Isolation and Characterization of RNA Polymerase B from the Larval Fat Body of the Tobacco Hornworm, Manduca sexta

Publisher Website
Google Scholar
Abstract
DNA-dependent RNA polymerase B was extensively purified from the larval fat body of the tobacco hornworm (M. sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels showed 1 main band and 1 minor band, both having enzyme activity sensitive to .alpha.-amanitin. The catalytic and physicochemical properties of the enzyme were similar to those of other eukaryotic B-type RNA polymerases. The enzyme had an apparent MW 530,000, is inhibited 50% by .alpha.-amanitin at 0.04 .mu.g/ml and showed maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacted with the pure enzyme and formed a precipitin line. This antibody did not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but did so with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.