Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection.
A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells.
D002462:Cell Membrane / Q000201:enzymology; D002871:Chromosome Banding; D002999:Clone Cells; D004351:Drug Resistance; D005784:Gene Amplification; D005796:Genes / Q000187:drug effects*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007621:Karyotyping; D007700:Kinetics; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology*; D010641:Phenotype; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase