Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells.
The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.
Hovanessian
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Unité d'Oncologie Virale (Unité associée Centre National de la Recherche Scientifique), Institut Pasteur, Paris, France.
Svab
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J
Marié
I
I
Robert
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N
Chamaret
S
S
Laurent
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AG
D015088:2',5'-Oligoadenylate Synthetase / Q000096:biosynthesis / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002846:Chromatography, Affinity; D006801:Humans; D007370:Interferon Type I / Q000502:physiology*; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007370:Interferon Type I; D007527:Isoenzymes; D011994:Recombinant Proteins; D015088:2',5'-Oligoadenylate Synthetase