IMMUNODETECTION AND ENZYMATIC CHARACTERIZATION OF THE ALPHA-3-ISOFORMOF NA,K-ATPASE IN DOG HEART

The expression of the canine alpha 2 and 3 subunit isoenzymes of NA,K- ATPase has been investigated in plasma membranes isolated from dog hea rt, brain and kidney by immunoblotting, employing polyclonal anti rat fusion protein, and enzymological techniques. Western blot analysis re vealed with purified dog membrane Na,K-ATPase preparations, one immuno reactive signal with rat specific alpha(3) antisera in cardiac tissues , and two immunoreactive signals with rat alpha(2) and alpha(3) antise ra in cerebral tissues. These findings suggested the specific expressi on of alpha(3) polypeptide in dog heart (99 kDa), whereas dog brain ex pressed the alpha 2 and 3 polypeptides. The stained bands were superim posed. The antibody to rat brain alpha(1) fusion protein did not cross -react with dog antigens whatever the three tissues tested. Expression of the alpha(3)-subunit isoform in dog heart membranes was consistent with a high affinity digitoxigenin-sensitive class of Na,K-ATPase (IC 50 = 7 +/- 2 nM). A single component with low affinity to digitoxigeni n (IC50 = 110 +/- 10 nM) characterized the alpha(1) kidney form. The m ixture of alpha(2) and alpha(3) isoforms in dog brain exhibited an app arent affinity for digitoxigenin (IC50 = 17 +/- 5 nM) lower than the h eart. The sodium dependences of the high affinity digitoxigenin sites were for the cardiac alpha(3) form (K-0.5= 10 +/- 1.9 mM) and for the cerebral alpha(2) and alpha(3) mixture (K-0.5 19.6 +/- 4.9 mM). The se nsitivities for Na+ of the low affinity sites (alpha(1)) were: 6.7 +/- 1.4 mM, 6.3 +/- 1.2 mM and 11.6 + 2.9 mM in heart, brain and kidney r espectively. This is the first report of the catalytic characteristics of the alpha 3 subunit isoenzyme in canine cardiac plasma membranes.