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Characterization of ouabain-resistant mutants of a canine kidney cell line, MDCK
Abstract:
Madin-Darby canine kidney (MDCK) cells were mutagenized and variants resistant to 10, 160, and 2000 times the ouabain lethal dose for wild type cells selected. The phenotypes were stable in the absence of selection. The frequencies with which variants were recovered were consistent with genetic alterations being responsible for drug resistance. It was shown that 50% of the (Na+, K+)-ATPase activity present in mutant cells had a higher Kd for ouabain than normal while 50% remained wild type for ouabain binding. Wild type MDCK cells were measured to have 2 X 10(6) ouabain binding sites per cell with a Kd for the drug of 0.6-1.0 X 10(-7) M. The novel (Na+, K+)-ATPase activities in the mutants demonstrated Kd values for ouabain of 10(-5) M, 3 X 10(-4) M, or 3 X 10(-3) M for the different mutant classes tested. The rate of synthesis of the (Na+, K+)-ATPase as well as the total amount of enzyme per unit of cell protein was unaltered in the mutants. Comparison of the alpha subunit of the enzyme, known to contain the ouabain-binding site, by sodium dodecyl sulfate-gel electrophoresis did not reveal any difference in the size of this subunit in mutant versus wild type cells.
Insights
Madin-Darby canine kidney (MDCK) cells developed stable genetic alterations conferring ouabain resistance. These mutations resulted in altered sodium-potassium adenosine triphosphatase ((Na+, K+)-ATPase) activity with higher ouabain binding affinity.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- The sodium-potassium adenosine triphosphatase ((Na+, K+)-ATPase) is a crucial ion pump involved in maintaining cell membrane potential.
- Ouabain is a cardiac glycoside that inhibits (Na+, K+)-ATPase) activity by binding to the enzyme.
- Understanding the mechanisms of ouabain resistance is important for both basic research and potential therapeutic applications.
Purpose of the Study:
- To investigate the genetic basis of ouabain resistance in Madin-Darby canine kidney (MDCK) cells.
- To characterize the biochemical properties of the (Na+, K+)-ATPase) in ouabain-resistant MDCK cell variants.
- To determine if genetic alterations affect the synthesis or structure of the (Na+, K+)-ATPase) alpha subunit.
Main Methods:
- Mutagenesis of MDCK cells followed by selection for ouabain resistance at various concentrations.
- Phenotypic characterization of stable ouabain-resistant variants.
- Biochemical assays to measure (Na+, K+)-ATPase) activity and ouabain binding affinity (Kd).
- Analysis of enzyme synthesis rates and total enzyme levels per cell protein.
- Sodium dodecyl sulfate-gel electrophoresis to compare the alpha subunit size between wild-type and mutant cells.
Main Results:
- Stable ouabain-resistant MDCK cell variants were successfully selected, with resistance levels up to 2000 times that of wild-type cells.
- Mutant cells exhibited altered (Na+, K+)-ATPase) activity, with 50% of the enzyme showing significantly higher dissociation constants (Kd) for ouabain (ranging from 10(-5) M to 3 X 10(-3) M) compared to wild-type cells (0.6-1.0 X 10(-7) M).
- The rate of enzyme synthesis and the total amount of (Na+, K+)-ATPase) per unit of cell protein remained unchanged in the mutants. Sodium dodecyl sulfate-gel electrophoresis revealed no difference in the size of the alpha subunit.
Conclusions:
- Genetic alterations are responsible for the observed ouabain resistance in MDCK cells.
- The resistance is mediated by modifications in the ouabain-binding site of the (Na+, K+)-ATPase), leading to altered drug affinity without affecting enzyme synthesis or alpha subunit structure.
- These findings provide insights into the structure-function relationship of the (Na+, K+)-ATPase) and mechanisms of drug resistance.