Demonstration that lysine-501 of the alpha polypeptide of native sodium and potassium ion activated adenosinetriphosphatase is located on its cytoplasmic surface.

Abstract

Evidence that the peptide HLLVMKGAPER, which can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion, is located on the cytoplasmic surface of the native enzyme has been obtained. An immunoadsorbent directed against the carboxy-terminal sequence of this tryptic peptide has been constructed. The peptide KGAPER was synthesized by solid-phase techniques. Antibodies against the sequence -GAPER were purified by immunoadsorption, using the synthetic peptide attached to agarose beads. These antibodies, in turn, were coupled to agarose beads to produce an immunoadsorbent. Sealed, right-side-out vesicles, prepared from canine kidneys, were labeled with pyridoxal phosphate and sodium [3H]borohydride in the absence or presence of saponin, respectively. A tryptic digest of these labeled vesicles was passed over the immunoadsorbent. Large increases in the incorporation of radioactivity into the peptides bound by the immunoadsorbent were observed in the digests obtained from the vesicles exposed to saponin. From the results of several control experiments examining the labeling reaction as applied to these vesicles, it could be concluded that this increase in incorporation resulted only from the access that the reagents gained to the inside of the vesicles in the presence of saponin and that the increase in the extent of modification was due to the cytoplasmic disposition of this segment in the native enzyme.