Methyl-deficient mammalian transfer RNA: II. Homologous methylation in vitro of liver tRNA from normal and ethionine-fed rats: ethionine effect on 5-methylcytidine synthesis in vivo

摘要

Following hydroxyapatite chromatography, rat liver tRNA methylase activity was assayed with liver tRNA from normal rats and with methyl-deficient liver tRNA from ethionine-fed rats. The difference in homologous methylation between normal and methyl-deficient tRNA was maximal in certain fractions in presence of cadaverine, and much less in presence of Mg++ or Mg++ plus cadaverine. These methylase fractions, which contained endogenous tRNA, were used for preparative homologous methylation of added normal and methyl-deficient tRNA in presence of 30 mM cadaverine. The 14C-methylated tRNA was digested with RNase T₂ and the resulting methylated mononucleotides were characterized and quantitated after twodimensional thinlayer chromatography and autoradiography. The major products of homologous tRNA methylation were m5C and m1A. However, the methylase fraction used here did not catalyze the formation of m₂6A with m₂6A-deficient tRNA as substrate-In addition to the previously described, analytically detectable m₂6A-deficiency, a partial m5C-deficiency was demonstrated in liver tRNA from ethionine-fed rats by measuring the methyl-acceptance in vitro. In presence of cadaverine, with the methylase fraction used here, methyl-deficient tRNA from ethionine-fed rats was a twofold more efficient methyl-acceptor in vitro than normal liver tRNA, while endogenous tRNA isolated from the methylase fraction was a threefold more efficient methyl-acceptor than normal liver tRNA. Homologous methylation of normal tRNA, as observed here, has not been described before.