Arthur Kahlenberg

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase: II. Characterization of the acyl phosphate intermediate as an l-glutamyl-γ-phosphate residue☆

Abstract The chemical nature of the acyl phosphate residue believed to be a phosphorylated intermediate in the (Na + + K + )-activated adenosinetriphosphatase has been investigated. The phosphorylated and nonphosphorylated forms of the enzyme were digested with pepsin, and the peptides were reacted with [2,3- 3 H] N -( n -propyl)hydroxylamine of high specific activity. A peptide fraction from each form of the enzyme was further degraded with pronase in the presence of carrier l -glutamyl-γ- N -( n -propyl)hydroxamate and carrier l -aspartyl-β- N ( n -propyl)hydroxamate. An increment in radioactivity migrated with authentic l -glutamyl-γ- N -( n -propyl)hydroxamate in a total of seven electrophoretic and Chromatographic systems. No increment in radioactivity was associated with authentic l -aspartyl-β- N -( n -propyl)hydroxamate in five of the seven systems. The increment associated with l -glutamyl-γ- N -( n -propyl)hydroxamate increased with successive purification, using some of the above systems. At the last stage of purification the radioactivity from the phosphorylated enzyme which migrated as l -glutamyl-γ- N -( n -propyl)hydroxamate was 2.5 times that from the nonphosphorylated enzyme. On gel filtration of partially purified radioactive material the major portion of the increment in radioactivity migrated with and gave the correct molecular weight for l -glutamyl-γ- N -( n -propyl)-hydroxamate. The final yield of increment in radioactivity, isolated as l -glutamyl-γ- N -( n -propyl)hydroxamate was low due to the resistance of the bulk of the peptides to complete enzymatic hydrolysis and to the instability of l -glutamyl-γ- N -( n -propyl)hydroxamate. However, stepwise cleavage of amino acids from the N -terminal ends of these peptides led to a loss in the increment in radioactivity; this would be expected of an l -glutamyl-γ- N -( n -propyl)hydroxamate residue on becoming N -terrminal, since it would then cyclize to form a pyrrolidonecarboxylic acid residue with loss of radioactive propylhydroxylamine. The response to Na + and to K + of the γ-activated glutamate, which was derivatized and eventually characterized as l -glutamyl-γ- N -( n -propyl)hydroxamate, was the same as that of the acyl phosphate previously described. From the above data it is concluded that the acyl phosphate intermediate in the (Na + + K + )-activated adenosinetriphosphatase is an l -glutamyl-γ-phosphate residue. It is suggested that a possible function of the phosphorylation and dephosphorylation of the glutamyl residue in the Na-K ATPase is to induce a cyclic conformational change in the enzyme, thus effecting the translocation of Na + and K + across the membrane.

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. V. Partial purification of the lubrol-solubilized beef brain enzyme.

Abstract The Lubrol-solubilized Na-K adenosinetriphosphatase from NaI-treated beef brain microsomes has been partially purified. This purification involves salt and isoelectric precipitation, ultracentrifugation at 114,000 g for 20 hr, and chromatography on carboxymethylcellulose. The final enzyme preparation is water-clear and is relatively stable if stored at 0 ° in the presence of Na and ATP. Most of the Lubrol is removed by the purification procedure. The partially purified enzyme has a specific activity which is ten times that of the original microsomes. A single, somewhat diffuse, ouabain sensitive ATPase band runs on electrophoresis in 3% polyacrylamide of Lubrol extracts of NaI-treated microsomes. However, partial aggregation occurs during purification, as attested by the appearance of a Na-K ATPase peak in the void volume (molecular weight > 2 × 10 6 ) on chromatography on 6% agarose and a ouabain-sensitive ATPase band which does not enter 3% polyacrylamide on disc gel electrophoresis. The enzyme can be disaggregated with Lubrol into a single species which, on polyacrylamide gel electrophoresis, runs as the enzyme in the original Lubrol extracts; however, concentrations of Lubrol required to disaggregate the enzyme lead to partial inactivation. The various proteins in the preparation do not separate as discrete bands on disc gel electrophoresis unless the proteins are solubilized in phenol-acetic acid-urea. It is estimated that the Na-K ATPase in the present partially purified preparation accounts for approximately 10% of the protein.