A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase

Abstract

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.

Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase

“…Representative traces are shown. Similar results have been obtained using a luciferase assay (Galkin et al 2006)…”

“…ATP synthesis by membranes was measured with pH-indicator phenol red at 37°C following scalar proton consumption at 557 nm. Membranes were prepared as described in (Galkin et al 2006; Ishmukhametov et al 2008). Reactions were initiated by adding 20 mM succinate to a 2 ml cuvette containing 200 μg membrane protein in 10 mM phosphate (potassium salts) at pH 8.0, 100 mM KCl, 5 mM MgCl 2 , 0,1 mM EDTA, 0.7 mM ADP.…”

ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase

“…F 1 F o -ATPase was purified from E. coli strain DK8 harboring plasmid pFV2, as previously described. Growth of cultures, preparation of membrane vesicles, purification of F 1 F o , and reconstitution into liposomes were also carried out as described previously [20,22]. Succinate minimal medium was made from minimal medium A [23], supplemented with 0.2% succinic acid (from a stock adjusted to pH 6.4 with KOH), and 0.2 mM L-valine, L-leucine and L-isoleucine.…”

“…For pH dependence ACMA-fluorescence quenching, the buffer was 5mM Tris/ 5 mM maleate, supplemented with 15 μM valinomycin. ATP synthesis was carried out using a luciferase assay as described previously [22]. Prior to measurement of ATP synthesis, membrane vesicles were resuspended in 1 ml of buffer (200 mM Tricine/HCl, pH 7.8, 100 mM KCl, 5 mM MgCl2, and 2.5% glycerol), passed through a 10-ml Sephadex G-50 column, equilibrated with the same buffer.…”

Functional Incorporation of Chimeric b Subunits into F 1 F o ATP Synthase

“…The uncF (b subunit) genes in these plasmids retained the designed chimeric b subunit gene and carried no other mutations. A recent publication suggested that a cold-stabilized form of the complex had a significant lag in ATP hydrolysis activity (12). Therefore, ATP hydrolysis was carefully reexamined under conditions that would account for a potential lag, and results essentially identical to those shown in Table 3 were obtained.…”