Characterization and solubilization of nucleotide-specific, Mg 2+-ATPase and Mg 2+-pyrophosphatase of tonoplast

Abstract

Nucleotide-specific, Mg 2+-dependent ATPase and Mg 2+-dependent pyrophosphatase were recovered with purified tonoplast obtained from isolated Tulipa petal vacuoles. Relative Mg 2+-dependent hydrolysis of ATP, GTP and pyrophosphate, the only substrates hydrolyzed to a substantial degree, was 1.0, 0.3, and 0.6, respectively. Tonoplast ATPase required Mg 2+, and essentially no Mg 2+-dependent or Mg 2+-independent p- nitrophenylphosphatase (which was associated with intact vacuoles) occurred with the membrane. Tonoplast ATPase was stimulated 10 to 30% by KCl, but was little effected by other cations (other than NH 4 +) or anions. No activity was observed with CaATP as substrate. The enzyme was cold stable and was inhibited by DCCD and Dio-9, but not by oligomycin. Its pH optimum was 7.0 and its specific activity was about 50 μmol P i/mg protein per h at 37°C. Properties of membrane-bound and Polidocanol (polyoxyethylene ether, 9 lauryl ether) solubilized enzyme were similar. Reduced activity of solubilized enzyme was partially restored with phospholipids. Tonoplast ATPase appears to be an integral membrane component which requires phospholipids for maximal activity. Tonoplast Mg 2+-pyrophosphatase had a pH optimum of ≥ 8.5, was stimulated 2.5-fold by 50 mM KCl, and was largely lost upon detergent treatment. Properties of tonoplast ATPase observed are consistent with the characteristics of proton transport exhibited by isolated, intact Tulipa vacuoles (Wagner, G.J. and Lin, W. (1982) Biochim. Biophys. Acta 689, 261–266). These observations suggest that tonoplast ATPase functions in proton transport.