Irena Landeka

Defining the Active Site of Yeast Seryl-tRNA Synthetase MUTATIONS IN MOTIF 2 LOOP RESIDUES AFFECT tRNA-DEPENDENT AMINO ACID RECOGNITION

The active site of class II aminoacyl-tRNA synthetases contains the motif 2 loop, which is involved in binding of ATP, amino acid, and the acceptor end of tRNA. In order to characterize the active site of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS), we performed in vitro mutagenesis of the portion of the SES1 gene encoding the motif 2 loop. Substitutions of amino acids conserved in the motif 2 loop of seryl-tRNA synthetases from other sources led to loss of complementation of a yeast SES1 null allele strain by the mutant yeast SES1 genes. Steady-state kinetic analyses of the purified mutant SerRS proteins revealed elevated Km values for serine and ATP, accompanied by decreases in kcat (as expected for replacement of residues involved in aminoacyl-adenylate formation). The differences in the affinities for serine and ATP, in the absence and presence of tRNA are consistent with the proposed conformational changes induced by positioning the 3′-end of tRNA into the active site, as observed recently in structural studies of Thermus thermophilus SerRS (Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 2834-2842). The crystal structure of this moderately homologous prokaryotic counterpart of the yeast enzyme allowed us to produce a model of the yeast SerRS structure and to place the mutations in a structural context. In conjunction with structural data for T. thermophilus SerRS, the kinetic data presented here suggest that yeast seryl-tRNA synthetase displays tRNA-dependent amino acid recognition.

Carbendazim impends hepatic necrosis when combined with imazalil or cypermethrin.

Imazalil, cypermethrin and carbendazim are detected in plants for human nutrition. To explore whether their combinations, applied orally in low doses, would induce changes in metabolic patterns and hepatotoxicity, a subchronic in vivo experiment was conducted. Doses of 10mg/kg of imazalil (im) and cypermethrin (cy) and 20 mg/kg of carbendazim (car) and their combinations (im, 10 mg/kg+cy, 10mg/kg; im, 10mg/kg+car, 20mg/kg; car, 20 mg/kg + im, 10 mg/kg) were given to Swiss mice daily over 28 days. After 24 hr from the last dose, the relationships of cytotoxicity biomarkers were analysed: serum lactate dehydrogenase, aspartate transaminase, alanine transferase, amylase, alkaline phosphatase, creatine kinase, creatinine and total proteins. Individual pesticides showed different toxic potential (cy>im car) generally characterized by increase in enzyme activities. Histological analysis showed that cypermethrin, but not imazalil or carbendazim, alone can cause mild necrosis. Combinations generally caused decrease in the activity of enzymes, indicating liver damage. Low doses of carbendazim in combination with low doses of imazalil or cypermethrin caused very pronounced hepatic necrosis, more than any of the three individually applied pesticides or combination of imazalil and cypermethrin. In fruits and vegetables for human consumption, residues of these three pesticides and prolonged combined intake of low doses, which by themselves acutely would not cause any effect, may have similar hepatotoxic effects.