Stabilization of aminoacyl-tRNA synthetases by sephadex and polyacrylamide gels

Several aminoacyl-tRNA synthetases from the yellow lupin (Lupinus luteus) were stabilized against inactivation during storage at 0–4°, by entrapment in Sephadex or Biogel matrices and drying over P₂O₅. The degree of stabilization depended on the rate of drying of the gel and the pH of the medium and to a lesser extent on the ionic strength and protein concentration. With the exception of prolyl-tRNA synthetase, a greater stability was achieved with those enzymes which were relatively stable to thermal denaturation. Aminoacyl-tRNA synthetases for glutamic acid, glutamine, methionine and arginine, which become inactivated during purification, were considerably stabilized by this procedure.     

Characterization of the tRNA and aminoacyl-tRNA synthetases of healthy and tumorous callus tissues from Nicotiana tabacum

Aminoacyl-tRNA synthetases extracted from healthy and crown gall tumor tissues (induced by Agrobacterium tumefaciens strain B6) from Nicotiana tabacum (strain Wisconsin 38) grown in vitro, showed the same ability to charge Phaseolus vulgaristRNA, for all the 15 amino acids tested. For each amino acid, optimal charging conditions (enzyme concentration, Mg²⁺/ATP ratios, K⁺ ion effects) have been determined with Phaseolus vulgaristRNA and were found to be the same whether aminoacyl-tRNA synthetases from healthy or tumor tissues were used. In each case, valyl- and glutamyl-tRNA synthetases were very sensitive to an excess of Mg²⁺ and K⁺ ions. Although tRNA's extracted from healthy and tumor tissues gave the same electrophoretic patterns, charging levels obtained with turner tRNAs were generally 45% higher than those obtained with tRNA's from healthy tissues.     

Purification and characterization of two tyrosyl-tRNA synthetase activities from soybean cotyledons

The tyrosyl-tRNA synthetases located in cytoplasm and chloroplasts of soybean cotyledons were purified to near homogeneity by ammonium sulfate precipitation, DEAE-cellulose chromatography, hydroxylapatite chromatography, and DEAE-Sephadex A-25 chromatography. Purified cytoplasmic tyrosyl-tRNA synthetase shows only a single band in acrylamide gel electrophoresis which corresponds to a MW of 126000. In SDS-acrylamide gel electrophoresis the enzyme again shows only a single band which corresponds to a MW of 61 000. Chloroplast tyrosyl-tRNA synthetase shows only one band in both acrylamide and SDS-acrylamide gel electrophoresis with MWs being 98 000 and 43 000, respectively. For cytoplasmic tyrosyl-tRNA synthetase the apparent K_ms determined are 6.8 μM L-tyrosine, 49 μM ATP, and 8.9 × 10^?8 M tRNA (as total tRNA). Apparent K_ms for chloroplast tyrosyl-tRNA synthetase are 4.9 μM L-tyrosine, 214 μM ATP and 2.2 × 10^?8 M tRNA (as BDC-ethanol fraction tRNA). Fractionation of soybean cotyledon-tRNA on RPC-5 columns gives 4 tyrosyl-tRNA species, the first two species (tRNA_{1 and 2}^Tyr) are acylated only by cytoplasmic tyrosyl-tRNA synthetase while the last two species (tRNA_{3 and 4}^Tyr) are acylated only by chloroplast tyrosyl-tRNA synthetase.     

Changes in leucyl-tRNAs and aminoacyl tRNA synthetases in developing and aging soybean cotyledons

Leucine specific tRNA of soybean cotyledons was frationated into six peaks (1–6). The relative amounts of Leu-tRNA 5 and 6 are lower in developing cotyledons than in germinating cotyledons. Leu-tRNA synthetase from developing cotyledons is less active in aminoacylating Leu-tRNA 5 and 6 compared to enzyme from 5-day-old germinating cotyledons. Leu-tRNA synthetase from cotyledons of germinating seedlings and developing cotyledons can be fractionated into three peaks (1–3). Peak 1 in the developing cotyledon is about 36% less than peak 1 from 5-day-old germinating cotyledons. Peaks 2 and 3 from developing cotyledons are about 10 and 18% higher than from germinating cotyledons, respectively. Peak 1 from developing cotyledons acylates all six species of Leu-tRNA in contrast with peak 1 from germinating cotyledons, which essentially acylates only Leu-tRNA 5 and 6. The specificity of peaks 2 and 3 towards Leu-tRNA 1–4 is identical in both the organs.     

