Abstract
The membrane-bound detergent-activated ATPase from Halobacterium saccharovorum was purified at a physiological salt concentration (4 M NaCl) in the presence of nonionic detergents. The preparation contains putative subunits of 110, 71, 31, 22, and 14 kDa. The enzyme activity required high salt concentration but was not dependent on any one specific monovalent cation or any anion. The hydrolysis of ATP was nonlinear with time; the data are consistent with a kinetic model where the enzyme is irreversibly converted from an initial into a final stable form during the first few minutes of the reaction. The model thus contains a rate constant (k) for the transition and hydrolytic rates, v1 and v2, for the two forms of the enzyme. We found that this hysteretic behavior was influenced differently by various conditions and inhibitors. The constant k was smaller with Mn2+ than with Mg2+ as the divalent cation, showed negative temperature dependence, and a distinct pH optimum between 7.5 and 8.5. Thiols decreased k, but nitrate, a specific inhibitor of archaebacterial ATPases, increased it. ADP showed competitive inhibition against both the initial and the final form of the enzyme. Nitrate reversibly inhibited only the latter and in a manner dependent on whether Mn2+ or Mg2+ was used. The kinetic data suggest that all agents tested, with effects on the hydrolytic activity, seem to act at or near the catalytic site of the enzyme.
Highlights
Themembrane-bound detergent-activated ATPase Konishi et al, 1987; Nanba and Mukohata, 1987) excludes a from Halobacterium saccharovorum was purified ata close relationship to FIFOand E1E2ATPases, one physiological salt concentration (4M NaCl) in the pres- report suggests that themorphology (Lubben et al, 1987) and ence of nonionic detergents
The model contairnaste constant (k)for the transition and hydrolytircates, V I and v2,for thetwo forms of the enzymeW. e found that this hysteretic behavior was influenced differently by the question of whetherarchaebacterialATPases form an entirely new family or constitute a subgroup of a known ATPase category is of interest
ATPase revealed that the two larger subhits were different in the H. halobium-enzyme; we found 86- and 64-kDa components, as reported by Nanba and Mukohata(1987)
Summary
SDS-Polyacrylamide Electrophoresis-To remove the salt, the purified ATPase wasprecipitatedby adding solid polyethylene glyctool. Decylmaltoside a final concentrationof 30%.After centrifugation at 44,000 X g for Nonidet P-40. System used is described by Naville (1971). The assaymixturewas incubated at 36 “C,and the enzyme reaction was initiated by the addition of ATP Tion).The liberatedPiwas measured accordingto the malachite green procedure (Lanzettaet al.,1979).A 0.1-ml aliquotof the assay mixture. The concentrations of Pi. Purfaifcitcoartion Yield were determined using a calibration curve. Protein was determined with the bicinchoninic acid procedure, as described by Smith et al (1986). Kinetic analysis was on an IBM-AT-type computer using a spreadsheet program
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