METHYLATION OF E. COLI TRANSFER RIBONUCLEIC ACIDS BY A tRNA ADENINE-l-METHYLTRANSFERASE FROM RAT BRAIN CORTEX AND BULK-ISOLATED NEURONS |
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Authors: | Carlos E Salas Otto Z Sellinger |
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Institution: | Laboratory of Neurochemistry, Mental Health Research Institute, University of Michigan Medical Center, 205 Washtenaw Place, Ann Arbor, MI 48109, U.S.A. |
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Abstract: | Abstract— Brain cortices or bulk-isolated neuronal cell bodies prepared from cortices of 8-day old male rats were used as the source of a l-methyl adenine-specific tRNA methyltransferase (tRNA-AMT). Ammonium sulfate fractionation and chromatography on spheroidal hydroxylapatite and Sephadex G-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 m= 6 μM) and the inhibitor, S -adenosyl- l -homocysteine (SAH) ( K i= 3.4 μ m ) were determined and several SAH analogs tested as inhibitors. S -Adenosyl- l -cysteine (SAC) ( 10 -4 m ) and S -adenosyl- d -homocysteine (SADH) (10-4 m ) produced a 35 and a 21% reduction in enzyme activity, respectively. The effects of Mg2+, NH4+ acetate and of the polyamines spermine, putrescine and spermidine on the brain tRNA-AMT mimicked the effects of these agents on hepatic tRNA-AMT (G lick et al , 1975). Comparing the ability of cerebral tRNA-AMT to methylate E. coli tRNAglu2, tRNAval, tRNAphe and bulk tRNA revealed tRNAglu2 as the best and tRNAphe 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. |
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