A SITE-DIRECTED ANTIBODY RECOGNIZES A COMPONENT OF THE OUABAIN-BINDING DOMAIN OF THE ALPHA(1) SUBUNIT OF RAT NA-ATPASE(,K+)

Antibodies (Ab(E1)) were raised against an oligopeptide derived from a component of the ouabain-binding site of the alpha(1) subunit of rat Na+,K+-ATPase, Preincubation of partially purified Na+,K+-ATPase from rat kidney with 1 mu M Ab(E1) partially inhibited the enzyme and reduc ed its sensitivity to 1 mM ouabain. Antibodies against an unrelated pr otein were ineffectual. Unexpectedly, the masses of the proteins detec ted on immunoblots of rat kidney by Ab(E1) a 160 kilodalton (kDa) doub let, was greater than the 97 kDa of the alpha(1) subunit. In lysates p repared with hot (85 degrees C) buffer constituted according to Laemml i's formulation for sample buffer (buffer L, a low chaotropic buffer), Ab(E1) always bound to the 160-kDa doublet. In contrast, Ab(E1) did n ot bind any proteins on blots of lysates that were prepared with buffe r that contained greater concentrations of detergent and reducing agen t (Laemmli's ''homogenization'' buffer, buffer H, a high chaotropic bu ffer), regardless of temperature. However, the presence of alpha(1) in lysates produced in homogenization buffer was confirmed by the presen ce of 97-kDa bands that were detected with two additional site-directe d antibodies that recognize the alpha(1) subunit. When homogenates pre pared with sample buffer L were examined, one of these antibodies boun d to bands of 160 kDa, indicating that alpha(1) is a component of the bands bound by Ab(E1) It is concluded that Ab(E1) recognizes a compone nt of the ouabain-binding site of Na+,K+-ATPase, but only under condit ions in which the alpha(1) subunit was associated into a higher molecu lar mass complex.