Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-[3H]ethylmaleimide.
Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-[3H]ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-[3H]ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate [Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., & Shively, J. E. (1984) J. Biol. Chem. 259, 9532-9535].
D000595:Amino Acid Sequence; D000818:Animals; D003488:Cyanogen Bromide; D004285:Dogs; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification*; D011865:Radioisotope Dilution Technique; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014316:Tritium; D000254:Sodium-Potassium-Exchanging ATPase; D005033:Ethylmaleimide; D003488:Cyanogen Bromide