Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B 3 - the true amatoxin receptor protein of multiple RNA polymerase B

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Abstract
Crude calf thymus DNA-dependent RNA polymerase, RNA polymerase B (ribonucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), was incubated with the 3H 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 2 radioactive components: PCI, the previously reported amatoxin binding protein, ABP, 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 1 main peak (> 2000 cpm) coinciding with the 550-nm absorption peak of subunit B 3 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.

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