33] Arylamine N-methyltransferase

Abstract

This chapter presents a procedure for the preparation of arylamine N -methyltransferase. The assay depends on the highly radioactive, methyllabeled —S -adenosylmethionine—which is readily available commercially. Tryptamine serves as the methyl group acceptor in the standard assay. After reaction, the methylated product is separated from the polar radioactive substrate by the extraction of the product into an organic solvent. The enzyme is purified from the livers of young adult New Zealand rabbits; the livers may be stored at – 80° for as long as 2 years without apparent effect. All procedures are conducted at ice-bath temperatures, including extraction by centrifugation; further process involve charging and washing of the filtrate on different columns, such as diethylaminoethyl (DEAE) cellulose I, DEAE-cellulose II, hydroxylapatite , aminohexyl-sepharose, and sephadex G-75. The enzyme is homogeneous by electrophoretic criteria: discontinuous polyacrylamide gels, pH 8.9, and SDS-polyacrylamide gels reveal one protein band; protein is coincident with enzyme activity in the nondenaturing gel system.