Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatically synthesized 2',5'-phosphorothioate dimer and trimer: unequivocal structural assignment and activation of 2',5'-oligoadenylate-dependent endoribonuclease
In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate-dependent endoribonuclease (RNase L), chirality has been introduced into the 2',5'-oligoadenylate (2-5A, p/sub 3/A/sub n/) molecule to give the R/sub P/ configuration in the 2',5'-internucleotide backbone and the S/sub P/ configuration in the ..cap alpha..-phosphorus of the pyrophosphoryl moiety of the 5'-terminus. This was accomplished by the enzymatic conversion of (S/sub P/)-ATP..cap alpha..S to the 2',5'-phosphorothioate dimer and trimer by the 2-5A synthetase from lysed rabbit reticulocytes. The most striking finding reported here is the ability of the 2',5'-phosphorothioate dimer 5'-triphosphate (i.e., p/sub 3/A/sub 2/..cap alpha..S) to bind to and activate RNase L. p/sub 3/A/sub 2/..cap alpha..S displaces the p/sub 3/A/sub 4/(/sup 32/P)pCp probe from RNase L with an IC/sub 50/ of 5 x 10/sup -7/ M, compared to an IC/sub 50/ of 5 x 10/sup -9/ M for authentic p/sub 3/A/sub 3/. Further, p/sub 3/A/sub 2/..cap alpha..S activates RNase L to hydrolyze poly(U)-3'-(/sup 32/P)pCp (20% at 2 x 10/sup -7/ M), whereas authentic p/sub 3/A/sub 2/ is unable to activate the enzyme. Similarly, the enzymatically synthesized p/sub 3/A/sub 2/..cap alpha..S at 10/sup -6/ M activated RNase L to degrade 18S and 28S rRNA, whereas authentic p/sub 3/A/sub 2/ was devoid of activity. p/sub 3/A/sub 3/..cap alpha..S was as active as authentic p/sub 3/A/sub 3/ in the core-cellulose and rRNA cleavage assays. The absolute structural assignment for p/sub 3/A/sub 2/..cap alpha..S is 5'-0-((S/sub p/)-1-P-thiotriphosphoryl(-(R/sub P/)-P-thioadenylyl(2'-5')-(R/sub P/)-P-thioadenylyl(2'-5')adenosine. These assignments confirm the previous suggestion of an R/sub P/ configuration at the 2',5'-internucleotide linkages of enzymatically synthesized p/sub 3/A/sub 3/..cap alpha..S.
- Research Organization:
- Temple Univ. School of Medicine, Philadelphia, PA
- OSTI ID:
- 5599705
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:22; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
62 RADIOLOGY AND NUCLEAR MEDICINE
ADENYLIC ACID
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOSYNTHESIS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DERIVATIZATION
ENZYME INDUCTION
ENZYMES
ESTERASES
EVEN-ODD NUCLEI
GENE REGULATION
HYDROLASES
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MAGNETIC RESONANCE
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS 31
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOASSAY
RADIOISOTOPES
RESONANCE
RNA-ASE
SEPARATION PROCESSES
STABLE ISOTOPES
SULFUR 35
SULFUR ISOTOPES
SYNTHESIS
THIOLS