Purification of the membrane-spanning tryptic peptides of the alpha polypeptide from sodium and potassium ion activated adenosinetriphosphatase labeled with 1-tritiospiro[adamantane-4,3'-diazirine].
Five long, membrane-spanning tryptic peptides from the alpha polypeptide of sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase] have been purified. (Na+ + K+)-ATPase, isolated from canine kidney, was exposed to ultraviolet light in the presence of a high concentration of 1-tritiospiro[adamantane-4,3'-diazirine], a carbene precursor that partitions into the bilayer of the membrane. The alpha polypeptide, modified with 1.2 mol of [3H]adamantylidene (mol of polypeptide)-1, was isolated and digested with trypsin. Digestion with trypsin ensures that membrane-spanning sequences remain intact during the digestion, since lysine and arginine, being extremely hydrophilic, rarely appear in the membrane-embedded regions of membrane proteins. This digestion produced radioactive tryptic peptides greater than 25 residues in length. The tryptic digest of the labeled alpha polypeptide was chromatographed on Sephadex LH-60 in ethanol-formic acid, 4:1. The majority of the radioactivity (87%) eluted with distribution coefficients corresponding to peptides longer than melittin (26 residues), whereas 73% of the protein traveled with distribution coefficients corresponding to peptides less than 30 residues in length. Five radioactive peptides were further purified by high-pressure liquid chromatography, and each peptide displayed a unique, hydrophobic amino-terminal sequence. No other candidates could be found when a search for additional membrane-spanning peptides was conducted. Gel filtration of the tryptic peptides from the alpha polypeptide of (Na+ + K+)-ATPase labeled with 5-[125I]iodo-1-naphthyl azide, a lipophilic nitrene precursor, produced no additional radioactive components. Amino-terminal sequences and amino acid compositions of the five purified peptides are presented.
D000218:Adamantane / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004285:Dogs; D007457:Iodine Radioisotopes; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014316:Tritium; D014357:Trypsin
D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014316:Tritium; C024080:adamantane diazirine; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D000218:Adamantane