N3p3A3 is covalently cross-linked to a single protein with a M/sub r/ ofmore » approx.80,000 in L929 cell extracts following 1 min. ultraviolet irradiation, 0C. This cross-linking is inhibited by p3A3 but not by ATP or 8-azidoATP suggesting that the protein that was photolabeled was RNase L. The use of the 2,5-azido analog provides a way to further characterize the binding and activation processes of RNase L. ( -TSP)8-AzidoATP crosslinked to 2-5A synthetase; 2,5-8-N3p3A3 did not compete with 8-azidoATP for this cross-linking. The mild conditions needed for the covalent linkage to 2-5A synthetase and RNase L makes it possible to determine the subcellular distribution and role of RNase L in cell growth in the intact cell.« less