Tryptamine-mediated stabilization of tryptophanyl-tRNA synthetase in human cervical carcinoma cell line.

Tryptamine is an endogenous neuroactive metabolite of tryptophan. Interpretation of the function of this bioamine, however, is restricted to manipulation with tryptamine synthetic pathways. Meanwhile, tryptamine is a potent inhibitor of protein biosynthesis, via the competitive inhibition of tryptophanyl-tRNA synthetase (TrpRS). The influence of the persistent tryptamine inhibition on the half-life and cellular content of TrpRS was examined by chase labeling of HeLa cells and the tryptamine-resistant subline with [35S]methionine. The results indicate that long-term tryptamine treatment of HeLa cells led to a significant increase in the half-life of TrpRS while the content, in vivo phosphorylation and gene dose of TrpRS were unchanged. These findings suggest that survival of drug-resistant cells may not be due to TrpRS gene amplification, but to stabilization of TrpRS. It was shown that tryptamine is an effective inhibitor of HeLa cell growth. In contrast to the well-characterized antineoplastic compounds, conferring a many hundred-fold elevated drug resistance to tumor cells, resistance to tryptamine at very low levels was difficult to achieve, i.e. the 2-fold resistant subline was selected after 19 months of treatment of HeLa cells with gradually increasing concentrations of tryptamine. The tryptamine-resistant HeLa subline exhibited a slower growth rate than the original HeLa line when similar concentrations of both cell populations were seeded on the plates. A low tryptamine resistance and a lack of TrpRS gene amplification were observed in two tryptamine-resistant HeLa sublines and three Chinese hamster sublines. The role of TrpRS in oncogenesis and the perspective for tryptamine as a potential anti-cancer drug are discussed.

Chemical modifications of Bacillus subtilis tryptophanyl-tRNA synthetase.

A concerted conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was evident from previous fluorescence on the quenching of the single Trp residue Trp-92 in the 4FTrp-AMP complexed enzyme. In this study, chemical modifications of the B. subtilis TrpRS were employed to further characterize this conformational change, with the single Trp residue serving as a marker for monitoring the change. Modifications of the enzyme by means of the Trp-specific agent N-bromosuccinimide (NBS) or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole) inactivated the enzyme in accord with the essential role of Trp-92, as identified previously by site-directed mutagenesis. ATP sensitized TrpRS toward inactivation by NBS and BNPS-skatole, which suggested a conformational change that resulted in greater accessibility of Trp-92 toward modifications. In contrast, the cognate tRNATrp substrate exerted a specific protective effect against inactivation by both of the reagents, indicating that the TrpRS-tRNATrp interaction reduces the accessibility of Trp-92 under our experimental conditions. By comparison, modification of sulfhydryl groups by means of iodoacetamide did not reduce TrpRS activity. Observations on Trp-specific modification and substrate protection effects are discussed in the context of the Bacillus stearothermophilus TrpRS crystal structure.