Isolation of Diploid Human Lymphoblast Mutants Presumably Homozygous for Ouabain Resistance

From a mass culture of diploid human lymphoblasts we have isolated a subline resistant to 1μ M ouabain, and from this, a subline resistant to 10 μ M ouabain. These sublines occurred spontaneously, but similar mutants were induced with 10-fold increased frequency by treatment with mutagens, in which case they could be selected as clones in soft agarose. Scatchard plot analyses of ouabain binding indicated that the subline resistant to 1 μ M ouabain retained an average of 39% of the high-affinity ouabain receptors of the parental lymphoblast line, and the subline resistant to 10 μ M ouabain retained an average of 8.4%. The ouabain binding site is known to be located in the Na+, K+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) molecule, an essential cell membrane enzyme that mediates ion transport. Studies on Na+, K+-ATPase activity, using86Rb in the absence and presence of ouabain, indicated that our parental lymphoblasts contained one population of Na+, K+-ATPase molecules highly sensitive to ouabain inhibition, the subline resistant to 10 μ M ouabain contained one population relatively insensitive to ouabain, and the subline resistant to 1 μ M ouabain contained both populations. Thus, the moderately resistant subline appears to be heterozygous for ouabain resistance, probably containing a structural mutation in the ouabain receptor region of the Na+, K+-ATPase molecule in one of the two homologous loci for this enzyme, whereas the highly resistant subline derived from it appears to be homozygous, containing an additional mutation in the other Na+, K+-ATPase locus.