Selective Extraction of Form I DNA Dependent RNA Polymerase from Rat Liver Nuclei and its Separation into Two Species.

DNA dependent RNA polymerase activity was extracted in a soluble form from rat liver nuclei by a mild, low salt, nonlytic technique. This technique preferentially extracts the form I polymerase which has been characterised by several laboratories. The enzyme was purified and separated into two species, present in approximately equal amounts, by DEAE-cellulose and phosphocelluose column chromatography. The two species, which have been designated form a and form Ib, were purified 40- and 100-fold, respectively, over the nuclear level with a recovery 0f 50-60%. Both forms have optimal activity at pH 8, sediment at 16 S in glycerol gradients, have a Km value for ATP of about 50 µM and are not inhibited by rifampicin or α-amanitin. No evidence could be obtained that the two forms represent a "core" and "complete" enzyme such as is produced by the bacterial enzyme on phosphocellulose. A standard competition hybridization technique could not distinguish between the RNA species produced by the two forms on a rat liver DNA template. [ABSTRACT FROM AUTHOR]