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At least 19 recordsLinked to original sources
PubMed1976

Subcellular localization of S-adenosyl-L-methionine:tRNA methyltransferases with aminoacyl-tRNA synthetases in human and mouse: normal and leukemic leukocytes.

The subcellular distributions of S-adenosyl-L-methionine:tRNA methyltransferases and aminoacyl-tRNA synthetases were investigated with the use of human and mouse normal and leukemic leukocyte cell lines. Differential centrifugation of homogenized cell suspensions produced three pelleted subcellular fractions (nuclear and membrane, microsomal, and postribosomal) and a supernatant fraction. Each fraction was assayed for both methyltransferase activity and synthetase activity. The largest amounts, 40-50%, of total methyltransferase and synthetase activities were localized in either the microsomal or the postribosomal fractions, depending on cell type. In addition, the highest specific activities of these two enzyme systems were found to be present in the microsomal and postribosomal fractions. The psotribosomal fraction from leukemic leukocytes had a methyltransferase specific activity higher than that of the microsomal fraction, while the same two fractions of normal leukocytes had approximately equal activities. Specific activities of aminoacyl-tRNA synthetases were found to be approximately equal for these two fractions, whether they were from normal or leukemic leukocytes. The activity of tRNA methyltransferases and synthetases within the postribosomal fraction of the cytoplasm suggests the existence of high-molecular-weight enzyme complexes for the modification as well as the aminoacylation of tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Characterization of a unique enzyme complex composed of S-adenosyl-L-methionine-tRNA-methyltransferase and aminoacyl-tRNA synthetase activities.

S-adenosyl-L-methionine-tRNA methyltransferases of a murine leukemia cell line were found to exist in a high molecular weight enzyme complex. Aminoacyl-tRNA synthetase activity always co-chromatographed and co-sedimented with methyltransferase activity in evidence of a unique association of these two groups of enzymes. Molecular weight studies showed a probable molecular weight of 9 X 10(5) daltsons for the intact complex which dissociates to complexes of 6 X 10(5) and 3 X 10(5) daltons. The complexes contain discrete polypeptides of 25,000-90,000 daltons as determined from SDS-gel electrophoresis. High resolution fatty acid analysis showed that only very small amounts of saponifiable lipids were associated with the purified enzyme complex. Similarly very little protein-bound sugars was found within the complex indicating that neither lipids nor sugars were involved in the protein-protein interactions of the complex. Analysis of tRNA methylated in vitro indicated the presence of most methyltransferase activities in the purified complex. Of note was the absence from the complex of the methyltransferase responsible for the production of ribo Tp.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Age-dependent changes in the specificity of tRNA methyltransferases in the cerebellum of the icteric and nonicteric Gunn rat.

The activity of tRNA methyltransferases present in the cerebellum of 6- and 21-day-old nonicteric and icteric Gunn rats was compared using purified E. coli tRNAs as substrates. At 6 days the tRNA methyltransferases of the icteric animals were significantly more effective in methylating tRNAGlu2 and tRNAPhe than were those of their nonicteric counterparts. This relationship reversed itself at 21 days. The action of the tRNA methyltransferases from the 6-day-old icteric animals led to higher proportions of 1-methyladenine in tRNAGlu2 and tRNAPhe than were obtained using the corresponding enzymes of the nonicteric animals. The proportion of N2-methylguanine was also higher, yet only in tRNAfMet and not in tRNAPhe. The study reveals much more extensive fluctuations in the activity and in the substrate recognition specificity among the cerebellar tRNA methyltransferases of the icteric than among those of the nonicteric controls during the crucial 6--21 day period of cerebellar development.

Aging↗
PubMed1976

7-Methylguanine specific tRNA-methyltransferase from Escherichia coli.

A 7-methylguanine (m7G) specific tRNA methyltransferase from E. coli MRE 600 was purified about 1000 fold by affinity chromatography on Sepharose bound with normal E. coli tRNA. The purified enzyme catalyzes exclusively the formation of m7G in submethylated bulk tRNA of E. coli K12 met- rel-. The purified enzyme transfers the methyl group from S-adenosyl-methionine to initiator tRNA of B. subtilis and 0.8 moles m7G residues are formed per mole tRNA. It is suggested that the enzyme specifically recognizes the extra arm unpaired guanylate residue.

