Binding of ouabain and human ouabainlike substance to different Na+,K+-ATPase isoforms

There is very little on the affinity of the human immunoreactive ouabainlike substance (OLS) to individual alpha-isoforms of Na+K+-ATPase, The present study addresses this issue by comparing ouabain and OLS binding to dog kidney alpha(1), rabbit kidney alpha(1) and porcine cerebral cortex alpha(3) Na+,K+-ATPase. OLS was initially isolated by solid phase extraction from human serum using cia columns. The extract was further purified by reverse phase HPLC in an acetonitrile/water (containing 0.1% TFA) step-up gradient (16-80%), In this system, two distinct ouabain immunoreactive peaks were resolved, Peak I demonstrated a polarity identical with that of authentic ouabain, In contrast, peak II was relatively non-polar and eluted later in the run. The final step in the purification of OLS involved immune-affinity chromatography of peak I using a specific sepharose immobilized mouse monoclonal anti-ouabain antiserum. Dose response curves (range 0-100 nmol/l) for ouabain with canine al and porcine alpha, Na+,K+-ATPase showed similar inhibitory profiles (IC50 = 15 nmol/l), whilst rabbit alpha(1) Na+,K+-ATPase was relatively insensitive to ouabain and purified peak I OLS, Two fold serial dilution of Peak I OLS, with subsequent analysis by canine and porcine Na+,K+-ATPase inhibition assays and RIA, demonstrated strong positive correlations between OLS determined by RIA and both canine (y = 0.945x-2.532, r(2) = 0.977) and porcine (y = 0.428x-1.685; r(2) = 0,993) Na+,K+-ATPase assays. The difference in the respective slopes suggests, however, that peak I OLS has a greater affinity for the canine derived enzyme compared to the porcine, In conclusion, these data suggest that like authentic ouabain, peak I OLS is alpha-isoform and species selective.