A Chinese hamster ovary leucyl-tRNA synthetase mutant with a uniquely altered high molecular weight leucyl-tRNA synthetase complex

The Chinese hamster ovary (CHO) cell culture temperature-sensitive mutant ts 025Cl with a defect in leucyl-tRNA synthetase (LeuRS) does not have an inherently more thermolabile LeuRS, but instead the mutation causes the complete loss of the LeuRS high molecular weight complexes which are present in normal wild-type cells. The mutant cell LeuRS has a single 8 S enzyme form which corresponds hydrodynamically to the 8 S free form of wild-type enzyme. Both 8 S forms have the same thermostability and the same K m for leucine, indicating that there is no inherent defect in the catalytic activity of the enzyme. The temperature-sensitive phenotype can be explained by the lack of thermostable high molecular weight forms of LeuRS.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44149/1/10528_2004_Article_BF00484233.pd

A methionyl-tRNA synthetase deficiency in cultured human fibroblasts from hypermethioninemic patients with normal methionine adeonslytransferase activity

Includes bibliographical references.Includes illustrations.Low methionine adenosyltransferase (MAT) activity has been observed in cultured fibroblasts from patients exhibiting elevated blood methionine levels (hypermethioninemia). Fibroblasts from two patients with both hypermethioninemia and either normal or elevated MAT levels have been found to contain lower methionyl-tRNA synthetase (MetRS) activity (~20% of control) when compared to appropriate controls. MetRS activity was determined on cell lysates of cultured human fibroblasts from a coded panel of hypermethioninemic patients and controls in a blind experiment. Enzyme assays were performed under optimized MetRS conditions and initial velocities were standardized to protein concentration. Of six hypermethioninemic patients tested, only the two with normal or elevated MAT activity exhibited a deficient MetRS, while four patients with low MAT activity and two normal controls exhibited comparable MetRS activity. In a control experiment testing for leucyl-tRNA synthetase (LeuRS) activity, there was no significant difference among the samples. Kinetic analysis and sedimentation experiments revealed that one patient had a MetRS form with a Kₘ for methionine not signifieantly different from control, but was-greatly reduced in quantity (~25% of normal) and lacked a large (22-25S) MetRS complex present in normal fibroblasts. The sedimentation profiles of leucyl-, alanyl-, and histidinyl-tRNA synthetases did not differ significantly between this MetRS deficient patient and normal cells. These results suggest that a primary gene defect in either the locus for methionyl-tRNA synthetase or a regulator of its activity could be associated with the hypermethioninemic condition. This finding implicates the aminoacyl-tRNA synthetases as potential regulators of blood amino acid levels in normal and aminoacidopathic conditions in humans.M.S. (Master of Science