Characterization of soluble and membrane-bound forms of a vanadate-sensitive ATPase from plasma membranes of the halotolerant alga Dunaliella salina

A plasma-membrane preparation, obtained by an osmotic lysis of cells of the halotolerant alga Dunaliella salina (Sheffer and Avron, 1986) was further purified on glycerol gradients. Two fractions of a vanadate-sensitive ATPase activity were resolved on the gradients: a soluble and a membrane-bound fraction. The enzymes exhibit identical sensitivities to vanadate, dicyclohexylcarbodiimide, diethylstilbestrol, SH reagents and phloridzin but are insensitive to molybdate, nitrate, cyanide, azide and quercetin. The two ATPase activities also have an identical K_m value for Mg-ATP (0.9 mM), optimal activity at pH 7, are stimulated by 200 mM KCl or NaCl but inhibited by high salt concentrations. Antibodies against yeast plasma membrane H⁺-ATPase cross-react with 92 kDa or with 60 kDa polypeptides in the plasma membrane and soluble ATPase fractions, respectively. Treatment of the purified plasma membranes with trypsin releases a soluble vanadate-sensitive ATPase from the membranes, and the release is protectable by ATP. It is suggested that the soluble ATPase, which is resolved on the glycerol gradients, is a proteolytic product of the plasma membrane ATPase which is a vanadate-sensitive H⁺-ATPase.