Isolation and characterization of DNA-dependent RNA polymerase III from Leishmania mexicana and inhibition by purine analogs.
A DNA-dependent RNA polymerase has been isolated and characterized from the parasitic flagellated protozoan Leishmania mexicana. The initial stages of purification utilized high-ionic-strength extraction and protamine sulfate treatment. The enzyme was further purified by differential elution by heparin-Sepharose, DEAE-Sephadex, and carboxymethyl-Sephadex chromatography. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms. The enzyme isolated had characteristics of true DNA-dependent RNA polymerase since it required DNA and all four nucleoside triphosphates for synthesis of RNase-sensitive products. Analysis of ammonium sulfate and metal ion optima, as well as relative activities of the enzyme with Mn2+ versus Mg2+, gave results similar to those reported for other RNA polymerase IIIs in eucaryotes. Formycin A triphosphate was found to be a noncompetitive inhibitor of RNA polymerase III, and cordycepin triphosphate was found to be inhibitory, although the exact mode of inhibition was not determined.
Nolan
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Division of Public Health, Morrill Science Center, University of Massachusetts, Amherst 01003-0081.
Fehr
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D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D002845:Chromatography; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology*; D005573:Formycins / Q000494:pharmacology*; D007894:Leishmania mexicana / Q000201:enzymology*; D012320:RNA Polymerase III / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D012265:Ribonucleotides / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D003838:Deoxyadenine Nucleotides; D005573:Formycins; D012265:Ribonucleotides; C032552:formycin triphosphate; C011746:3'-deoxyadenosine 5'-triphosphate; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III