Methylation of E. coli transfer ribonucleic acids by a tRNA adenine-1-methyltransferase from rat brain cortex and bulk-isolated neurons.

Brain cortices or bulk-isolated neuronal cell bodies prepared from cortices of 8-day old male rats were used as the source of a I-methyl adenine-specific tRNA methyltransferase (tRNA-AMT). Ammonium sulfate fractionation and chromatography on spheroidal hydroxylapatite and Sephadex (3-200 yielded an 80-fold purified enzyme, as determined by using E. coli bulk tRNA as substrate. The kinetic parameters of tRNA-AMT for the substrate S-adenosyl-L-methionine (SAM) ( K , = 6 PM) and the inhibitor, S-adenosyl-L-homocysteine (SAH) (Ki = 3.4 PM) were determined and several SAH analogs tested as inhibitors. S-Adenosyl-L-cysteine (SAC) ( 1 0 4 ~ ) and S-adenosyl-o-homocysteine (SADH) ( I W 4 ~ ) produced a 35 and a 21% reduction in enzyme activity, respectively. The effects of Mg", NH; acetate and of the polyamines spermine, putrescine and spermidine on the brain tRNAAMT mimicked the effects of these agents on hepatic tRNA-AMT (GLICK et al., 1975). Comparing the ability of cerebral tRNA-AMT to methylate E. coli tRNAE'"', tRNA'"', tRNAPh' and bulk tRNA revealed tRNAgIu2 as the best and tRNAph' as the least effective substrate. tRNA-AMT prepared from neuronal cell bodies showed closely similar characteristics to the cortical enzyme. A comparison of the activities of tRNA-AMT in neurons and glial cells revealed higher values in the former. tRNA adenine I-methyltransferase [EC 2.1.1.36] (tRNA-AMT) is an enzyme or group of closely related enzymes that methylates the 1 position of adenine residues in a tRNA polynucleotide chain. Unlike the recently reported non-specific RNA adenine1methyltransferase extracted from the nuclear fraction of the dinoflagellate Cripthecodinium cohnii (WERNER et al . , 1976). tRNA-AMT seems to recognize specific sequences within the tRNA molecule for the insertion of the methyl group (KUCHINO & NISHIMURA. 1974). In the past years several attempts have been made to purify this enzyme using biological material from different sources (e.g. rat liver, HeLa cells and prokaryotic cells) (BAGULEY & STAEHELIN, 1968~1,b; KUCHINO & NISHIMURA, 1970; AGRIS ef a!., 1974; KERR, 1974; CLICK et al., 1975). The activity was shown to be affected by polyamines, divalent cations, ammonium acetate. SAM and SAH (YOUNG & SRINIVASAN, 1971; PEGG, 1971; HACKER, 1973; LEBOY & GLICK, 1976). In a previous study we reported a relative decrease in the methylation of E. col i bulk tRNA adenine residues when 2.5 mM-spermidine was present ' Present address: Institut de Biologie Moleculaire et Cellulaire du C.N.R.S.. Laboratoire de Biochimie. 1 5 rue Rene Descartes, 67084 Strasbourg, France. ' To whom correspondence should be addressed Ahhreoiations used: tRNA-AMT, tRNA adenine-Imethyltransferase: SAM. S-adenosyl-L-methionine; SAH, S-adenosyl-r-homocysteine: SAC. S-adenosyl-L-cysteine: SADH. S-adenosyl-o-homocysteine; in the incubation mixture containing a crude enzyme preparation from rat brain cortex (SALAS et a/., 1976). At least two possibilities were suggested to explain the observed results: (a) spermidine inhibits the methylation of adenine residues in position 1 ; or (b) the methylation of all other bases is proportionately higher than that of adenine in position 1, a n d this is reflected in an apparent, relative decrease in the percentage of I-methyladenine formed. The same study (SALAS et a/., 1976) also showed a relative enrichment in tRNA-AMT activity in extracts derived from bulk-isolated nerve cell bodies (SELLINGER et a/., 1971) as compared to similar preparations obtained from the brain cortex (neurons + glial cells). To clarify some of these issues, we decided t o examine the cellular localization of tRNA-AMT and compare some of the properties of the partially purified enzyme obtained independently from the cortex and its nerve cell bodies. EXPERIMENTAL PROCEDURES