Cysteine Activation in Cultured Cystinotic Cells THE SPECIFIC ACTIVITY OF CYSTEINYL-tRNA SYNTHETASE AND tRNACys AND THE DETERMINATION OF THE MICHAELISMENTEN CONSTANTS FOR CYSTEINYL-tRNA SYNTHETASE

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This study explored the possibility whether an altered cysteinyl-tRNA synthetase might lead to the faulty regulation of cyst(e)ine levels in cystinotic cells. This hypotheses is attractive, since amino acid activation is important in the regulation of amino acid metabolism in microorganisms. By using cultured fibroblasts from patients with cystinosis, those cell components responsible for cysteine activation were examined: cyst(e)ine, the cysteinyl-tRNA levels, cysteinyl-tRNA synthetase activity, and the Km of cysteine, ATP, and tRNACys for cysteinyl-tRNA synthetase, Fibroblasts from two patients with the infantile form of cystinosis were labeled for three days with [35S]-cystine. In comparison with normal cells, these cells contained high levels of free cysteine and cystine. Labeled fibroblasts from a patient with the adolescent form of the disease contained elevated levels of cystine, although elevated cysteine levels were not detected. The ratio of acceptor activity of tRNACys to tRNALeu in cystinotic cells was 0.46 in cystinotic cells and 0.54 in normal cells. The specific activity of cysteinyl-tRNA synthetase measured in fibroblasts of two infantile and one adolescent form was: 6.1, 2.2, and 2.1 pmol of [14C]aminoacyl-tRNA formed/μg protein/10 min, respectively. In addition, the cysteine Km's for the same cells, respectively, were: 3.1 μM, 1.5 μM, and 1.2 μM. The corresponding data for specific activities of two normal cell lines were 2.0 and 5.1 pmol [14C]aminoacyl tRNA formed/μg protein/10 min, with Km's of 3.0 μM and 1.7 μM. These data indicate that cystinotic cells contain levels of tRNACys and Cys-tRNA synthetase comparable to normal cells. In addition, within the cystinotic cells, the relative level of the Cys-tRNA synthetase and tRNACys to those of leucine and alanine are comparable to normal cells. Finally, the Km of Cys-tRNA synthetase for ATP and tRNA is similar in normal and cystinotic cells.

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