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Hypertens Res ; 23 Suppl: S45-50, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11016819

RESUMO

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.


Assuntos
Cardiotônicos/metabolismo , Digoxina , Isoenzimas/metabolismo , Ouabaína/metabolismo , Saponinas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Anticorpos Monoclonais , Ligação Competitiva , Cardenolídeos , Cardiotônicos/imunologia , Cardiotônicos/farmacologia , Córtex Cerebral/enzimologia , Reações Cruzadas , Cães , Humanos , Isoenzimas/antagonistas & inibidores , Rim/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Ouabaína/imunologia , Ouabaína/farmacologia , Coelhos , Saponinas/imunologia , Saponinas/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Especificidade da Espécie , Suínos
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