INVITRO REPLICATION OF COWPEA MOSAIC-VIRUS RNA .2. SOLUBILIZATION OF MEMBRANE-BOUND REPLICASE AND PARTIAL-PURIFICATION OF SOLUBILIZED ENZYME

A method for the solubilization of membrane-bound Cowpea mosaic virus RNA replicase was developed bypassing the use of detergents. Solubilization was achieved by washing the 31,000 .times. g-pellet containing the bound replicase with a Mg2+-deficient buffer. This procedure had several advantages as compared to treatments with nonionic or ionic detergents: the solubilized enzyme was stable at 4.degree. C, more than 80% of the replicase could be solubilized without loss of total enzyme activity, the replicase was rather selectively released resulting in a 2-3-fold increase in specific activity, and most of the green color from chloroplast fragments present in the crude replicase fraction remained membrane bound resulting in only slightly colored preparations of solubilized enzyme. The solubilized replicase was further purified by DEAE-Bio Gel column chromatography. RNA synthesis directed by the DEAE-purified enzyme was template dependent and proceeded at a linear rate for at least 9 h.