The optimal conditions for the assay in vitro of nucleic acid methylases in extracts of mammalian cells were investigated. Methylation of tRNA catalysed by extracts of rat liver or kidney was markedly increased in the presence of the diamines, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane or the polyamines, spermidine and spermine. Mg2+ produced only a much smaller stimulation of the reaction. Partially purified preparations of nucleic acid methylases were almost entirely dependent on the addition of these amines for activity unless assayed in solutions of very high ionic strength. In the presence of polyamines, extracts from rat liver catalysed the formation of 1-methyladenine, 1-methylguanine, 7-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 5-methylcytosine and 5-methyluracil. Some evidence that the effect of amines on the methylation of tRNA was due to combination with the nucleic acid substrate rather than a direct effect on the enzyme was obtained. The effects of other substances known to bind to nucleic acids on the enzymic methylation of tRNA were also studied. Ethidium bromide, acridine orange and proflavin were potent inhibitors of methylation, but streptomycin and 3,3′-diaminobenzidine produced a small stimulation.
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