Fractionation and characterization of 2',5'-oligoadenylates by polyacrylamide gel electrophoresis: an alternative method for assaying 2',5'-oligoadenylate synthetase.
2',5'-Linked oligoadenylates of varying chain lengths (2,5As) are formed from ATP by an interferon (IFN)-induced enzyme, 2',5'-oligoadenylate synthetase (2-5A synthetase). To identify these multiple forms, a method was devised utilizing electrophoretic separation of 32P-labeled 2,5As in a thin 20% polyacrylamide gel containing 7 M urea. A mixture of 2,5As synthesized from rat liver nuclear suspension was fractionated by this method. Each species was eluted from the gel for characterization by specific nucleotidylic enzymes. All major species in the gel were identified, including dimeric, trimeric, and tetrameric forms with either 5' tri- or diphosphates, as well as dephosphorylated species. Thus, in a single step, this method produced a more complete assessment of newly synthesized 2,5As and their degradative products than conventional, multistep DEAE-cellulose chromatography. It also allowed more rapid screening of multiple samples than high-pressure liquid chromatography (HPLC). This method, used to assay 2-5A synthetase induction by IFN-alpha in human T-cell H9 and CEM-CM3 lines, should be applicable for routine analysis of clinical specimens.
Miele
M B
MB
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033.
Liu
D K
DK
Kan
N C
NC
D015088:2',5'-Oligoadenylate Synthetase / Q000032:analysis; D000227:Adenine Nucleotides / Q000032:analysis*; D000818:Animals; D005591:Chemical Fractionation; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D007372:Interferons / Q000494:pharmacology; D009843:Oligoribonucleotides / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013601:T-Lymphocytes / Q000187:drug effects
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; C023505:2',5'-oligoadenylate; D007372:Interferons; D015088:2',5'-Oligoadenylate Synthetase