Topological disposition of the sequences -QRKIVE- and -KETYY in native (Na+ + K+)-ATPase.
The dispositions with respect to the plane of the membrane of lysine-905 in the internal sequence -EQRKIVE- and of lysine-1012 in the carboxy-terminal sequence -RRPGGWVEKETYY of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase have been determined. These lysines are found in peptides released from the intact alpha-polypeptide by the extracellular protease from Staphylococcus aureus strain V8 and by trypsin, respectively. Synthetic peptides containing terminal sequences of these were used to prepare polyclonal antibodies, which were then used to prepare immunoadsorbents directed against the respective peptides. Sealed, right-side-out membrane vesicles containing native (Na+ + K+)-ATPase were labeled with pyridoxal phosphate and sodium [3H]borohydride in the absence or presence of saponin. The labeled alpha-polypeptide was isolated from these vesicles and digested with appropriate proteases. The incorporation of radioactivity into the peptides binding to the immunoadsorbent directed against the sequence pyrERXIVE increased 3-fold in the presence of saponin as a result of the increased accessibility of this portion of the protein to the reagent when the vesicles were breached by saponin; hence, this sequence is located on the cytoplasmic face of the membrane. It was inferred that the carboxy-terminal sequence -KETYY is on the extracytoplasmic face since the incorporation of radioactivity into peptides binding to the immunoadsorbent directed against the sequence -ETYY did not change when the vesicles were breached with saponin.
Bayer
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Department of Chemistry, University of California, San Diego, La Jolla 92093.
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D008239:Lysine; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase; D013211:Staphylococcus aureus / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
D010447:Peptide Hydrolases; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008239:Lysine