The prime plasmalemma ATPase of the halophilic alga Dunaliella bioculata: purification and characterization

The prime plasmalemma ATPase of the halophilic green alga Dunaliella bioculata has been solubilized by Triton X-100 from a plasmalemma-rich membrane fraction and purified by anion-exchange chromatography. Vanadate-sensitive ATPase activity was totally enriched about 230-fold to a specific activity of approx. 250 nkat·mg protein-1. The presence of Mg2+ or Mn2+ is essential for ATP hydrolysis by the enzyme. In addition to an equimolar requirement (1:1 Mg2+:ATP), there is further stimulation by Mg2+ (up to 20 mM) and by (100 mM) monovalent cations (${\mathrm{K}}^{+}\simeq \mathrm{N}{\mathrm{H}}_{4}^{+}>\mathrm{R}{\mathrm{b}}^{+}\simeq \mathrm{N}{\mathrm{a}}^{+}>\mathrm{C}{\mathrm{S}}^{+}>\mathrm{L}{\mathrm{i}}^{+}\simeq \mathrm{c}\mathrm{h}\mathrm{o}\mathrm{l}\mathrm{i}\mathrm{n}{\mathrm{e}}^{+}$). Most anions have no or little effect. With a molecular mass of about 105 kDa for the single subunit, sensitivity to vanadate and N,N′-dicyclohexylcarbodiimide (50% inhibition at about 1 μM and 0.3 mM, respectively), strict ATP-specificity, and an acidic pH optimum, this enzyme shows the typical characteristics of the common type of H+-ATPase in the plasmalemma of higher plants and fungi. These results undermine the hypothesis of a wider distribution of a special (high salt) type of plasmalemma ATPase as found in the marine alga Acetabularia.