A Membrane-Bound ATPase from Halobacterium halobium; Purification and Characterization1

Abstract

An ATPase was newly identified on the inner face of the plasma membrane of the extremely halophilic archaebacterium Halobacterium halobium. The enzyme was released into an alkaline EDTA solution and purified by several chromatographic steps in the presence of sulfate at 1 M or over. The molecular weight of the native enzyme was around 320,000; it is most likely composed of two pairs (alpha 2 beta 2) of 86,000 (alpha) and 64,000 (beta) subunits. The enzyme hydrolyzed ATP and other nucleoside triphosphates but neither ADP nor AMP. The enzyme required divalent cations, among which Mn2+ was most effective (Mg2+ activated 35% of Mn2+). The ATPase activity was optimum at pH between 5.5 and 6, particularly in a nearly saturated Na2SO4 (or Na2SO3) solution, while it was very low in a chloride salt solution even at 4 M at any pH. The Km value for ATP was 1.4 mM and the K1 value for ADP (competitive to ATP) was 0.08 mM. Neither azide (a specific inhibitor for F0F1-and F1-ATPase) nor vanadate (for E1E2-ATPase) inhibited the enzyme. The ATPase was stable at high concentrations of sulfate. At low concentrations of salts, or at low temperatures even in high NaCl concentrations, the enzyme was inactivated. Although the ATPase isolated here from halobacterial membrane has such unusual characteristics, it is the most probable candidate for the (catalytic part of) halobacterial ATP synthase, which differs from F0F1-ATPase/synthase (Mukohata et al. (1986) J. Biochem. 99, 1-8; Mukohata and Yoshida (1987) J. Biochem. 101, 311-318).

Evolution of the vacuolar H+-ATPase: implications for the origin of eukaryotes.

“…This phylogenetic scheme also explains the presence of similar ferredoxins and vacuolar (gas vacuole) proteins in halobacteria and cyanobacteria, as well as the similarity between the ferredoxins of Sulfolobus and eukaryotes (29,30). On the other hand, the membrane H+-ATPases of two species of Halobacterium appear to be similar to the V-ATPases of Sulfolobus and eukaryotes, based on inhibitor profiles and immunological cross-reactivity (32)(33)(34) …”

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

“…In a variety of haloarchaea, including H. volcanii, internally-oriented membrane-bound ATPases have been described and extensively characterized in terms of kinetics, cofactor requirements and inhibitor sensitivities [4,10,22,33]. The enzymatic hydrolysis of externally added ATP can be measured colorimetrically in an assay which reflects the concentration of liberated orthophosphate [26].…”

V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

“…V o V 1 -ATPases are also found in the plasma membranes of most archea (7-9) and some kinds of eubacteria (10 -12). Several studies indicate that the physiological role of V o V 1 -ATPases of some archea and the thermophilic eubacterium Thermus thermophilus is ATP synthesis coupled to proton flux across the plasma membranes (7,9,(13)(14)(15). Precise understanding of V o V 1 -ATPases would allow the comparison to F o F 1 -ATPases and the elucidation of the common essential mechanism for the coupling of proton translocation across a membrane with ATP formation.…”

“…They are responsible for vacuolar acidification, which plays an important role in a number of cellular processes (1). V o V 1 -ATPases are also found in the plasma membranes of most archea (7)(8)(9) and some kinds of eubacteria (10 -12). Several studies indicate that the physiological role of V o V 1 -ATPases of some archea and the thermophilic eubacterium Thermus thermophilus is ATP synthesis coupled to proton flux across the plasma membranes (7,9,(13)(14)(15).…”