Competition Binding Assay Using o-Methyl-[ 3 H]-Demethyl-γ-Amanitin for Study of RNA Polymerase B

Summary

An improved method permitting the synthesis of a radioactive derivative of α-amanitin from a small amount of the commercially available parent compound has been developed. The labeled derivative was used in an amatoxin competition binding assay designed to detect eukaryotic RNA polymerase B in either purified form or in crude homogenates. Both compounds are shown to compete for the same binding site and with approximately the same affinity. The competition assay proves to be both sensitive and highly selective for RNA polymerase B and provides a new, direct method for studying the enzyme-amanitin interaction over a much broader range of concentration than previously reported.

We greatly appreciate the assistance of Ms. Aphia Abdou in preparation of illustrations, Ms. Donna Chambers for secretarial assistance, and Drs. John A. Armstrong, Patricia A. Craven and Mary Edmonds for critically reviewing the manuscript. This work was supported through the Medical Research Service of the U. S. Veterans Administration.