Any of several lysines can react with 5'-isothiocyanatofluorescein to inactivate sodium and potassium ion activated adenosinetriphosphatase.
Determinations of reaction stoichiometry demonstrate that the covalent incorporation of one molecule of 5'-isothiocyanatofluorescein can inactivate one molecule of sodium and potassium ion activated adenosinetriphosphatase in agreement with earlier determination of this stoichiometry. Several different modified peptides are produced, however, when the modified enzyme is digested with trypsin. One of these peptides has been identified as HLLVMK (thioureidylfluorescein)GAPER by use of a specific immunoadsorbent. The modified lysine is lysine 501 in the amino acid sequence of the alpha polypeptide of (Na+ + K+)-ATPase. This peptide has been previously isolated from such digests [Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., & Shively, J. E. (1984) J. Biol. Chem. 259, 9532-9535]. The other specifically modified peptides have been purified and identified by amino acid sequencing. Their sequences identify lysine 480 and lysine 766 from the alpha polypeptide as amino acids modified by 5'-isothiocyanatofluorescein in reactions sensitive to the addition of ATP and responsible for inactivation of the enzyme.
Xu
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Department of Chemistry, University of California, San Diego, La Jolla 92093.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004285:Dogs; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology*; D066298:In Vitro Techniques; D008239:Lysine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology*
D005452:Fluoresceins; D010446:Peptide Fragments; D013861:Thiocyanates; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine