[Lipid components of aminoacyl-tRNA synthetase complex in eukaryotes and their function]. / Lipidni komponenti u skladi visokomolekuliarnogo kompleksu aminoatsil-tRNA-cintetaz eukariot ta ïkx funktsiia

This review includes data about lipids composition of eukariotic high molecular complex of aminoacyl-tRNA synthetases under normal conditions and under the effect of ionizing radiation. The role of different lipids in formation of the structure of high molecular complex and functioning is discussed. The role of different groups of phospholipids, prostaglandins in providing the normal work of high molecular complex and some synthetases in control and pathological state is considered. A conclusion has been made that different groups of lipids composed the high molecular complex aminoacyl-tRNA synthetases. Some of them play structural role and other (prostaglandins, some phospholipids) are the regulatory components of the complex.

Effect of continuous-wave and amplitude-modulated 2.45 GHz microwave radiation on the liver and brain aminoacyl-transfer RNA synthetases of in utero exposed mice.

Investigations have been carried out concerning the effects of microwave (MW) exposure on the aminoacyl-transfer ribonucleic acid (tRNA) synthetase of the progeny of females that were exposed during their entire period of gestation (19 days). The changes caused by continuous-wave (CW) and amplitude-modulated (AM) MW radiation have been compared. CFLP mice were exposed to MW radiation for 100 min each day in an anechoic room. The MW frequency was 2.45 GHz, and the amplitude modulation had a 50 Hz rectangular waveform (on/off ratio, 50/50%). The average power density exposure was 3 mW/cm2, and the whole body specific absorption rate (SAR) was 4.23 +/- 0.63 W/kg. The weight and mortality of the progeny were followed until postnatal day 24. Aminoacyl-tRNA synthetase enzymes and tRNA from the brains and livers of the offspring (461 exposed, 487 control) were isolated. The aminoacyl-tRNA synthetase activities were determined. The postnatal increase of body weight and organ weight was not influenced by the prenatal MW radiation. The activity of enzyme isolated from the brain showed a significant decrease after CW MW exposure, but the changes were not significant after 50 Hz AM MW exposure. The activity of the enzyme isolated from liver increased under CW and 50 Hz modulated MW.

[Cyclic nucleotides in a high molecular weight aminoacyl-tRNA synthetase complex from the liver of normal and irradiated rats]. / Tsyklichni nukleotydy u skladi vysokomolekyliarnogo kompleksu aminoatsyl-tRNK-syntetaz iz pechinky normal'nykh ta oprominenykh shchuriv.

The content of cAMP and cGMP in the composition of the high-molecular complex aminoacyl-tRNA-synthetase from the rat liver has been studied. The ionizing radiation decreases the level of the cyclic nucleotides in the composition of the complex. A problem on interregulation of the cyclic nucleotides and prostaglandins in the complex has been examined.

[Effect of ionizing radiation on the lipid composition of aminoacyl-tRNA-synthetase complexes]. / Vliianie ioniziruiushchei radiatsii na lipidnyi sostav kompleksov aminoatsil-tRNK-sintetaz.

The effect of X-radiation (0.21 C/kg) on a lipid component of aminoacyl-tRNA-synthetase complexes from rat liver (for instance, phospholipids, neutral lipids, and prostaglandins) has been studied. The content of prostaglandins and lysophosphatidyl choline increases and that of phospholipids and neutral lipids decreases 60 min after irradiation. In 24 h, the content of prostaglandins, fatty acids, cholesterol, and phosphatidyl ethanolamine approaches the control level.

[Effect of ionizing radiation on the properties of rat liver phosphoprotein phosphatase]. / Vliianie ioniziruiushchei radiatsii na svoistva fosfoproteidfosfatazy iz pecheni krys.

Phosphoproteidphosphatase (3.1.3.16) of high specificity for lysil-tRNA-synthetase (6.1.1.6) and proteins of high-molecular-weight multienzyme complex of aminoacyl-tRNA-synthetases (6.1.1.) was isolated from rat liver. Irradiation of animals with an absolutely lethal dose of 0.21 C/kg decreased phosphoproteidphosphatase activity: a 3-4-fold decrease was noted 1 hr following irradiation. The activity of the enzyme isolated 24 hr after irradiation increased but did not reach the control level.

[Study of the kinetics of thermoinactivation of lysyl-tRNA-synthetase from the liver of x-irradiated rats]. / Izuchenie kinetiki termoinaktivatsii lizil-tRNK-sintetazy iz pecheni krys pri deistvii rentgenovskikh luchei.

A study was made of the kinetics of thermoinactivation of lysyl-tRNA-synthetase isolated from rat liver at early times of radiation damage development after the effect of a minimum absolutely lethal X-radiation dose (0.21 C/kg). The thermostability of a dimer form of the enzyme was shown to be higher than that of a monomer. It was established that substrates had a stabilizing effect on the enzyme during thermoinactivation. On the basis of the data obtained from the studies in the kinetic properties of the enzyme and the thermoinactivation a conclusion is made that lysyl-tRNA-synthetase is stabilized during subunit aggregation. The thermostability of the enzyme was decreased by irradiation.

Directly photocrosslinked nucleotides joining transfer RNA to aminoacyl-tRNA synthetase in methionine and tyrosine systems.

We have used ultraviolet photocrosslinking and 32P post-labeling to help define the contact surface between transfer RNAs and aminoacyl-tRNA synthetases for the methionine and tyrosine systems. Photocrosslinking between tRNAs and synthetases is shown to occur only in cognate complexes. The increased sensitivity of our procedures reduces the amounts of interacting macromolecules and permits lower ultraviolet light doses, thereby minimizing radiation damage. These procedures have detected crosslinks only within the 3'-terminal RNase T1 fragments in yeast tRNAMeti and Escherichia coli tRNATyr2; and although the photoadducts were unstable, we have identified the crosslinked nucleotides. These crosslinks occur at positions C74 and A76 in yeast tRNAMeti and position U64 in E. coli tRNATyr1&2 (conventional tRNA numbering system of Gauss & Sprinzl, 1981). This work demonstrates that even labile photocrosslinks can be exploited for mapping crosslinked nucleotides.

[Effect of roentgen irradiation on the luminescent properties of rat liver lysyl-tRNA synthetase]. / Vliianie rentgenovskogo oblucheniia na liuminestsentnye svoistva lizil-tRNK-sintetazy iz pecheni krys.

The parameters of proper fluorescence of lysil-tRNA-synthetase from rat liver were determined in normal conditions and after X-irradiation. Irradiation of animals with a minimal absolutely lethal dose (0.21 C/kg) caused conformational transformations of the protein molecule accompanied by a change in the microenvironment of the chromophores: 60 min after irradiation it became less polar and after 24 h more polar than normal. The changes in the conformational status of the enzyme molecules after irradiation were connected with a change in the specific activity of lysil-tRNA-synthetase.

Difference in radiosensitivity of valyl-tRNA synthetases isolated from chick embryo liver and brain.

The sensitivity in vitro against gamma-radiation of valyl-tRNA synthetase (VRS) from chick embryo brain proved to be higher than that of liver VRS. In order to study this phenomenon properties of VRS isolated from both organs were compared. Both enzymes have the same molecular weight (Mr = 110 000) determined by equilibrium sedimentation and by gel filtration. Electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate showed no evidence for subunit structure. The optimal reaction conditions in valyl-tRNA formation were found to be the same, except the pH values. Km values of the reactions were also similar. Both enzymes reacted with the tRNAs isolated from the other organ. The stability during storage of the liver VRS was higher than that of the brain VRS. In the brain VRS molecule one buried and eleven free SH groups could be detected and divided into three classes with different reactivities. In 1 mol of liver VRS one buried and nine free thiol groups were determined and these could be divided only into two classes. These observations suggest the existence of two VRS isoenzymes with different radiosensitivities: the more sensitive occurs mainly in brain and the less sensitive mainly in the liver of chick embryo.

Effect of gamma radiation on E. coli ribosomes, tRNA and aminoacyl-tRNA synthetases.

Gamma-irradiated E coli ribosomes and tRNA, in aerated solutions, were inactivated with D37 doses of 144 and 77 Gy, respectively. Aminoacyl-tRNA-synthetases were only slightly inactivated under comparable conditions. Effects of additives to ribosome and tRNA solutions suggest that hydroxyl radicals were the major damaging species, that superoxide anions were not damaging and that radiolytically-formed hydrogen peroxide was also unimportant. Part of the damage by hydroxyl radicals is expressed through secondary radicals produced from additives and buffers. Results obtained with three different buffers suggest that (1) acetate ions provide protection by competing for hydroxyl radicals, (2) chloride ions are without effect and (3) inactivation of ribosomes and aminoacyl-tRNA-synthetases in Tris-HCl/MgCl2 and phosphate/MgCl2 buffered solutions was similar but the tRNA inactivation was lower in Tris-HCl/MgCl2 buffer.

[Comparative analysis of affinity modification of several aminoacyl-tRNA synthetases with gamma-(p-azidoanilide)-ATP]. / Sravnitel'nyi analiz affinnoi modifikatsii riada aminoatsil-trnk-sintetaz s pomoshch'iu-gamma-(p-azidoanilida)-ATP.

