AN IMPROVED METHOD FOR PURIFYING 2',5'-OLIGOADENYLATE SYNTHETASES

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  • 1 January 1984
    • journal article
    • research article
    • Vol. 259  (2) , 1363-1370
Abstract
A new, rapid and convenient procedure is described for purifying 2'',5''-oligoadenylate synthetases, using precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) .cntdot. poly(C) cellulose and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 .times. g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. The enzymes from 2 sources were purified to apparent homogeneity, about 2500-fold from the cytoplasm of [human cervical carcinoma] HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2'',5''-oligoadenylate synthetase from rabbit reticulocytes, mouse [neoplastic fibroblast] L cells and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) .cntdot. poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) .cntdot. poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) .cntdot. poly(C) paper and activates the bound enzyme about 3 times better.