Cysteine Activation in Cultured Cystinotic Cells THE SPECIFIC ACTIVITY OF CYSTEINYL-tRNA SYNTHETASE AND tRNA<sub>Cys</sub> AND THE DETERMINATION OF THE MICHAELISMENTEN CONSTANTS FOR CYSTEINYL-tRNA SYNTHETASE

John R. Waterson; William P. Winter; Roy D. Schmickel

doi:10.1172/JCI107741

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

John R. Waterson, William P. Winter, and Roy D. Schmickel

Published July 1, 1974 - More info

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Abstract

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 K_m of cysteine, ATP, and tRNA_Cys 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 tRNA_Cys to tRNA_Leu 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 K_m'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 K_m's of 3.0 μM and 1.7 μM. These data indicate that cystinotic cells contain levels of tRNA_Cys and Cys-tRNA synthetase comparable to normal cells. In addition, within the cystinotic cells, the relative level of the Cys-tRNA synthetase and tRNA_Cys to those of leucine and alanine are comparable to normal cells. Finally, the K_m of Cys-tRNA synthetase for ATP and tRNA is similar in normal and cystinotic cells.