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The diadenosine oligophosphates (Ap(n)A) were discovered in the mid-sixties in the course of studies on aminoacyl-tRNA synthetases (aaRS). Now, more than 30 years later, about 300 papers have been published around these substances in attempt to decipher their role in cells. Recently, Ap(n)A have emerged as intracellular and extracellular signalling molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. Great variety of physiological and pathological effects in mammalian cells was found to be associated with alterations of Ap(n)A levels (n from 2 to 6) and Ap3A/Ap4A ratio. Cell differentiation and apoptosis have substantial and opposite effects on Ap3A/Ap4A ratio in cultured cells. A human Ap3A hydrolase, Fhit, appeared to be involved in protection of cells against tumourigenesis. Ap3A is synthesised by mammalian u synthetase (TrpRS) which in contrast to most other aaRS is unable to synthesise Ap4A and is an interferon-inducible protein. Moreover, Ap3A appeared to be a preferred substrate for 2-5A synthetase, also interferon-inducible, priming the synthesis of 2' adenylated derivatives of Ap3A, which in turn may serve as substrates of Fhit. Tumour suppressor activity of Fhit is assumed to be associated with involvement of the Fhit.Ap3A complex in cytokine signalling pathway(s) controlling cell proliferation. The Ap(n)A family is potentially a novel class of signal-transducing molecules whose functions are yet to be determined.

Bovine tryptophanyl-tRNA synthetase (TrpRS, E.C.6.1.1.2) is unable to catalyze in vitro formation of Ap4A in contrast to some other aminoacyl-tRNA synthetases. However, in the presence of L-tryptophan, ATP-Mg2+ and ADP the enzyme catalyzes the Ap3A synthesis via adenylate intermediate. Ap3A (not Ap4A) may serve as a substrate for TrpRS in the reaction of E.(Trp approximately AMP) formation and in the tRNA(Trp) charging. The Km value for Ap3A was higher than the Km for ATP (approx. 1.00 vs. 0.22 mM) and Vmax was 3 times lower than for ATP. The Zn(2+)-deficient enzyme catalyzes Ap3A synthesis in the absence of exogenous ADP due to ATPase activity of Zn(2+)-deprived TrpRS. The inability of mammalian TrpRS to synthesize Ap4A, might be considered as a molecular tool preventing the removal of Zn2+ due to chelation by Ap4A and therefore preserving the enzyme activity.

Bovine tryptophanyl-tRNA synthetase (ECC 6.1.1.2.; WARS) is one of the best studied synthetases from multicellular organisms. Previous assignment of the WARS gene to the long arm of human Chromosome (Chr) 14 has been made by somatic cell hybrid techniques (Denney and Craig 1976; Shimizu et al. 1976). Francke and co-workers (1977) mapped it on 14q21q32. We describe here the localization of the WARS gene to the bovine and human chromosomes with specific cDNA probes (Frolova et al. 1991) for in situ hybridization. Detailed protocols for the in situ hybridization, chromosome staining, and silver grain distribution analysis have been given previously (Graphodatsky et al. 1992). Standard nomenclatures of the human and cattle chromosomes were used (ISCN 1985; ISCNDA 1989). Our results confirm the previously reported assignment of the tryptophanyl-tRNA synthetase gene to Chr 14 (Denney and Craig 1976; Shimizu et al. 1976; Francke et al. 1977). Twenty-four percent of all grains (202 grains from 104 total metaphases) were mapped on Chr 14 (• = 185.0; P < 0.001). Sixty-three percent of the grains over Chr 14 were localized to the 14q23q31 region; 33% to the 14q24 band. These results demonstrate the location of the WARS gene in the 14q23Lq31 region and presumably more precisely in the 14q24 band. Unexpected results were obtained with bovine chromosomes probed with cloned human cDNA. Thirteen percent of all the grains (353 grains per 103 metaphases) were mapped on Chr 3 (• = 53.6; P < 0.001). However, these grains were not condensed in one locus but were concentrated in two separate loci: 38% of grains on BTA3 were found over the qll--q21 region and 36% over the q42-45 region. Figure 1 shows the Gand RBG-banding patterns of cattle 3 (BTA3) Chr and human Chr 14 (HSA14).

4137 Background: Tryptophanyl-tRNA synthetase (trpRS) is an aminoacyl-tRNA synthetase involved in protein synthesis, regulation of transcription and translation of RNA and an inhibitor of angiogene...

Brown MX-5BR specifically and irreversibly inactivates tryptophanyl-tRNA synthetase from Bacillus stearothermophilus at pH 8.5. The enzyme is protected from inactivation by the substrates tryptophan and ATP and to lesser extents by ADP, AMP, the product inorganic pyrophosphate and other nucleotides such as GTP. The Kd of the pure reactive dye for the enzyme was measured to be 6.7 X 10(-6) M. The Km values of the two substrates tryptophan and MgATP were found to be 1 x 10(-5) M and 5 x 10(-5) M respectively. The aminated dye is a competitive inhibitor of tryptophanyl-tRNA synthetase with respect to both tryptophan and MgATP with Ki values of 2 x 10(-4) M against both substrates. The use of this dye as an active-site-directed affinity label is discussed.

