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Tryptophanyl-tRNA synthetase in cell lines resistant to tryptophan analogs
E L Paley1, V N Baranov, N M Alexandrova
1Engelhardt Institute of Molecular Biology, USSR Academy of Sciences, Moscow.
Abstract:
Bovine kidney cell lines resistant to tryptamine and tryptophanol (tryptophan analogs) were selected. The content of tryptophanyl-tRNA synthetase (WRS, EC 6.1.1.2) was assayed by measuring the binding of monospecific polyclonal antibodies to the 35S-labeled enzyme in detergent-soluble and -insoluble forms and measuring the enzyme activity. Both the enzyme content and activity were elevated in the resistant cells. As was found by immunoelectron microscopy, the initial and resistant cells contained WRS in most of their cellular compartments: on free polyribosomes, as large conglomerates in the cytoplasm, on polysomes bound to the rough endoplasmic reticulum membranes and to the outer nuclear membrane, on the cytoskeleton, and in the detergent-insoluble nuclear matrix. Immunochemically stained tangles of filaments were found in the resistant cells, but not in the control cells. WRS was less phosphorylated in the resistant than in the original Madin Darby bovine kidney cells. Karyological and morphometric analysis revealed that, in tryptamine-resistant cells, the marker acrocentric chromosome was longer and the frequency of its duplication rose to 96%. The results of this work indicate that the cultivated cells have become resistant to tryptophan analogs because of an elevated WRS concentration in the cells, possibly due to amplification of the WRS gene.
Insights
Bovine kidney cells developed resistance to tryptophan analogs due to increased tryptophanyl-tRNA synthetase (WRS) levels. This elevation in WRS content and activity, potentially from gene amplification, confers resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Tryptophan analogs can induce cellular resistance mechanisms.
- Tryptophanyl-tRNA synthetase (WRS) is crucial for protein synthesis.
- Understanding resistance mechanisms in cell lines is vital for biochemical research.
Purpose of the Study:
- To investigate the molecular basis of resistance to tryptophan analogs in bovine kidney cells.
- To determine the role of tryptophanyl-tRNA synthetase (WRS) in this resistance.
- To analyze cellular and genetic changes associated with analog resistance.
Main Methods:
- Selection of resistant bovine kidney cell lines.
- Assay of tryptophanyl-tRNA synthetase (WRS) content and activity using antibody binding and enzyme measurements.
- Immunoelectron microscopy to localize WRS within cellular compartments.
- Analysis of WRS phosphorylation levels.
- Karyological and morphometric analysis of chromosomes.
Main Results:
- Resistant cells exhibited elevated WRS content and activity compared to control cells.
- WRS was localized in various cellular compartments, including polyribosomes, endoplasmic reticulum, cytoskeleton, and nuclear matrix.
- Immunochemically stained filament tangles were observed in resistant cells.
- WRS was less phosphorylated in resistant cells.
- Tryptamine-resistant cells showed chromosomal alterations, including a longer marker acrocentric chromosome and increased duplication frequency.
Conclusions:
- Elevated WRS concentration is a key factor in the resistance of cultivated bovine kidney cells to tryptophan analogs.
- The increased WRS levels may result from WRS gene amplification.
- Chromosomal changes and altered WRS phosphorylation are associated with analog resistance.