Isolation and characterization of RNA polymerase B from the larval fat body of the tobacco hornworm, Manduca sexta.
DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca 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 shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.
D000273:Adipose Tissue / Q000201:enzymology*; D000546:Amanitins / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005779:Immunodiffusion; D007700:Kinetics; D007814:Larva; D007915:Lepidoptera / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology*; D011233:Precipitin Tests; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D013698:Templates, Genetic
D000546:Amanitins; D046911:Macromolecular Substances; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases