PMC

Effect of AMP-PNP on ATP and GTP synthesis by authentic TF_oF₁. A, analytical conditions were the same as in , except that IMV was prepared from thermophilic Bacillus PS3 cells. The reactions were carried out at 45 °C (A) or 58 °C (B). C, effect of AMP-PNP on the specificity of substrate nucleotide of authentic TF_oF₁. mU, milliunits.

Effect of AMP-PNP on ATP and GTP synthesis. Effect of AMP-PNP on ATP synthesis activity (A) and GTP synthesis activities (B) of WT- and ϵΔc-TF_oF₁. The analytical conditions were the same as described in the legend to except that AMP-PNP (0, 0.1, 0.3, 1, 2 mm and in some cases, 0.05 mm) was supplemented to the reaction mixture. C, effect of AMP-PNP on the specificity of substrate nucleotide of WT-TF_oF₁ and ϵΔc-TF_oF₁. Ratios of GTP to ATP synthesis activity were plotted against AMP-PNP concentration. D, effect of other NTPs (GTP, UTP, and CTP) on AMP-PNP-induced activation of ATP synthesis. ATP synthesis was monitored at 37 °C with luciferase. G/U/CTP, GTP+UTP+CTP (each 0.3 mm); AMP-PNP, 0.3 mm AMP-PNP; mU, milliunits. Experimental details are as described under “Experimental Procedures.”

NTP synthesis in the presence of NDP-regenerating system. A, time courses of NTP-synthesis at 45 °C by IMV prepared from E. coli cells expressing WT- and ϵΔc-TF_oF₁. The reaction was initiated by addition of 5 mm sodium succinate into the reaction mixture containing 1 mm NDP, 25 mm potassium phosphate, IMV (72 μg proteins/ml), and the NDP-regenerating system (hexokinase, glucose-6-phosphate dehydrogenase, glucose, and NADP). NTP synthesis was monitored by NADPH production (A₃₄₀). Dotted lines show the time courses in the presence of 1 μg/ml FCCP. B, synthesis activities at 0.3 mm NDP (left panel) and 1 mm NDP (right panel). Experimental details are as described under “Experimental Procedures.”

Nucleotide specificity in hydrolysis and proton-pumping activities of WT-TF_oF₁ and ϵΔc-TF_oF₁. A, activities of ATP hydrolysis and GTP hydrolysis of WT-TF_oF₁ and ϵΔc-TF_oF₁ at 45 °C. B, ATP-dependent activation of ATPase of WT-TF_oF₁ and the up-to-down conformational transition of ϵ probed by FRET change. Closed circles, ratios of ATPase activities (WT-TF_oF₁/ϵΔc-TF_oF₁) obtained from the data in A; Open circles, FRET represented by donor fluorescence intensity. High donor fluorescence reflected the down-state ϵ. Lines represent the best fit of the data points. C, activities of UTP hydrolysis and CTP hydrolysis of WT-TF_oF₁ and ϵΔc-TF_oF₁ at 45 °C. Note the different y-scale in A and C. D, comparison of the NTPase activities at 1 mm NTP. E, proton-pumping activity of WT-TF_oF₁ and ϵΔc-TF_oF₁ driven by 1 mm NTP. The proton pumping was monitored by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence (ACMA) at 42 °C (10% quenching is shown in the figure). The reaction was initiated by adding 1 mm NTP and terminated by 1 μg/ml FCCP.

Analysis of NTP synthesis with anion-exchange HPLC. A, elution profiles of NTPs and NDPs. NT(D)P (8 nmol) in 10 mm CDTA was applied to the column. B, NTP synthesis by IMV prepared from E. coli cells expressing WT-TF_oF₁ (left panels) and ϵΔc-TF_oF₁ (right panels). The reaction mixture contained 0.3 mm NDP, 50 mm potassium phosphate, and IMV (72 μg proteins/ml). The reaction was initiated by addition of 5 mm sodium succinate. After incubation for 500 s at 45 °C, the reaction was terminated by addition of 10 mm CDTA, and the nucleotides in the reaction mixtures were analyzed with HPLC (red trace). In the control samples, 10 mm CDTA (black traces) or 1 μg/ml FCCP (green traces) were further included in the mixtures from the start of the reaction. The amount of synthesized NTP was determined from the net increase of the peak areas (red and black). C, comparison of NTP synthesis activities of WT-TF_oF₁ and ϵΔc-TF_oF₁. Detailed experimental conditions are as described under “Experimental Procedures.” abs, absorbance.