Amino acid incorporation on plant ribosomes: The transfer system

Pre-formed Vicia faba phenylalanyl-tRNA was active in a TYMV-RNA-directed Transfer System, whereas a similar tRNA preparation from yeast was not. Thus, lack of charging of yeast tRNA by enzymes from Phaseolus was not the only reason why yeast tRNA would not function in this Transfer System. In the poly U-directed Transfer System; where both types of tRNA were active, the pH and ionic parameters governing the reaction with yeast tRNA were more stringent.     

Effect of seed germination on levels of tRNA aminoacylation

In ungerminated seeds of Lupinus luteustRNAs are aminoacylated 10% or less depending on species of tRNA. The levels of tRNA aminoacylation for specific tRNAs increase steadily during seed germination. Specific tRNAs in cotyledons and axes of 3-day-old seedlings are aminoacylated to a similar extent. No significant changes are observed in the tRNA population during germination.     

Amino acid substrate specificity of asparaginyl-,aspartyl- and glutaminyl-tRNA synthetase isolated from higher plants

AspNH₂-, Asp- and GluNH₂-tRNA synthetases were purified from Phaseolus aureus; their optimum assay conditions, substrate specificities and salt sensitivities were investigated. AspNH₂-tRNA synthetase from β-cyanoalanine-producing (Vicia sativa), and non-producing (P. aureus and V. faba) species was able to utilize the analogue as a substrate irrespective of the source of the enzyme. Asp-tRNA synthetase from P. aureus was able to utilize α-aminomalonate and threo-β-hydroxy Asp as a substrate. The transfer of ¹⁴C-GluNH₂ to tRNA, catalyzed by GluNH₂-tRNA synthetase, was only inhibited by high concentrations of those analogues tested; albizziine was the most efficient, but no difference could be demonstrated between the substrate specificities of the enzyme isolated from an albizziine-producer (A. julibrissin and a non-producer (P. aureus) species.     

tRNA species in the developing grain of triticum aestivum

The level of lysyl- and prolyl-tRNA in various stages of the maturing wheat grain was measured by the aminoacylation procedure. The levels of these tRNAs changed only slightly during the maturation period. Several species of lysyl- and prolyl-tRNA were obtained from different parts of the developing grain by fractionation on benzoylated-DEAE cellulose (BD-cellulose). The embryo contained three discrete species of prolyl and at least three species of lysyl isoacceptor tRNA throughout development, whilst the tRNA obtained from the endosperm gave more complicated elution profiles on chromatography on BD-cellulose. Small changes were noted in the levels of aminoacylation of individual isoacceptor tRNA species for lysine or proline during seed maturation. However, these were insufficient to account for the changing pattern of lysine and proline in the storage protein during the development of the endosperm.     

Leucine specific transfer ribonucleic acids and synthetases in the cotyledons of mature and germinating pea seeds

Changes in isoaccepting species of tRNA^Leu were determined in germinating pea seedlings and in developing pods. Leucine specific transfer ribonucleic acids of pea cotyledons can be fractionated into four isoaccepting species by reversed-phase chromatography (RPC-5) on a Plaskon column. In contrast, only two species of tRNA^Leu were observed in developing seed pods. Leucyl-tRNA synthetase purified by ammonium sulfate precipitation and DEAE cellulose column chromatography retained the full range of specificity towards all four tRNA^Leu species of pea cotyledons. This partially purified pea cotyledon enzyme could be further separated on a hydroxylapatite (HA) column into two peaks of leucyl-tRNA synthetase activity. Enzyme 1 is dominant in seed pods while 2 is predominant in cotyledons. Enzymes 1 and 2 from cotyledons were examined for the amino acid acceptor activity of twelve different amino acids. Both these fractions showed less than 3% acceptor activity for eleven other amino acids as compared to leucine-tRNA synthetase activity. Preliminary characterization of enzyme 2 from cotyledon, by isoelectric focusing and polyacrylamide gel electrophoresis indicates at least three subspecies.     

Differences in the level of plastid-specific tRNA's in chloroplasts and etioplasts of phaseolus vulgaris

Comparison of the chromatographic profiles of chloroplast and etioplast leucyl-tRNA's and valyl-tRNA's shows that the levels of plastid-specific tRNA species are relatively higher in the chloroplasts. This suggests that light can stimulate the synthesis of plastid-specific tRNA's in higher plants.     