The covalent derivative of the tryptophanyl-tRNA synthetase obtained under the action of32PPi contains one mole of the covalently bound pyrophosphate (or 2 moles of orthophosphate) per mole of dimeric enzyme. Dephosphorylation with alkaline phosphatase causes practically no changes of enzymatic activity although the enzyme looses its ability to bind PPi.

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.

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 tRNA Trp and that the transfer reaction is not the rate-limiting step in the overall aminoacylation process.

The biological role of Ap3A synthesized in cells by tryptophanyl-tRNA synthetase (WRS) is unknown. Previously we have demonstrated that the cellular level of Ap3A significantly increases after interferon treatment. Here we show that the human 46 kDa 2-5A synthetase efficiently utilizes Ap3A as a primer for oligoadenylate synthesis. The Km for Ap3A is several-fold lower than for Ap4A and 100-fold lower than for ATP. This implies that Ap3A might be a natural primer for the 2′-adenylation reaction catalysed by 2-5A synthetase. Since WRS and 2-5A synthetase are both interferon-inducible proteins, a new link between two interferon-dependent enzymes is established.

Neither tryptophanyl-tRNA nor tryptophanyl-tRNA synthetase is required in vitro for regulation of transcription of the tryptophan operon of Escherichia coli.

The aim of this paper is to review briefly the recent results obtained with beef pancreas tryptophanyl-tRNA synthetase (E.C.6.1.1.2) in our laboratory. This enzyme has been the subject of intensive investigations during the last decade and the data are summarized elsewhere (Kisselev and others, 1979; Kisselev and others, in press; Knorre and Kisselev, 1979). The covalent derivative between tryptophanyl-tRNA synthetase and tryptophan is characterized. The tryptophanyl residue is bound to the native enzyme via anhydride bond whereas after denaturation it is transferred preferentially to the SH-group of the protein. The functional role of the tryptophanyl enzyme is discussed. The immunochemical properties of the enzyme are studied; comparison with other synthetases of the same specificity is undertaken.

A previously constructed trp-S-lacZ fusion encoding a hybrid protein with beta-galactosidase activity was subcloned from a multicopy plasmid onto a lambda vector. Single-copy lysogens of lambda trpS-lacZ were used to determine whether trpS was regulated in a manner similar to that of other aminoacyl-tRNA synthetases. trpS regulation was found to resemble that of the majority of synthetases, in that expression of the lysogen-encoded hybrid beta-galactosidase varied with growth rate; beta-galactosidase activity increased 2.5-fold as the generation time decreased from 150 to 37 min. This regulatory response was confirmed by DNA/RNA hybridization experiments, which also suggested that this form of metabolic regulation occurred at the transcriptional level. No alteration in the level of hybrid beta-galactosidase was observed, however, when cells were starved for tryptophan.

Publisher Summary This chapter describes the isolation of tryptophanyl-tRNA synthetase [L-tryptophan: tRNA Trp ligase (AMP-forming), EC 6.1.1.2] from beef pancreas. The purification procedure comprises of five steps and yields about 130 mg of the enzyme per kilogram of tissue. The yield of the enzyme is considerably higher than that reported by others. Various properties and inhibitors of the given enzyme are described as well. From the mentioned data on beef pancreas tryptophanyl-tRNA synthetase it is concluded that this enzyme is characterized enzymologically rather intensively. Development of primary and three-dimensional structure investigations in the nearest future is expected because of the convenient and rapid procedure for enzyme purification. The availability of the specific inhibitors of different chemical nature makes it possible to evaluate various functional groups involved in the formation of active center. The mechanism of enzymic action needs to be investigated by means of fast kinetic methods.

Highly purified tryptophanyl-tRNA synthetase from beef pancreas forms an enzymetryptophanyladenylate complex which has been isolated from reaction mixtures by gel filtration. The stoichiometry of the complex is 2 moles of tryptophanyladenylate per mole of native enzyme of 108000 molecular weight. The complex reacts with inorganic pyrophosphate to form 1 mole of ATP per mole of bound tryptophanyladenylate and with yeast-tRNA to form tryptophanyl-tRNA. An enzyme-tryptophan complex can also be isolated by gel filtration. Its stability is lower than that of the enzyme-tryptophanyladenylate complex. When the protein has been specially treated to remove bound tryptophan, no enzyme-ATP complex can be isolated from a mixture of enzyme and ATP. The bound tryptophan can react stoichiometrically with ATP to give tryptophanyladenylate which remains bound to the enzyme.