Electrophoresis, Disc↗
PubMed1976

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.

Ammonia↗
PubMed1978

In vitro methylation of tobacco mosaic virus RNA with ribothymidine-forming tRNA methyltransferase. Characterization and specificity of the reaction.

A novel method has been developed for the detection and study of tRNA-like moieties in viral RNAs. Tobacco mosaic virus RNA is an acceptable substrate for crude Escherichia coli ribothymidine-forming tRNA methyltransferase. Under optimum reaction conditions at least 85% of the methylation product is ribothymidine (rT). The reaction is essentially quantitative, 1 mol of rT being formed per mol of tobacco mosaic virus RNA. The optimum reaction conditions include the presence of 6.6 micrometers S-adenosyl-L-[Me-3H]methionine, 25 micrometers spermine, 25 mM ammonium acetate, and 50 mM HEPES, pH 8.0. Sequence analysis of (Me-3H)-labeled tobacco mosaic virus RNA shows that all of the methylation occurs at a single site and strongly suggests that this site is the 32nd residue from the 3'-end of tobacco mosaic virus RNA. This site closely resembles the normal position of rT in transfer RNA.

Base Sequence↗
PubMed1979

Selective changes in tRNA methyltransferase activity in confluent monolayers of WI-38 cells stimulated to proliferate.

In quiescent confluent monolayers of WI-38 cells, the specific activity of the tRNA methyltransferases falls to 20% of the level found in log phase cells. When the resting cells are stimulated to proliferate by a change to fresh medium, the enzyme show a rapid rise in specific activity which correlates with early increases in the rate of tRNA synthesis. The specific activity of the enzymes continues to rise throughout the period of DNA synthesis, at the end of which it is somewhat higher than that of log phase cells. The increases in enzyme activity could be blocked by exposure of the stimulated cells to Actinomycin D (2 microgram/ml). The increases in activity were not equivalent for the different base-specific enzymes. The contribution of the N2-methylguanine specific enzyme remained relatively constant, while that of the N2,N2-dimethyl-guanine specific and 1-methyladenine specific enzymes doubled and tripled, respectively, by late S phase. The contributions of the 1-methylguanine and the 7-methylguanine specific enzymes fell to a few percent of the total by late S phase. This indicates non-coordinate variations in the expression of the different base-specific enzymes after stimulation of resting cells and may be related to altered isoaccepting tRNA profiles observed in resting and growing cells.

Cell Division↗
PubMed1979

[In vitro inhibition of tRNA methyltransferases by queen substance, a pheromone of queen honeybees].

The Queen Substance 1, a pheromone of the queen Honeybee Apis mellifica is an in vitro inhibitor of E. coli B tRNA methylations. This activity is not specific of the methylase source, as inhibitions have been observed with preparations from queen honeybee ovaries, Rat liver or a Mouse plasmocytoma 1-adenine methylase. These results, together with preceding ones concerning t, t-farnesyl-acetone 3, are discussed.

Animals↗
PubMed1977

Methylation of an adenosine in the D-loop of specific transfer RNAs from yeast by a procaryotic tRNA (adenine-1) methyltransferase.

tRNA (adenine-1) methyltransferase occurs in Bacillus subtilis. Eucaryotic tRNAThr and tRNATyr from yeast in which 1-methyladenosine (m1A) is already present in the TpsiC loop, can be methylated in vitro with S-adenosylmethionine and B. subtilis extracts. Each of the specific tRNAs accepts 1 mol of methyl groups per mol tRNA. The enzyme transforms into m1A the 3'-terminal adenylic acid residue of the dihydrouridine loop, a new position for a modified adenosine residue in tRNA. Both tRNAs have the sequence Py-A-A-G-G-C-m2(2)G in the D-loop and D-stem region. Other tRNAs with the same sequence in this region also serve as substrates for the tRNA (adenine-1) methyltransferase.

