2- 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.
The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP 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 p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-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-32P]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 [alpha-32P]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 p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)
Suhadolnik
R J
RJ
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Karikó
K
K
Sobol
R W
RW
Li
S W
SW
Reichenbach
N L
NL
Haley
B E
BE
D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis*; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007202:Indicators and Reagents; D007739:L Cells / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D008970:Molecular Weight; D009843:Oligoribonucleotides / Q000378:metabolism*; D010777:Photochemistry
D000227:Adenine Nucleotides; D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; D009843:Oligoribonucleotides; C059245:5'-O-triphosphoryl-2-azidoadenylyl-(2'-5')-2-azidoadenylyl-(2'-5')-2-azidoadenosine; C059244:5'-O-triphosphoryl-8-azidoadenylyl-(2'-5')-8-azidoadenylyl-(2'-5')-8-azidoadenosine; C017040:8-azidoadenosine 5'-triphosphate; C023505:2',5'-oligoadenylate; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D015088:2',5'-Oligoadenylate Synthetase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease