Glycine Activating Enzyme in the Posterior Silkgland of Silkworm

1. Aminoacyl tRNA synthetase was extracted from the silkgland of silkworm (Bombyxmori Linné) and fractionated on a DEAE-cellulose column. Activities were estimated by ATP-PP_i exchange reaction as well as glycyl tRNA formation.

2. Two peaks, A and B, having ATP-PP_i exchange activity were found in the separated fractions, respectively. There was also observed a marked difference between the both peaks with respect to the pH optimum and activity dependence on MgCl₂ concentration.

3. Peak A showed no activity of glycyl tRNA formation. Only a part of peak B coincided with the activity of glycyl tRNA formation. The activities of both the ATP-PP_i exchange reaction and glycyl tRNA formation were found to be dependent on MgCl₂ concentration, and the optimum concentration was different between two peaks.

4. It also seemed to exist two peaks of activities, a and b, in glycyl tRNA formation which could be separated with a DEAE-cellulose column.     

Gram-scale purification of arginine, formylmethionione, glutamic acid and the nylalanine transfer ribonucleic acids from E. coli K-12MO7

Gram-sized quantities of purified arginine, formylmethionine, glutamic acid, and phenylalanine-2 tRNAs have been prepared from pools of E. coli K–12 MO7 mixed tRNAs by reversed-phase chromatography after preliminary fractionation on DEAE-cellulose. Purified formylmethionine tRNA and partially purified arginine tRNA and glutamie acid tRNA were obtained from large-scale RPC–3 runs (4 × 36 in. column). The arginine tRNA was further purified by rechromatography on RPC–4 columns, and the gluatmic acid tRNA by rechromatography on an RPC–3 column. Two phenylalanine tRNAs were resolved on large-scale (2 × 96 in. column) RPC–3 runs; only the second phenylalanine tRNA reached a satisfactory degree of activity. About 0.88 g of arginine tRNA, 70% activity; 3.32 g of formylmethionine tRNA, 97% activity; 0.80 g of glutamic acid tRNA, 83% activity and O.92 g of phenylalanine-2 tRNA, 78% activity, were produced. The processing steps employed are reliable and reproducible and the procedure is amenable to routine production of these tRNAs.     

A simple method for large-scale preparation of chick embryo tRNA

A simple method for preparing bulk quantities of tRNA from chick embryo has been developed. In this method chick embryos were homogenized in a buffer of pH 4.5, followed by deproteinization with phenol. The aqueous layer was allowed to separate under gravity. The resulting aqueous layer, after two more phenol treatments, was directly passed through a DEAE-cellulose column and the tRNA eluted therefrom with 1 m NaCl. The tRNA prepared by this method was as active as the one prepared at neutral pH.     

Trace 5-Methylaminomethyl-2-selenouridine in bovine tRNA and the selenouridine synthase activity in bovine liver

We measured the amount of Se in bovine liver tRNA. tRNA was chromatographed on a BD-cellulose column and Se-rich tRNA was eluted from the column in front of a main tRNA peak. There was 0.3 mmol Se/mol of tRNA and this level is about one tenth that of Escherichia coli tRNA. This suggests the presence of an enzyme that modifies tRNA with Se in bovine liver. We isolated the activity of this enzyme (selenouridine synthase) by chromatography of bovine liver extracts on a DEAE-cellulose column. ATP and selenophosphate synthetase, as well as selenouridine synthase and tRNA, were necessary for the reaction. ⁷⁵Se was used to label the reaction products, which were analyzed by TLC after digestion with ribonuclease T₂. The position of the ⁷⁵ Se-nucleotide on a TLC plate was identical to that of the Se-nucleotide, 5-methylaminomethyl-2-seleno-Up, prepared from ⁷⁵Se-tRNA in E. coli.     

A rapid assay technique for RNA ribose methylases.

A rapid technique for quantitative separation of ribose-methylated nucleosides from base-methylated and non-methylated nucleosides by chromatography on DEAE-cellulose paper in the presence of borate is described. The method has been used as an assay for tRNA ribose methylases from yeast, using under methylated Escherichia coli tRNA as substrate. The main product formed with a partly purified yeast enzyme was characterized as 2'-O-methylcytidine.     

Plant tRNA nucleotidyltransferase

A method for purification of tRNA nucleotidyltransferase from Lupinus luteus L. seeds on a preparative scale is presented. This involved ammonium sulfate fractionation and chromatography on DEAE-cellulose, hydroxylapatite, and QAE-Sephadex A-50. The final enzyme prepartion was apparently homogeneous and a 2000-fold purification was achieved. Data presented suggest the existence of only one form of tRNA nucleotidyltransferase in dry lupin seeds.     

EFFECTS OF CHRONIC ETHANOL INGESTION ON BRAIN AMINOACYL-tRNA SYNTHETASES AND tRNA

The effects of chronic ethanol ingestion on the in vivo aminoacylation of brain transfer RNA (tRNA) were examined in C57BL/6J mice. A pronounced inhibition in the formation of [¹⁴C]leucy]-tRNA and [¹⁴C]phenylalanyl-tRNA was observed in the ethanol drinking mice. Properties of aminoacyl-tRNA synthetases and tRNA were examined following their separation and isolation on a DEAE-cellulose column. Synthesis of [¹⁴C]leucyl-tRNA was found to have a complete dependence on ATP and Mg²⁺. Incubations were carried out by cross-matching tRNA from control rat brain with synthetases obtained from the brains of control or ethanol-drinking mice. Under these conditions, a decreased ability for aminoacylation could be demonstrated when the source of enzyme was derived from ethanol-treated brain. The data indicate that the major effect of ethanol ingestion on the aminoacylation reaction is exerted on aminoacyl-tRNA synthetases.     

Improved separation of modified nucleosides from tRNA hydrolysates: the patterns of tRNA methylation in rat tissues

A sensitive and reproducible method for the isolation of minor nucleosides derived from tRNA is described. The nucleosides obtained from enzymatic digestion of tRNA are separated into several groups using a QAE Sephadex column and increasing concentrations of boric acid in a step-wise manner. The nucleosides in each group are separated by isocratic elution from a preparative Partisil 10-SCX column and high-performance liquid chromatography at ambient temperature. With this method we have determined the patterns of tRNA methylation in vitro with extracts from rat bone, liver, kidney and adrenal glands. Although different tissues appear to contain the same tRNA methyltransferases, the patterns of methylated nucleosides are different.     

Simple and rapid procedure for chromatographic identification of multiple transfer RNA species from Escherichia coli

A modification of the benzoylated DEAE-cellulose method of tRNA fractionation has been developed to provide a rapid and highly efficient method for the quantitative identification of multiple tryptophan, tyrosine, and phenylalanine tRNA species from E. coli aminoacylated in vivo. This method should be of particular use in physiological studies of these tRNA species.     

Purification of peptidyl-tRNA on benzoylated DEAE-cellulose columns.

The o-nitrophenylsulfenyl group, and amino protecting group in the chemical synthesis of peptidyl-tRNA was used to attach newly synthesized peptidyl-tRNA to a benzoylated DEAE-cellulose column. This facilitated the isolation of a highly purified specific tRNA with a well defined peptide chain.     

Affinity chromatography of aminoacyl-tRNA synthetases on specific tRNA columns without prior purification of tRNA

A method is described for the purification of aminoacyl-tRNA synthetases by affinity chromatography, using a column of tRNA lacking the cognate tRNA, followed by a column of the cognate tRNA. The ability of the enzyme to discriminate between cognate and non-cognate tRNA is exploited in a novel and rapid preparation of the two columns.     

Method for isolation of aminoacyl-tRNA synthetases from plants: purification and some properties of methionyl,phenylalanyl and arginyl tRNA synthetases from yellow lupin seeds

A method for the simultaneous purification of methionyl-, phenylalanyl- and arginyl-tRNA synthetases from yellow lupin seeds (Lupinus luteus) is described. The method uses ammonium sulphate fractionation, and DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. Molecular weight and kinetic parameters of the pure enzymes are reported.     

Isolation and binding properties of leucyl-tRNA synthetase from Escherichia coli MRE 600

Summary A procedure for the large-scale isolation of leucyl-tRNA synthetase from E. coli MRE 600 is described: The enzyme was purified about 320-fold to homogeneity by precipitation with cetyl-trimethyl-ammonium bromide, two consecutive chromatographies on DEAE-cellulose and three on hydroxyapatite with an over-all yield of 4%.The molecular weight of leucyl-tRNA synthetase from E. coli MRE 600 was found to be 99 000 daltons. Binding studies by ultracentrifugation and equilibrium partition showed that the enzyme binds leucine, leucyl-adenylate and tRNA^Leu, each in a 1 : 1 stoichiometry. For ATP only a very weak binding to the enzyme could be observed, which did not allow the evaluation of the complex stoichiometry. The presence of ATP was not required for the binding of leucine or tRNA to leucyl-tRNA synthetase from E. coli MRE 600.     

Simple and rapid chromatographic purification of type V collagen from a pepsin digest of porcine intestinal connective tissue,an unmanageable starting material for conventional column chromatography

A chromatographic method is described for purification of type V collagen, a minor constituent in extracellular matrix, from a pepsin digest of porcine intestinal connective tissue. The starting material was a viscous and turbid solution even after centrifugation. Direct application of the sample to a commercially available DEAE-cellulose column resulted in clogging. On the other hand, type V collagen, [alpha1(V)](2)alpha2(V) form, was successfully captured by a filter paper-based DEAE-cellulose column chromatography and purified by a subsequent commercially available cation-exchange medium without clogging. This is a vast improvement over previously described salt fractionation methods.     

Isolation of Deoxyfructosazine and Its 6-Isomer from the Nondialyzable Melanoidin Hydrolyzate

The nondialyzable melanoidin prepared from glucose-ammonia system (kept in pH 5.3~6.0 during the reaction) was hydrolyzed. The hydrolyzate was fractionated by DEAE-cellulose column and Dowex 50 W column. Deoxyfructosazine and its 6-isomer were respectively isolated from main two fractions, and identified. Even on boiling the melanoidin in aqueous solution, these pyrazines as well as imidazoles and β-hydroxy pyridines in the melanoidin were liberated.

Furthermore, amounts of these heterocyclic compounds liberated from the nondialyzable melanoidin and the fractionated melanoidins (fractionated into five fractions on DEAE-cellulose column according to the method described previously) were examined.

The results obtained seem to suggest that these heterocyclic compounds are not present probably as a molecular skelton or an inclusion compound in the melanoidin, but as a small moiety of the melanoidin molecule with loose chemical bond.     

The mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena are indistinguishable.

The mitochondrial and cytoplasmic valyl tRNA synthetases from Tetrahymena pyriformis are indistinguishable. These synthetases cannot be differentiated through hydroxylapatite, DEAE-cellulose, or phosphocellulose column chromatography. Both enzymes show the same mean sedimentation coefficient of 5.9 S in sucrose gradient centrifugation analysis; when bound with tRNA, they are relatively stable and sediment at 7.8 S. The temperature optimum for aminoacylation reaction is 27.5 °C, the optimum Mg²⁺ concentration is 4.4 mm, and substrate affinities (K_m values) for valine and ATP in aminoacylation are the same for both enzymes at 1.0 μm and 2.5 mm, respectively. These enzymes show identical specificities for acylation of different tRNA species, i.e., Tetrahymena and rat liver tRNAs can be equally well recognized, but no significant acylation can be observed with Escherichia coli and Saccharomyces tRNAs. These observations suggest the probable molecular identity of mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena.     

Purification of human placenta phenylalanine, valine, methionine, glucine, and serine transfer ribonucleic acids.

By using column chromatography on varied media, the purification of several individual tRNAs from human placenta has been achieved. The crude human placenta tRNA was isolated using phenol extraction at pH 4.5 followed by DEAE-cellulose chromatography (B. Roe (1975), Nucleic Acids Res. 2, 21-42) and initially fractionated on BD-cellulose at neutral pH. Subsequent chromatography of the partially purified tRNA using high-speed, high-pressure liquid chromatography on RPC-5 and Aminex A-28 coupled with chromatography on BD-cellulose at acidic pH and on DEAE-Sephadex A-50 significantly shortened isolation time for milligram quantities of several pure tRNA species. Those tRNAs from human placenta obtained in a purity greater than 1.2 nmol/A260 unit are tRNAPhe, tRNAMet(i), tRNAVal(1a), tRNAVal(1b), and tRNAGly(1), while those obtained at purity of at least 0.8 nmol/A260 unit are tRNASer2 and tRNASer3. In addition, the use of Aminex A-28 as a chromatographic system for the isolation of tRNA is discussed.     

Recovery of erythropoietin from anemic sheep plasma

A method is described for the large-scale preparation of erythropoietin from anemic sheep plasma. DEAE-cellulose and carboxymethylcellulose column chromatography was used to prepare Step II erythropoietin. A total of 168 sheep yielded 499 liters of plasma from which 323,000 IU of Step II erythropoietin was obtained.     

Purification and characterization of Chlamydomonas reinhardtii chloroplast glutamyl-tRNA synthetase, a natural misacylating enzyme

Glutamyl-tRNA synthetase from Chlamydomonas reinhardtii was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, Mono Q, and Mono S. The apparent molecular mass of the protein when analyzed under both denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 62,000 Da; this indicates that the active enzyme is a monomer. The purified glutamyl-tRNA synthetase was identified as the chloroplast enzyme by its tRNA charging specificity. Reversed-phase chromatography of unfractionated C. reinhardtii tRNA resolved four peaks of glutamate acceptor RNA when assayed with the purified enzyme. The enzyme can also glutamylate Escherichia coli tRNA(2Glu), but not cytoplasmic tRNA(Glu) from yeast or barley. In addition, the enzyme misacylates chloroplast tRNA(Gln) with glutamate. A similar mischarging phenomenon has been demonstrated for the barley chloroplast enzyme (Sch?n, A., Kannangara, C.G., Gough, S., and S?ll, D. (1988) Nature 331, 187-190) and for Bacillus subtilis glutamyl-tRNA synthetase (Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M., and Lapointe, J. (1983) J. Biol. Chem. 258, 753-759).