A facile procedure for purifying maize chloroplast RNA polymerase from whole cell homogenates.
Two DNA-dependent RNA polymerases have been purified from homogenates of maize leaves by a relatively rapid procedure involving Sepharose 4B and DEAE-cellulose chromatography followed by resolution of two RNA polymerasies on phosphocellulose. The RNA polymerase eluting from phosphocellulose at 0.11 M (NH4)2SO4 is inhibited strongly by low levels of alpha-amanitin and possesses catalytic properties and polypeptide subunits like those of maize nuclear RNA polymerase II. The RNA polymerase eluting from phosphocellulose at 0.15 M (NH4)2SO4 resembles the RNA polymerase solubilized from isolated maize chloroplasts in many ways. This enzyme and that isolated from chloroplasts are resistant to alpha-amanitin and rifamycin-SV at high concentrations. Both RNA polymerases have virtually the same Mn2+ and Mg2+ optima, Mg2+/Mn2+ activity ratios, (NH4)2SO4 sensitivity, kinetics of UMP incorporation, and temperature optima. Electrophoresis of this phosphocellulose-purified RNA polymerase on denaturing polyacrylamide slab gels reveals 14 heavily stainable polypeptides that are identical in number and molecular mass to those from chloroplast RNA polymerase. Moreover, two-dimensional tryptic maps of the 14 polypeptides from the phosphocellulose-purified RNA polymerase are very similar to the maps of corresponding polypeptides from chloroplast RNA polymerase. Using this method, relatively large quantities (0.5 mg/kg leaves) of a form of chloroplast RNA polymerase can be prepared in a few days.
D002499:Centrifugation, Density Gradient / Q000379:methods; D002736:Chloroplasts / Q000201:enzymology; D002845:Chromatography / Q000379:methods; D002847:Chromatography, Agarose / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010455:Peptides / Q000032:analysis; D003313:Zea mays / Q000201:enzymology
D010455:Peptides; D012321:DNA-Directed RNA Polymerases