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2',5'-8-azidoadenylate trimer 5'-triphosphate (2,5-8-N3p3A3): enzymatic synthesis, biological properties and photoaffinity labelling of RNase L

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:6285210
; ; ; ;

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/ of 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.

Research Organization:
Temple Univ. School of Medicine, Philadelphia, PA
OSTI ID:
6285210
Report Number(s):
CONF-870644-
Journal Information:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 46:6; ISSN FEPRA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOSYNTHESIS
CHEMICAL PROPERTIES
CHEMICAL REACTIONS
CHROMATOGRAPHY
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DISTRIBUTION
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
ENZYMES
ESTERASES
HYDROLASES
HYDROLYSIS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
LYSIS
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
POLYMERIZATION
RADIOISOTOPES
REACTION KINETICS
RNA-ASE
SEPARATION PROCESSES
SOLVOLYSIS
SUBCELLULAR DISTRIBUTION
SYNTHESIS
TRACER TECHNIQUES