[Phosphorylation of tryptophanyl-tRNA-synthase in extracts of bovine pancreas]. / Fosforilirovanie triptofanil-tRNK-sintetazy v ékstraktakh podzheludochnoi zhelezy krupnogo rogatogo skota.

The polypeptide with a mobility of the tryptophanyl-tRNA-synthetase subunit can be labeled in bovine pancreas extracts from [gamma-32P]ATP. Immunoprecipitation analysis with monospecific polyclonal antibodies against the enzyme as well as identification of [32P]phosphoamino acids in the immunoprecipitate revealed that in bovine pancreas extracts tryptophanyl-tRNA-synthetase undergoes phosphorylation at serine residues. The level of phosphorylation does not change in the presence of activity modulators of cAMP-, cGMP- and Ca2(+)-dependent protein kinases, decreases after addition of phosphoseryl/phosphothreonyl-protein phosphatase inhibitors and increases in the presence of their activators. It was supposed that phosphorylation of tryptophanyl-tRNA-synthetase catalyzed by seryl/threonyl-specific protein kinase depends on the activity of phosphoseryl/phosphothreonyl-phosphatase.

[Immunocytochemical localization of tryptophanyl-tRNA-synthetase in a line of bovine kidney cells and in sublines with elevated enzyme levels]. / Immunotsitokhimicheskaia lokalizatsiia triptofanil-tRNK-sintetazy v linii kletok pochki byka i v subliniiakh s povyshennym soderzhaniem fermenta.

Intracellular localization of tryptophanyl-tRNA synthetase (E. C. 6.1.1.2) has been studied immunocytochemically using monospecific antibodies in cultured bovine kidney cells (strain MDBK) and in substrains with elevated enzyme levels. Both light and electron microscopy were used and native or detergent-treated cells were examined. The product of cytochemical reaction was revealed on free polyribosomes, polyribosomes attached to membranes of granular endoplasmic reticulum, on cytofilaments and in the nucleus as well.

[Karyological and morphological analysis of bovine kidney cells resistant to tryptophan analogs--inhibitors of tryptophanyl-tRNA synthetase]. / Kariologicheskii i morfologicheskii analiz kletok pochki byka, rezistentnykh k analogam triptofana--ingibitoram triptofanil-tRNK-sintetazy.

Karyological and morphological analysis of the wild type MDBK cell line (spontaneously transformed bovine kidney cells) was undertaken. The results were compared with the same data obtained with resistant lines derived from the wild type line after prolonged cultivation with increasing quantities of tryptophanol and tryptamine, competitive analogues of tryptophan. Tetraploids are much more abundant in the resistant lines than in the initial one. In tryptamine-resistant cells a large marker acrocentric chromosome is duplicated in 96% of cells and elongated, due to appearance of an additional segment. In the population of resistant cells bi- and multinuclear cells are abundant as well as giant cells; the nuclei are enlarged and the number of nucleoli is increased. A hypothesis is proposed that resistance to tryptophan analogues is associated with amplification of tryptophanyl-tRNA synthetase gene.

[Selection of cell lines resistant to tryptophan analogs]. / Poluchenie kletochnykh linii, rezistentnykh k analogam triptofana.

After prolonged cultivation in the presence of increasing amounts of carboxyl-substituted tryptophan analogs (tryptamine and tryptophanol), cell lines resistant to high concentrations of these compounds were obtained. The initial culture was the Madin-Darby line of spontaneously transformed bovine kidney cells. In the resistant lines the amount of tryptophanyl-tRNA synthetase (E. C. 6.1.1.2) is manyfold increased as shown by two criteria: (i) enzymatic activity (ATP-PPi isotopic exchange) per mg of protein, (ii) binding of in vivo 35S-labeled proteins to polyclonal antibodies against tryptophanyl-tRNA synthetase. It was shown that tryptophanyl-tRNA synthetase is phosphorylated in vivo, and the degree of phosphorylation of the enzyme in initial cells seems to be higher then in the resistant ones. The Km value for tryptophan is not significantly changed for the enzyme from resistant cells. The permeability for tryptophan and its analogs is reduced in the resistant cells. It is proposed that the acquisition of the resistance against tryptophan analogs are due to alterations at the genomic level (for example, gene amplification etc.).