Purification and properties of a plasma membrane H⁺-ATPase from the extremely acidophilic alga Dunaliella acidophila

This paper describes partial purification and characterization of a vanadate-sensitive H⁺-ATPase from plasma membranes of Dunaliella acidophila, an extremely acidophilic unicellular alga (I. Sekler, H.U. Gläser, U. Pick [1991] J Membr Biol 121: 51-57). Purification is based on the insolubility in and stability of the enzyme in Triton X-100. The purified enzyme is highly enriched in a polypeptide of molecular mass 100 kD, which cross-reacts with antibodies against the plant plasma membrane H⁺-ATPase. Upon reconstitution into proteoliposomes, the enzyme catalyzes an ATP-dependent electrogenic H⁺ uptake. ATP hydrolysis is stimulated by lipids, is inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide, erythrosine, and mercurials, and shows a sharp optimum at pH 6. Unusual properties of this enzyme, by comparison with plant plasma membrane H⁺-ATPases, are a higher affinity for ATP (K_m = 40 μM) and a larger stimulation by K⁺, which interacts with the enzyme from its cytoplasmic side. Comparative studies with cross-reacting antibodies, prepared against different domains of the plant H⁺-ATPase, suggest that the central hydrophilic domain containing the catalytic site is more conserved than the C- and N-terminal ends. The high abundance and stability of the plasma membrane H⁺-ATPase from D. acidophila make it an attractive model system for studies of the structure-function relations and regulation of this crucial enzyme.