The inhibitory action of gamma-(p-azidoanilide)-ATP on the reactions of tRNA aminoacylation catalysed by several aminoacyl-tRNA synthetases was investigated. This compound was shown to be a competitive inhibitor with respect to ATP in the case of arginyl-, valyl-, isoleucyl-, leucyl-, threonyl-, phenylalanyl-tRNA synthetases of E. coli MRE-600 and tryptophanyl-tRNA synthetase of beef pancreas. The Ki value of this analog changes from 3 x 10(-5) up to 4 x 10(-3) M depending on the enzyme specificity. In the case of methionyland lysyl-tRNA synthetases from E. coli the non-competitive and mixed inhibition accordingly was observed. The activity of isoleucyl-, valyl-, leucyl-, threonyl-, phenyl-alanyl- and tryptophanyl-tRNA synthetases in the reaction of tRNA aminoacylation is decreased as a result of UV-irradiation of the enzymes in the presence of gamma-(p-azidoanilide)-ATP. ATP and aminoacids protect these enzymes against irreversible inactivation. These results confirm the affinity labelling of the substrate binding sites of these enzymes. However, the appreciable inactivation of enzymes with UV-irradiation in the presence of gamma-(p-azidoanilide)-ATP was not detected in the cases of hystidyl-, lysyl-, methionyl-, seryl-, tyrosyl- and phenylalanyl-tRNA synthetases of E. coli MRE-600. The data obtained enable one to suggest the difference in the structure of the amino acid activating sites of different aminoacyl-tRNA synthetases.

Studies on the valyl-h-tRNA synthetase of chick embryo brain irradiated with 60Co gamma-rays, in vivo.

18-day-old chick embryos (Leghorn) were irradiated in vivo with 1,2,3,4,5 or 7 Gy 60Co gamma-rays (dose rate = 0.9 Gy/min). Twenty-four hours after irradiation the activity of valyl-tRNA synthetase isolated from the brains was determined and compared with that of the non-irradiated control. The aminoacylation activity was found to decrease exponentially as a function of the dose (D37 = 6 Gy). Irradiation caused a more pronounced decrease in valyl-tRNA synthetase activity in 18-day-old than in 14-day-old chick embryos and induced in the valine-dependent ATP-PPi exchange reaction less change than in the valyl-tRNA FORMATion. The exposure of 15-day-old chick embryos to a dose of 4 or 5 Gy induced, respectively, a 40 or 20 per cent increase in enzyme activity of VRS (prepared on day 19 of embryonic life) relative to the control. In these experiments a decrease of Km value for tRNA has been found. The change in the number of sulfhydryl groups was also investigated.

Yeast phenylalanyl-tRNA synthetase. Affinity and photoaffinity labelling of the stereospecific binding sites.

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The RNAPhe was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (S4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate-oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the beta subunit (Mr 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to eigher type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.

Photocross-linking analysis of the contact surface of tRNA Met in complexes with Escherichia coli methionine:tRNA ligase.

Photoinduced covalent cross-linking has been used to identify a common surface of four methionine-accepting tRNAs which interact specifically with the Escherichia coli methionine:tRNA ligase (EC 6.1.1.10). tRNA--ligase mixtures were irradiated, and the covalently linked complexes were isolated and digested with T1 RNase (Schimmel & Budzik, 1977). The fragments lost from the elution profile of the T1 RNase digest were considered to have been cross-linked to the protein and therefore in intimate contact with the enzyme. Only specific cognate tRNA--ligase pairs produce covalently linked complexes. The four substrate tRNAs used in this study have substantially different sequences, but all showed a common cross-linking pattern, supporting the view that the sites cross-linked to the enzyme reflect the functionally common contact surface rather than particularly photoreactivity regions of tRNA. The cross-linked contact surface is comprised of three regions: (1) the narrow groove of the anticodon stem and its extension into the anticodon loop; (2) the 3' terminal residues; and (3) the 3' side of the "T arm". Unlike previous studies with other tRNAs, the D arm is not involved and significant radiation damage is suffered by the tRNA which must be taken into account in the analysis. The results are consistent with and complement chemical modification studies [Schulman, L. H., & Pelka, H. (1977) Biochemistry 16, 4256].

Effect of 60Co gamma radiation on the valyl-tRNA synthetase isolated from chick embryo brain.

The effect of 60Co gamma irradiation on the activity of valyl-tRNA synthetase isolated from chick embryo brain was studied. The enzyme activity exponentially decreased in the dose range 10--200 krad. The first step of the enzyme action, i.e. the amino acid activation, was found to be less sensitive to irradiation than the whole reaction, the formation of valyl-tRNA, 2-Mercapto ethanol and/or glycerol had a significant radioprotective effect. The lesion caused by radiation in the enzyme was also influenced by its concentration during exposure (dilution effect). According to gel-electrophoretic experiments, no chain rupture occurred in the enzyme molecule. Not even a change in Km was observed; however, the maximum velocity of the reaction was found to decrease with increasing radiation dose.