TY - JOUR
T1 - Fractionation and Characterization of 2',5'-O1igoadenylates by Polyaecrylamide Gel Electrophoresis
T2 - An Alternative Method for Assaying 2',5'-O1igoadenylate Synthetase
AU - Miele, Mary Beth
AU - Liu, Dai Kee
AU - Kan, Nancy C.
PY - 1991/2
Y1 - 1991/2
N2 - 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-α in human T-cell H9 and CEM-CM3 lines, should be applicable for routine analysis of clinical specimens.
AB - 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-α in human T-cell H9 and CEM-CM3 lines, should be applicable for routine analysis of clinical specimens.
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U2 - 10.1089/jir.1991.11.33
DO - 10.1089/jir.1991.11.33
M3 - Article
C2 - 1709417
AN - SCOPUS:0026381212
SN - 0197-8357
VL - 11
SP - 33
EP - 40
JO - Journal of Interferon Research
JF - Journal of Interferon Research
IS - 1
ER -