Studies on the Characterization of the Sodium-Potassium Transport Adenosine Triphosphatase XV. DIRECT CHEMICAL CHARACTERIZATION OF THE ACYL PHOSPHATE IN THE ENZYME AS AN ASPARTYL β-PHOSPHATE RESIDUE

Abstract Phosphoenzyme and dephosphoenzyme from the purified (sodium + potassium)-activated adenosine triphosphatase from Squalus acanthias were reduced with sodium [3H]borohydride followed by acid hydrolysis and treatment with NaOH. Standard homoserine and α-amino-δ-hydroxyvaleric acid were added, and amino acid analysis was carried out in an amino acid analyzer. In a modified buffer, homoserine and α-amino-δ-hydroxyvaleric acid were clearly separated without any overlapping of any of the other amino acids. There was a strong radioactive peak coinciding with the homoserine peak. The radioactivity in the homoserine peak from the dephosphoenzyme was less than 5% of that from the phosphoenzyme. No radioactivity was associated with any other amino acid. The yield of radioactivity from the 32P-labeled acyl phosphate recovered as radioactive homoserine ranged from 19 to 34%. In the complete amino acid analysis, the remainder of the tritium radioactivity was eluted with neutral material and in a small peak emerging at about 25 min. There was no difference in these peaks between dephospho- and phosphoenzyme. Since reduction of an aspartyl β-phosphate residue gives rise to homoserine and reduction of a glutamyl γ-phosphate residue gives rise to α-amino-δ-hydroxyvaleric acid, which can be recoverd after acid hydrolysis of the protein and alkaline treatment, we conclude that the acyl phosphate residue in the (sodium + potassium)-activated adenosine triphosphatase is an aspartyl β-phosphate residue. Under conditions of standard protein hydrolysis in acid for 36 hours, 13% of the α-amino-δ-hydroxyvaleric acid was quantitatively converted to proline. The remainder of the α-amino-δ-hydroxyvaleric acid was recovered unchanged.