Bernard Labouesse

Tryptophanyl-tRNA synthetase is a major soluble protein species in bovine pancreas

Besides their central role in protein synthesis, aminoacyl-tRNA synthetases have been found or thought to be involved in other processes. We present here a study showing that tryptophanyl-tRNA synthetase has a surprising tissular distribution. Indeed, immunochemical determinations showed that in several bovine organs such as liver, kidney and heart, tryptophanyl-tRNA synthetase constitutes, as expected, about 0.02% of soluble proteins. In spleen, brain cortex, stomach, cerebellum or duodenum, this amount is about 10-times higher, and in pancreas it is 100-fold. There is no correlation between these amounts and the RNA content of the organs. Moreover, the concentration of another aminoacyl-tRNA synthetase (methionyl-tRNA synthetase) is higher in liver than in pancreas, while the amount of tRNATrp is not higher in pancreas than in liver as compared to other tRNAs. Among several interpretations, it is possible that tryptophanyl-tRNA synthetase is involved in a function other than tRNA aminoacylation. This unknown function would be specific to the differentiated organs, since fetal cerebellum and fetal pancreas contain the same amount of tryptophanyl-tRNA synthetase as adult liver.

Tryptophanyl-tRNA synthetase from beef pancreas. Spectroscopic analysis of the stoichiometry of formation of the enzyme-tryptophanyl-adenylate complex

The dimeric enzyme tryptophanyl-tRNA synthetase from beef pancreas catalyses the stoichiometric formation of one mole of tryptophanyl-adenylate per subunit. This formation is associated with optical changes (absorbance, fluorescence, optical rotation) and is confirmed by analytical ultracentrifugation. An equal amplitude of the change is observed for each adenylation site at pH 8.0, 25 degrees C, regardless of the optical method used. The formation of two tryptophanyl adenylates per dimer corresponds to a molar absorbance change delta epsilon 291 = 12000 +/- 500 cm-1 M-1, to a fluorescence quenching of 24 per cent at 340 nm and to a variation in optical rotation of 6 per cent at 313 nm. The circular dichroic band of the adenosine moiety of ATP is strongly increased. The addition of sodium pyrophosphate to the tryptophanyl-adenylate-enzyme complex restores the absorbance and fluorescence amplitude observed prior to the addition of ATP to the enzyme. Magnesium ions are necessary to the reaction. A pertubation of the environment of both the protein and the substrates (tryptophan and ATP) have to be taken into account to explain the magnitude of the observed changes.

Tryptophanyl-tRNA synthetase is found closely associated with and stimulates DNA polymerase α-like activity from wheat embryos

Abstract DNA polymerase α-like from wheat embryos is found to purify closely associated with a tryptophanyl-tRNA synthetase activity. No other aminoacyl-tRNA synthetases were present. A purified preparation of wheat tryptophanyl-tRNA synthetase free of polymerase activity was able to stimulate plant DNA polymerase of the α-like type, while the γ-like polymerase from wheat embryos was not affected by the enzyme. We have not been able to find a diadenosine 5′, 5′′′-P 1 ,P 4 -tetraphosphate binding activity associated to the polymerase-synthetase complex. We have also observed a specific inhibition by beef tRNA Trp of DNA polymerase α-like activity, while other tRNAs will not change the enzyme activity.

Kinetics of formation of tryptophanyl-adenylate by tryptophanyl-tRNA synthetase from beef pancreas

The formation of an aminoacyl-adenylate has been shown to occur for most aminoacyl-tRNA synthetases as a first step in the tRNA aminoacylation reaction [ 1,2]. In the particular case of tryptophanyl-tRNA synthetase from beef pancreas a tryptophanyl-adenylate-enzyme complex has been evidenced by gel filtration [3] and spectroscopic changes [4]. Two moles of adenylate can be bound per mole of dimeric enzyme, though under some conditions it has been suggested that tryptophan can also be convalently linked to the protein in a 1: 1 ratio when the enzyme is obtained after fast purification procedure [5]. In the case of the non-covalent complex of the enzyme with L-tryptophan 2 mol tryptophan are bound in an apparent anti-cooperative way to the protein [6]. The formation of the adenylate-enzyme complex has not been studied under prestationary conditions which could show whether or not it is rate determining in the overall ATP-PP, isotope exchange and in the tRNA aminoacylation reactions. Since the formation of the adenylate-enzyme complex can be evidenced by spectroscopic changesof the enzyme [4], this reaction can be followed kinetically using the fluorescence changes of the system. This formation can also be studied by the depletion of [j’P]ATP according to [7] and by the measurement of the stoichiometry of appearance of [‘4C]tryptophanyl-adenylate [8] in order to ascertain that the recorded variations of the spectroscopic signal correspond really to the chemical step of carboxyl activation of tryptophan. This paper presents a preliminary report of the study of such a pre-stationary phase.

Binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase from bovine pancreas under pH conditions of maximum activity. Analysis by ultracentrifugation, fluorescence quenching and chemical modification

The binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef is examined by three approaches, under pH conditions of maximum activity (pH 8.0). (1) Analytical ultracentrifugation evidences the binding of a single mol of tRNATrp in a 2.5-10 microM concentration range. (2) tRNATrp quenches the fluorescence of the enzyme. The dependence of this fluorescence quenching on the tRNATrp concentration (0.1-4 microM) reflects also the binding of 1 mol of tRNA per mol of enzyme, with a Kd value of 0.19 +/- 0.02 microM. (3) tRNATrp protects the enzyme against derivatization by oxidized ATP. Out of the two fast-reacting lysine residues of the native enzyme, only one is prevented from reacting by tRNATrp in the 0.5-110 microM concentration range. This protection can be significantly analyzed only by assuming a one-to-one complex between the enzyme and tRNA. These results, obtained at pH 8.0 and 25 degrees C, are in contrast with the stoichiometry of 2 mol of tRNA to 1 mol of enzyme, previously observed at pH 6.0 and 4 degrees C.

On the mechanism of tRNATrp aminoacylation catalysed by beef tryptophanyl-tRNA synthetase using presteady-state kinetics

The dimeric tryptophanyl‐tRNA synthetase from beef pancreas has been found to activate 2 tryptophans/mol enzyme [Eur. J. Biochem. (1982) 128, 389–398]. By using quenched‐flow and stopped‐flow methods under presteady‐state conditions, we show that only one enzyme subunit operates at a time in the aminoacylation of tRNATrp and that the transfer reaction is not the rate‐limiting step in the overall aminoacylation process.

The adenosine triphosphate-pyrophosphate isotopic exchange reaction: A tool for determination of tryptophan

Quantitative determination of tryptophan at the picomole level is described, using the ATP-[32P]PPi isotopic exchange reaction catalyzed by tryptophanyl-tRNA synthetase. Sensitivity limits of 500 fmol were obtained. The presence of other amino acids at a 1000-fold excess over tryptophan did not interfere significantly with the quantitative determination of tryptophan. The specificity of the reaction was checked using five tryptophan analogs. These analogs did not prevent the determination of tryptophan when present in the same concentration range as tryptophan. When sensitive determination of a single amino acid is needed, the ATP-[32P]PPi exchange reaction catalyzed by aminoacyl-tRNA synthetases is suggested as a general method and as an alternative to HPLC procedures.