Adenine Nucleotides↗
PubMed1978

Purification and characterization of two tRNA-(guanine)-methyltransferases from rat liver.

tRNA(guanine-1-)-methyltransferase (EC 2.1.1.31) and tRNA(N2-guanine)-methyltransferase I (EC 2.1.1.32) were isolated from rat liver. The (guanine-1-)-methyltransferase preparation is 6800-fold purified and is free from contaminating methyltransferases or ribonuclease. The molecular weight of (guanine-1-)-methyltransferase is 83 000. Of seven purified Escherichia coli tRNAs examined, only tRNAMetf was utilized as substrate by (guanine-1-)-methyltransferase. The methylation of tRNAMetf is maximally stimulated by 40 mM putrescine with a pH optimum of 8.0. Using E. coli K-12 tRNA, the Km for S-adenosylmethionine is 3 micrometer and Ki for S-adenosylhomocysteine is 0.11 micrometer for (guanine-1-)-methyltransferase. (N2-Guanine-)-methyltransferase is 6200-fold purified and is also free of interfering enzymes. It has a molecular weight of 69 000. E. coli tRNAPhe, tRNAVal and tRNAArg are substrates for this enzyme which introduces a methyl at the 2-amino group of the guanine at position 10 from the 5'-terminus of these tRNAs. The methylation of tRNAPhe is maximally stimulated by 100 micrometer spermidine with a pH optimum of 8.0. (N2-Guanine-)-methyltransferase has a Km for S-adenosylmethionine of 2 micrometer and a Ki for S-adenosylhomocysteine of 23 micrometer with E. coli K-12 tRNA as methyl acceptor.

Animals↗
PubMed1978

S-adenosylhomocysteine analogues as inhibitors of specific tRNA methylation.

Of 17 base- or amino acid-modified analogues of S-adenosylhomocysteine, six were found to produce at least 50% inhibition of the activity of an unfractionated tRNA methyltransferase extract at concentrations of 200 micron. The inhibitory effects of these six analogues on five purified rat liver tRNA methyltransferases were examined. The purified enzymes differed greatly in their sensitivity to the analogues. Ki values for the inhibitory analogues were determined for the three most highly purified methyltransferases. The kinetic analyses indicated that inhibition is competitive for nearly all enzyme/inhibitor combinations. The Ki values for good enzyme/inhibitor pairs were in the range of 0.11--2 micron. Each analogue appears to inhibit one methylation more strongly than others; e.g. the Ki values obtained for N6-methyl-S-adenosyl-L-homocysteine are approx. 0.4 micron for guanine-1 tRNA methyltransferase, 6 micron for adenine-1 tRNA methyltransferase and 100 micron for N2-guanine tRNA methyltransferase I. Structural features which are important for inhibitory activity are presence of a terminal amino group on the amino acid and the presence of adenosine rather than any other base. Ring nitrogens, a terminal carboxyl group and conformation at the asymmetric carbon appear to be important for some but not all of the tRNA methyltransferases examined.

Animals↗
PubMed1976

Transfer RNA methyltransferase activity in paramecium aurelia.

The tRNA methyltransferases from Paramecium aurelia were investigated. The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C. The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of tRNA in P. aurelia is similar to that observed in other eukaryotes.

Ammonia↗
PubMed1977

The effect of vitamin A deficiency on testicular transfer RNA methyltransferase activity.

Testicular transfer RNA methyltransferase activity was examined in normal and vitamin A-deficient rats. The specific activity was reduced by 50% in the vitamin A-deficient rats. In addition, a 5-fold decrease in the extent of tRNA methylation was observed with enzyme preparations from deficient testes. Both the rate and extent of tRNA methylation returned to control levels in vitamin A-repleted rats. In contrast, retinoic acid repletion did not reverse the effect of vitamin A deficiency on testicular tRNA methyltransferase activity. The methylated nucleoside composition of tRNA methylated by extracts of vitamin A-deficient testes was altered dramatically compared to that of tRNA methylated by control testicular enzymes. Decreased testicular tRNA methyltransferase activity was noted in midly deficient rats before the onset of testicular degeneration suggesting that the decreased tRNA methyltransferase activity in the testes is primarily the result of vitamin A deficiency.

Animals↗