tRNA METHYLTRANSFERASE ACTIVITY IN NEONATAL AND MATURE MAMMALIAN NEURAL TISSUE

Abstract

Abstract— The activity and kinetic characteristics of tRNA methyltransferases were measured with enzyme preparations obtained from neonatal and adult mouse brain tissue. Both neonatal and mature brain enzyme preparations were shown to contain a considerable amount of protein methylase activity which could interfere with the measurement of the tRNA methyltransferases. When increasing amounts of the unfractionated enzymes from young and adult neural tissue were added to reaction mixtures, the saturation kinetics were found to be considerably different. However, fractionation of the samples by precipitation at pH 5 resulted in an increase in the enzyme activity of preparations obtained from adult brain. Although the precipitation at pH 5 allowed a quantitative recovery of the enzyme activity of immature brain samples, this partial purification step led to an apparent activation of the tRNA methyltransferases in adult preparations. This activation was shown to be independent of differential changes in the thermolability of the enzymes but rather to be associated with an increase in the sites methylated and the measured affinity of the adult enzyme preparations with the tRNA substrate. Nicotinamide, a potent inhibitor of tRNA methyltransferase activity in other tissues, was shown to be ineffective in modulating brain tRNA methyltransferase activity. The results are discussed in light of the possible modulation of the activity of specific enzyme species and the alterations in the synthesis of nucleic acid precursors during neural development.

The homologous methylation of tRNA in rat brain

“…These workers stated that all of the methylation observed in the absence of E. coil tRNA was 'presumabl~y the result of protein methylation', even though they failed to examine the nature of the [14C-methyl] products so produced. Our results do not support the conclusion of Johnson et al 6, inasmuch as they demonstrate that, on the contrary, a sizable portion of the 'endogenous methylation' in rat brain cortex involves homologous tRNA. These findings supplement the recent report by Jank and Gross 5, who found that homologous tRNA methylation in adult rat liver involves principally the transfer of [14C-methyl] groups to cytidine and adenine residues to form 5-methylcytosine and 1-methyladenine.…”

“…These workers also showed that rat brain contains 'more than one methylating activity' and that cerebral methylases are about 7 times more active in fetal than in adult rat brain. More recently, Johnson et al 6 compared tRNA methyltransferases in neonatal and adult mouse brain and noted significant methylation of E. coli tRNA but little, if any, homologous tRNA methylation. These workers stated that all of the methylation observed in the absence of E. coil tRNA was 'presumabl~y the result of protein methylation', even though they failed to examine the nature of the [14C-methyl] products so produced.…”

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine

“…The present study shows that the immature rat brain cortex contains methyltransferase enzymes that effectively catalyze the methylation of endogenous macromolecular acceptors, an observation that contrasts with previous reports (4, 6) of little, if any, endogenous methylation in the adult kidney (4) and brain (6). More recently, Johnson et al (9) reported identical levels of endogenous protein methylation in neonatal and adult mouse brain.…”

“…In a most recent study, Johnson et al (9) compared these activities in neonatal and adult whole mouse brain, while we demonstrated homologous methylation of tRNA in rat brain (10) and showed the resulting [Me-14C] tRNA to contain chiefly [Me-14C] 1-methyl guanine and [Me-14C] N2-methyl guanine (10,11).…”

Radioactive labelling of ribosomal proteins with reductive alkylation and its use in studying ribosome-cytosol interactions

“…20ml) was dialysed overnight at 4°C against 200vol. of lOmM-Tris/HCl buffer (pH 7.5) containing 10mM-2-mercaptoethanol and I0mM-MgCl2 as described by Johnson et al (1974). Batches of the dialysed cytosol, containing 4-5mg of protein/ml, were stored at -70°C for up to 3 months.…”