tRNA methyltransferases from rat liver. Differences in response of partially purified enzymes to polyamines and inorganic salts.
Three tRNA methyltransferases, purified from rat liver, have been compared for their activity in the presence of various amines and Mg2+. The enzymes differ with respect to the ion which permits maximal activity; they also differ with respect to the concentration of a given ion necessary for maximal activity. The methyltransferase which forms N2-methylguanine in the region between the dihydrouridine loop and the acceptor stem (2mG I), when assayed using purified tRNA as substrate, shows high activity with 3--5 mM sperimidine or 20 mM putrescine and significantly lower rates of methylation with 200--350 mM ammonium acetate or 1--10 mM magnesium acetate. The enzyme responsible for forming N2-methylguanine between the dihydrouridine and anticodon loops (2mG II) works well in the presence of 0.2--0.5 mM spermidine, 10 mM putrescine or 200--300 mM ammonium acetate and shows slightly lower activity with 1 mM magnesium acetate. The optimal conditions for assaying 1-adenine methyltransferase (1mA) with purified tRNAs are either 200--300 mM ammonium acetate or 30 mM putrescine; spermidine is slightly less effective and magnesium acetate permits less than 25% of maximal activity. The addition of 10 mM Mg2+, in combination with polyamines or NH4+, depresses slightly the activity of the guanine methyltransferases but completely abolishes the polyamine or ammonium-stimulated activity of the adenine methyltransferase. When unfractionated (Escherichia coli) tRNA is used as substrate, the concentrations of polyamines required for optimal methyltransferase activity are increased but the patterns of response of the three enzymes do not differ significantly from those obtained with purified tRNA substrates. Based on the studies with these three enzymes, unfractionated tRNA and 40 mM putrescine should provide the most reliable system for detecting methylating activity if the nature of the tRNA methyltransferase is unknown.
D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D011700:Putrescine / Q000494:pharmacology; D012343:RNA, Transfer; D051381:Rats; D013095:Spermidine / Q000494:pharmacology; D012359:tRNA Methyltransferases / Q000378:metabolism*
D000641:Ammonia; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; D008274:Magnesium; D013095:Spermidine; D011700:Putrescine