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 alpha1, rabbit kidney alpha1 and porcine cerebral cortex alpha3 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 0-100 nmol/l) for ouabain with canine alpha1 and porcine alpha3 Na+,K+-ATPase showed similar inhibitory profiles (IC50=15 nmol/l), whilst rabbit alpha1 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 a-isoform and species selective.
Butt
A N
AN
Department of Chemical Pathology, GKT Medical and Dental School, London, UK.
Tennant
B P
BP
Gillingwater
S D
SD
Shepherd
P S
PS
Swaminathan
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002298:Cardenolides; D002316:Cardiotonic Agents / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D002540:Cerebral Cortex / Q000201:enzymology; D003429:Cross Reactions; D004077:Digoxin; D004285:Dogs; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D010042:Ouabain / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D012503:Saponins / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013045:Species Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D002298:Cardenolides; D002316:Cardiotonic Agents; D007527:Isoenzymes; D012503:Saponins; C034504:digoxin-like factors; D010042:Ouabain; D004077:Digoxin; D000254:Sodium-Potassium-Exchanging ATPase