title: Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of alpha-amanitin-resistant and -sensitive enzymes.
abstract: We have isolated, characterized and substantially purified two distinct RNA polymerase activities from the flagellate protozoan parasite Trypanosoma brucei. RNA polymerases from this organism were resolved poorly on DEAE-Sephadex, but could be separated with CM-Sephadex. One form was totally resistant to alpha-amanitin, whereas the second was 50% inhibited by 10-20 micrograms of the drug/ml. The enzymes had different salt optima, but both were of high Mr (greater than 480,000) and demonstrated the template preference: poly[d(A-T)] greater than denatured DNA greater than native DNA, and both were more active with Mn2+ than with Mg2+. The amanitin-resistant enzyme, polymerase R, was partially purified by chromatography on CM-Sephadex, DEAE-Sephadex and heparin-Sepharose. This enzyme was very labile, and activity yields were around 9%; after purification, one or two protein bands could be discerned after electrophoresis under non-denaturing conditions, but about 20 polypeptides were resolved on denaturing gels, including a major component (not thought to be part of the enzyme) of Mr 65,000. Polymerase S, sensitive to low alpha-amanitin concentrations, was more extensively purified, with an 18% recovery, and yielded a single major band with two minor ones after native gel electrophoresis. Analysis under denaturing conditions permitted a possible subunit structure for this enzyme to be ascribed.
mesh_terms: D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D000546:Amanitins; D007527:Isoenzymes; D012321:DNA-Directed RNA Polymerases