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.

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.

Valyl-tRNA synthetase from chick embryo brain. Properties of the sulfhydryl groups.

The number of thiol groups in valyl-tRNA synthetase [L-valine tRNA ligase (AMP) E.C.6.1.1.9] isolated from chick embryo brain was determined. Titration with 5.5'-dithiobis(2-nitrobenzoic) acid showed 11 free SH groups calculated on the basis of a molecular weight of 110000 daltons. In 8M urea reaction with DTNB revealed 1 additional SH-group. Binding of substrates to the active site of the molecule decreased the number of titratable SH-groups. The modification of enzyme activity was studied by the use of thiol reagents: metal ions Ag+, Cu2+, Hg2+ and pCMB. The ATP-PPi exchange activity of valyl-tRNA synthetase was significantly less inhibited by metal ions than the aminoacylation activity. SH-groups essential for tRNA acylation were not required for the activation of valine. After gamma irradiation of valyl-tRNA synthetase the number of SH-groups diminished parallel with the decrease in enzyme activity.

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.

Three photo-cross-linked complexes of yeast phenylalanine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases.

Yeast tRNA-Phe has been cross-linked photochemically to three aminoacyl-tRNA synthetases, yeast phenylalanyl-tRNA synthetase, Escherichia coli isoleucyl-tRNA synthetase, and E. coli valyl-tRNA synthetase. The two non-cognate enzymes are known to interact with tRNA-Phe. In each complex, three regions on the tRNA are found to cross-link. Two of these are common to all of the complexes, while the third is unique to each. Thus, the cognate and non-cognate complexes bear considerable similarity to each other in the way in which the respective enzyme orients on tRNA-Phe, a result which was also established for the complexes of E. coli tRNA-Ile (BUDZIK, G.P., LAM, S.M., SCHOEMAKER, H.J.P., and SCHIMMEL, P.R. (1975) J. Biol. Chem. 250, 4433-4439). The common regions include a piece extending from the 5'-side of the acceptor stem to the beginning of the dihydrouridine helix, and a segment running from the 3' side of the extra loop into the TpsiC helix. These two regions overlap with and include some of the homologous bases found in eight tRNAs aminoacylated by yeast phenylalanyl-tRNA synthetase (ROE, B., SIROVER, M., and DUDOCK, B. (1973) Biochemistry 12, 4146-4153). Although well separated in the primary and secondary structure, these two segments are in close proximity in the crystallographic tertiary structure. In two of the complexes, the third cross-linked fragment is near to the two common ones. The picture which emerges is that the enzymes all interact with the general area in which the two helical branches of the L-shaped tertiary structure fuse together, with additional interactions on other parts of the tRNAas well.