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Published on: November 15, 2013
Inverse relationship between galactokinase activity and 2-deoxygalactose resistance in Chinese hamster ovary cells
Abstract:
Galactokinase activity is reduced in 12 independent clones of Chinese hamster ovary cells resistant to 2-deoxygalactose. The frequency of resistant colonies is increased with chemical mutagens. The resistant phenotype is stable in the absence of selection. There is an inverse correlation between the levels of galactokinase activity and the cloning efficiency in deoxygalactose. Cells with high resistance have 1% or less of the enzyme activity observed in the parental cells; while cells with low resistance have 10-30% galactokinase activity. Studies with tetraploid hybrid cells reveal that resistance to deoxygalactose is a recessive trait and that cells with high resistance do not complement those with low resistance. In cell lines with low resistance, the Km for galactose, Ki for deoxygalactose, Km for ATP, and thermolability were not significantly altered compared to sensitive parental cells. Although the possibility of mutation at the structural gene locus has not been ruled out, the reduced enzyme activity may also be due to mutation at a regulatory site which affects the number of galactokinase molecules per cell.
Insights
Chinese hamster ovary cells resistant to 2-deoxygalactose exhibit reduced galactokinase activity. This resistance is a stable, recessive trait, potentially caused by mutations affecting enzyme regulation.
Area of Science:
- Biochemistry
- Genetics
- Cell Biology
Background:
- Galactokinase (GALK) is crucial for galactose metabolism.
- Understanding resistance mechanisms provides insights into enzyme function and regulation.
- Chinese hamster ovary (CHO) cells are a model for genetic and biochemical studies.
Purpose of the Study:
- To investigate the molecular basis of 2-deoxygalactose resistance in CHO cells.
- To characterize the galactokinase (GALK) activity and genetic properties of resistant cell lines.
- To determine if mutations affect the structural gene or regulatory elements of GALK.
Main Methods:
- Isolation and characterization of 2-deoxygalactose-resistant CHO cell clones.
- Measurement of galactokinase enzyme activity in parental and resistant cells.
- Analysis of genetic complementation in hybrid cells.
- Biochemical characterization of GALK kinetics and stability.
Main Results:
- 12 independent CHO cell clones resistant to 2-deoxygalactose showed significantly reduced galactokinase activity.
- Resistance is a stable, recessive genetic trait.
- High-resistance cells had <1% of parental GALK activity; low-resistance cells had 10-30%.
- Kinetic parameters (Km, Ki) and thermolability of GALK were largely unchanged in low-resistance cells.
Conclusions:
- Reduced galactokinase activity is the primary cause of 2-deoxygalactose resistance in these CHO cells.
- The recessive nature and lack of complementation suggest mutations in a single gene or regulatory pathway.
- Mutations may occur at the structural gene locus or, more likely, at a regulatory site affecting GALK expression levels.
