Binding of Ouabain and Human Ouabainlike Substance to Different Na⁺, K⁺-ATPase Isoforms

• BUTT Asif N.

  the Departments of Chemical Pathology

• TENNANT Brian P.

  the Departments of Chemical Pathology

• GILLINGWATER Scott D.

  the Departments of Chemical Pathology

• SHEPHERD Philip S.

  Immunology, GKT Medical and Dental School, St. Thomas' Campus

• SWAMINATHAN R.

  the Departments of Chemical Pathology

Bibliographic Information

Other Title

• Binding of Ouabain and Human Ouabainlike Substance to Different Na+, K+-ATPase Isoforms

Abstract

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)

Journal

• Hypertension Research

  Hypertension Research 23 (Supplement), S45-S50, 2000

  The Japanese Society of Hypertension

Keywords

• ouabain
• ouabainlike substance
• Na⁺,K⁺-ATPase isoforms

Details

• CRID: 1390282679695824384
• NII Article ID: 10008741550
• NII Article ID: 130003456500
• NII Book ID: AA10847079
• DOI: 10.1291/hypres.23.supplement_s45
• ISSN: 13484214
• ISSN: 09169636
• PubMed: 11016819
• Web Site: https://search.jamas.or.jp/link/ui/2001001522
• Text Lang: en
• Data Source:
  • JaLC
  • Crossref
  • PubMed
  • CiNii Articles
• Abstract License Flag: Disallowed