A site-directed antibody recognizes a component of the ouabain-binding domain of the alpha 1 subunit of rat Na+,K(+)-ATPase.
Antibodies (AbE1) 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 microM AbE1 partially inhibited the enzyme and reduced its sensitivity to 1 mM ouabain. Antibodies against an unrelated protein were ineffectual. Unexpectedly, the masses of the proteins detected on immunoblots of rat kidney by AbE1, a 160 kilodalton (kDa) doublet, was greater than the 97 kDa of the alpha 1 subunit. In lysates prepared with hot (85 degrees C) buffer constituted according to Laemmli's formulation for sample buffer (buffer L, a low chaotropic buffer), AbE1 always bound to the 160-kDa doublet. In contrast, AbE1 did not bind any proteins on blots of lysates that were prepared with buffer that contained greater concentrations of detergent and reducing agent (Laemmli's "homogenization" buffer, buffer H, a high chaotropic buffer), regardless of temperature. However, the presence of alpha 1 in lysates produced in homogenization buffer was confirmed by the presence of 97-kDa bands that were detected with two additional site-directed antibodies that recognize the alpha 1 subunit. When homogenates prepared with sample buffer L were examined, one of these antibodies bound to bands of 160 kDa, indicating that alpha 1 is a component of the bands bound by AbE1. It is concluded that AbE1 recognizes a component of the ouabain-binding site of Na+,K(+)-ATPase, but only under conditions in which the alpha 1 subunit was associated into a higher molecular mass complex.
Scully
R R
RR
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77225.
Pressley
T A
TA
O'Neil
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D000939:Epitopes / Q000276:immunology*; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology*; D010042:Ouabain / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology*; D011487:Protein Conformation; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology*
D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase