Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B3-the true amatoxin receptor protein of multiple RNA polymerase B.
Crude calf thymus DNA-dependent RNA polymerase, RNA polymerase B (ribonucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), was incubated with the tritium labeled, potent inhibitor [3H]amanin, in order to form the enzymatically inactive [3H]amanin-polymerase complex ([3H]A-P complex). Subsequent purification procedures for the [3H]A-P complex were based on radioactive assays. Phosphocellulose chromatography separated two radioactive components: PCI, the previously reported amatoxin binding protein, ABP (Brodner and Wieland, 1976), and PC II, the [3H]A-P complex. Sodium dodecyl sulfate gel electrophoresis of the complex showed the presence of a new heavy band very close to subunit B 1 and a decreased intensity of subunit band B 3. These were the only differences noted in the subunit structure of RNA polymerase B. [3H]Amanin was covalently coupled to the enzyme, affinity labeling, by a water-soluble carbodiimide and the resultant conjugate submitted to sodium dodecyl sulfate gel electrophoresis. The profile of radioactivity showed one main peak (greater than 2000 cpm) coinciding with the 550-nm absorption peak of subunit B 3 on a stained parallel gel. Since no other protein band contains any significant radioactivity, the binding site for [3H]amanin and most probably for all amatoxins is localized on the B 3 subunit SB 3.
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D009183:Mycotoxins / Q000494:pharmacology; D011485:Protein Binding; D011955:Receptors, Drug; D013950:Thymus Gland / Q000201:enzymology
D000345:Affinity Labels; D046911:Macromolecular Substances; D009183:Mycotoxins; D011955:Receptors, Drug; D012321:DNA-Directed RNA Polymerases