Development of aminoacyl tRNA synthetases in cultured Nicotiana tabacum cells

Changes in the activity of aminoacyl tRNA synthetases during growth of tobacco XD cells in suspension culture have been determined by the pyrophosphate exchange assay. Alanyl, arginyl, glutamyl, glutaminyl and seryl tRNA synthetases showed the lowest activity, whilst lysyl, histidyl, leucyl, isoleucyl, phenylalanyl threonyl and valyl tRNA synthetases were most active. Most synthetases, and total protein, increased to a maximum, at around 7 days, just before mid-exponential phase, and then fell.     

Substrate discrimination by prolyl-tRNA synthetase from various higher plants

Prolyl-tRNA synthetase from plants (e.g. Delonix regia) containing azetidine-2-carboxylic acid (A2C), activated imino acid analogues larger than proline (Pro) more efficiently than did the enzyme from plants lacking A2C. The reverse situation was observed for analogues, including A2C itself, that are smaller than Pro. The enzyme from A2C-producing species was quite labile and salt-sensitive, with a high pH optima for the ATP-³²PPi exchange reaction, whereas the enzyme from non-producer species was stable and insensitive to salts, with a lower pH optimum. Certain analogues of Pro, which failed to stimulate ATP-³²PPi in the presence of a particular type of Pro-tRNA synthetase, nevertheless could bind to the enzyme and inhibit the esterification of tRNA by Pro. In the absence of tRNA, no significant ATP-³²PPi exchange was catalyzed by the Delonix enzyme on addition of A2C; the addition of tRNA resulted in a low but real level of activation of the analogue relative to Pro. These findings are discussed in relation to the ability of the enzyme from A2C-producing plants to discriminate against the analogue.     

The activation of amino acid analogues by phenylalanyl- and tyrosyl-tRNA synthetases from plants

Partially purified preparations of Phe- and Tyr-tRNA synthetases were obtained from seed or seedlings of Phaseolus aureus, Delonix regia and Caesalpinia tinctoria, and the ability of a variety of structural analogues of Phe or Tyr to act as alternative substrates or inhibitors was tested. 3-Hydroxymethylphenylalanine, a natural product of C. tinctoria, formed a particularly effective substrate for the Tyr-tRNA synthetase from P. aureus. The structural features commensurate with substrate activity in an analogue molecule are discussed.     

Extraction and purification of tRNA from fruit tissues

Readily measurable yields of undamaged tRNA were obtained from tomato, pear and apple fruits by phenolic extraction at pH 8.8, removal of interfering alcohol insoluble substances by precipitation with 0·2 volumes of iso-PrOH and final purification by DEAE chromatography. Various other commonly used extraction and purification procedures were tested and found to be less effective. Active synthetases were isolated from acidic fruit tissues by adequate control of pH and maceration in the frozen state. After acylation with a radioactively labelled amino acid, fruit isoacceptor tRNA species were separated by reverse phase chromatography.     

Cold-lability of prolyl-tRNA synthetase from higher plants

Pro-tRNA synthetase from D. regia and P. aureus lost enzymic activity more rapidly at 0° than at room temperature. The enzyme from a number of higher plants that produce azetidine-2-carboxylic acid (A2C) was more rapidly inactivated in the cold than the enzyme from plants which do not contain A2C. The rate of cold inactivation was dependent on temperature and on the concentration of glycerol, protein and sulphydryl-reducing reagents. Substrates of Pro-tRNA synthetase also stabilized the enzyme against cold inactivation. Enzyme which had been completely inactivated by storage in the cold, could be reactivated by warming in the presence of a sulphydryl-reducing reagent. The rate of reactivation was dependent on temperature, pH and the concentration of sulphydryl-reducing reagent. Kinetic analysis indicated the existence of more than one molecular form of the enzyme. It is suggested that the cold-lability of Pro-tRNA synthetase may be due to dissociation of the active enzyme molecule into inactive subunits.     

Transfer ribonucleic acid from Scenedesmus obliquus D3: Purification of the major formylatable methionine-accepting species

The major formylatable methionine tRNA of Scenedesmus obliquus has been purified using two column chromatographic steps: (1) chromatography on     

Amino acid incorporation on 80s plant ribosomes: The complete system

A cell-free system directed by poly U or turnip yellow mosaic virus (TYMV)-RNA was obtained from imbibed seeds of Phaseolus aureus; this in vitro system was dependent upon exogenous tRNA. The poly U-directed system functioned in the presence of tRNAs from P. aureus, Vicia faba and yeast, whereas TYMV-RNA was translated only in the presence of tRNAs from P. aureus or V. faba. The pH and Mg²⁺ optima for aminoacylation of tRNAs of P. aureus, V. faba and yeast by leucine and phenylalanine were related to the overall pH and ionic concentration optima for the complete system.