Purification and Properties of a Plasma Membrane H+-ATPase from the Extremely Acidophilic Alga Dunaliella acidophila

Abstract

This paper describes partia1 purification and characterization of a vanadate-sensitive H+-ATPase from plasma membranes of Dunaliella acidophila, an extremely acidophilic unicellular alga (I. Sekler, H.U. Glaser, 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. l h e purified enzyme is highly enriched in a polypeptide of molecular mas 100 kD, which cross-reacts with antibodies against the plant plasma membrane H+-ATPase. Upon reconstitution into proteoliposomes, the enzyme catalyzes an ATPdependent 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, = 40 ~IM) 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 Nterminal ends. l h e high abundance and stability of the plasma membrane H+-AlPase from D. acidophila make it an attractive model system for studies of the structure-function relations and regulation of this crucial enzyme.

Sterol Modulation of the Plasma Membrane H+-ATPase Activity from Corn Roots Reconstituted into Soybean Lipids

“…In the present study sterol modulation of the PM H+-ATPase activity from corn roots was assessed after reconstitution of the partially purified enzyme into proteoliposomes by means of either freeze-thaw sonication or octylglucoside dilution. The first procedure, which has been largely used for transport studies, was previously applied to the reconstitution of the PM H'-ATPase from corn roots (St. MartyFleurence et al, 1988;Gibrat et al, 1990) and from other plant materials such as tomato roots (Anthon and Spanswick, 1986), oat roots (Vara and Serrano, 1982;, or the alga Dunaliella acidophila (Sekler and Pick, 1993). The second method first implies a co-solubilization of the enzyme and lipids in a detergent (octylglucoside or sodium cholate or deoxycholate or Zwittergent 3-14).…”

Primary structure of the plasma membrane H+-ATPase from the halotolerant alga Dunaliella bioculata

“…port systems in Dunaliella deserve special interest. P-type ATPases in the plasma membrane of several Dunaliella species have been characterized biochemically and are believed to be involved in cytosolic Na + homeostasis [8,23,25,31,38,42]. In this investigation we have isolated the gene for the primary ion pump in the plasma membrane of D. bioculata.…”

Life at acidic pH imposes an increased energetic cost for a eukaryotic acidophile

“…This indicates that H + can be taken up into an intracellular compartment and released without the use of a plasma-membrane-bound H + transporter. Dunaliella acidophila possesses a wellcharacterized P-type H + ATPase to maintain cytosolic neutrality (Sekler et al, 1991). The H + -ATPase transcripts increase with decreasing extracellular pH (Weiss and Pick, 1996).…”