Abstract:
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Brodner OG,Wieland Tdoi
10.1021/bi00661a013subject
Has Abstractpub_date
1976-08-10 00:00:00pages
3480-4issue
16eissn
0006-2960issn
1520-4995journal_volume
15pub_type
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