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 a-isoforms of Na+, K+-ATPase. The present study addresses this issue by comparing ouabain and OLS binding to dog kidney α1, rabbit kidneyα1 and porcine cerebral cortex α3 Na+, K+- ATPase. OLS was initially isolated by solid phase extraction from human serum using C18 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 immuno-affinity chromatography of peak I using a specific sepharose immobilized mouse monoclonal anti-ouabain antiserum. Dose response curves (range 0-100nmol/l) for ouabain with canineα1, and porcine α3 Na+, K+-ATPase showed similar inhibitory rofiles (IC50=15nmol/l), whilst rabbit α1 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, r2=0.977) and porcine (y=0.428x-1.685; r2=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 α-isoform and species selective. (Hypertens Res 2000; 23 Suppl: S45-S50)