Abstract
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.
Highlights
2-5A-synthetase in addition to the smalle(r40 and 46 endoribonucleaseresponsiblefor degradation of viral and kDa) forms has recently been established
Tion, synthesis, and activityof 69- and 100-kDa2’,5’- lo), several authors have suggested the presence of different oligoadenyla(t2e-5Asy) nthetaseinsterferonforms of 2-SA-synthetase [11,12,13,14,15,16,17,18].The existenceof such forms treated human Daudicells
Induction of these synthetases is detectable incells treated with as little as 1-5 units/ml of human a-interferon, higherconcentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein
Summary
In the presence of low concentrations of to as mAb 56/3-Sepharose and mAb 25/11-Sepharose These dsRNA, the 69- and 100-kDa enzymes synthesize mainly the immunoadsorbants bind 69- and 100-kDaproteins dimeric form of 2-5A. Enhanced synthesis of 69- and 100- the different forms of 2-5A-synthetase might be specific to kDa proteins occurs with 1 unit/ml of a-interferon, with a each form and might vary according to the activation maximal enhancement at 100 units/ml. Seem t o be slightly different: maximal rate of 69-kDa protein the concentration of the RNA-activator in different synthesis occurring between 8 to 12 h whereas that of 100- organs of rabies virus-infected mice might have been low, the kDa protein occurs between 12 to 16h Such differences might reason for which only dimeric form of 2-5A was synthesized either be due to a change in the kinetics of transcription of in such infected mice. Cystein el abeled enzyms were used i n ordertoestimatether&overy o f 69 and 100 KO. proteins .Elute dm aterials were collectedinatest tubecontainingbovine plasma albumin(Signa, 991 pure a t 0.2 mgllnl
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
