Using KpnI/ SacI. The resulting fragment was introduced into the plasmid pBluescript II SK (Agilent Technologies, Santa Clara, USA) for mutagenesis-PCR. After verification of the introduced mutation via sequencing, the resulting plasmid was cut using KpnI/SacI, whereas the desired fragment was ligated into the corresponding template plasmid. Mutations were again verified by sequencing. The resulting plasmid was named pFH4 (aP281C, cK109C, cI279C). The plasmid pKG11 (aE284C, cK109C, cL276C) was kindly provided by K. Gumbiowski (University of Osnabruck, ?Germany). An additional mutation (cA213C) was introduced in pKG11 as described above, resulting in the plasmid pGH19.of gamma cross-links were determined from the changes of intensity of the c-bands in the oxidized state in comparison to the reduced state.ImmunoblotFor immunoblotting proteins were transferred to a PVDFmembrane after SDS-PAGE, applying 500 mA for 1 h in a blotting tank (Carl Roth, Karlsruhe, Germany). Polyclonal primary mouse and Title Loaded From File rabbit 18325633 antibodies against EF1-a and EF1-c were used at dilutions of 1:200,000 and 1:400,000, respectively. Peroxidase-conjugated secondary monoclonal antibodies against primary antibodies (diluted 1:10,000) and the Lumi-LightPlus Western Blotting-Kit (Roche, Mannheim, Germany) were used for visualization by chemiluminescence. Films were developed on a Konica SRX-101A (Konica, Munchen, Deutschland). ?Expression and PurificationPlasmids for EF1 were transformed into E. coli DK8 cells [20] and grown overnight on minimal medium. Purification of EF1 was performed as described before [17]. Membranes were isolated and purified essentially according to [21]. EF1 was extracted by treatment with 1 mM EDTA in the presence of 0.5 mM DTT and applied to an anion-exchange column (Tosoh Fractogel TSKDEAE 650(S), Toyo Soda, Darmstadt, Germany) equilibrated with Title Loaded From File buffer A (50 mM Tris/H2SO4, 10 (v/v) methanol, pH 7.4). EF1 was eluted from the column using a stepwise gradient of 0.5 M Na2SO4 in buffer A. Enzyme-containing fractions (eluted at about 150 mM Na2SO4) were combined, stabilized with 0.1 mM Mg2+-ATP, and reduced with 1 mM DTT. Aliquots were further purified by size exclusion chromatography, using PD-10 columns (Amersham Pharmacia Biotech), which were equilibrated with buffer B (20 mM MOPS, 5 mM MgCl2, 50 mM KCl, 10 (v/v) glycerol, pH 7.5). For the rotation assay an equimolar amount of biotin-PEAC5-maleimide was used for the biotinylation of mutants (20 min incubation at room temperature) at residue cC109 and cA213C. The reaction was stopped by adding 2 mM of N-acetylcysteine. Then samples were applied to a Ni-NTA affinity chromatography column (5 mg of protein/ml Ni-NTAagarose) equilibrated with the same buffer. After washing with buffer B (containing 20 mM imidazole), pure EF1 was eluted with 200 mM imidazole in buffer B.ATPase hydrolysis activityATPase activity was determined colorimetrically as described previously [17,23]. Proteins (10 mg/ml) in 50 mM Tris-HCl, pH 8.0, 3 mM MgCl2, and 10 mM sodium-ATP were incubated for 5 min at 37uC, before the reaction was stopped by the addition of trichloroacetic acid, and the released Pi was determined colorimetrically. The activity of reduced and oxidized samples was determined after exposure overnight at room temperature to 20 mM DTT and 200 mM DTNB, respectively. For control experiments re-reduction was achieved by incubating the oxidized samples with 20 mM DTT for 2 hours at room temperature.Rotation Assay and.Using KpnI/ SacI. The resulting fragment was introduced into the plasmid pBluescript II SK (Agilent Technologies, Santa Clara, USA) for mutagenesis-PCR. After verification of the introduced mutation via sequencing, the resulting plasmid was cut using KpnI/SacI, whereas the desired fragment was ligated into the corresponding template plasmid. Mutations were again verified by sequencing. The resulting plasmid was named pFH4 (aP281C, cK109C, cI279C). The plasmid pKG11 (aE284C, cK109C, cL276C) was kindly provided by K. Gumbiowski (University of Osnabruck, ?Germany). An additional mutation (cA213C) was introduced in pKG11 as described above, resulting in the plasmid pGH19.of gamma cross-links were determined from the changes of intensity of the c-bands in the oxidized state in comparison to the reduced state.ImmunoblotFor immunoblotting proteins were transferred to a PVDFmembrane after SDS-PAGE, applying 500 mA for 1 h in a blotting tank (Carl Roth, Karlsruhe, Germany). Polyclonal primary mouse and rabbit 18325633 antibodies against EF1-a and EF1-c were used at dilutions of 1:200,000 and 1:400,000, respectively. Peroxidase-conjugated secondary monoclonal antibodies against primary antibodies (diluted 1:10,000) and the Lumi-LightPlus Western Blotting-Kit (Roche, Mannheim, Germany) were used for visualization by chemiluminescence. Films were developed on a Konica SRX-101A (Konica, Munchen, Deutschland). ?Expression and PurificationPlasmids for EF1 were transformed into E. coli DK8 cells [20] and grown overnight on minimal medium. Purification of EF1 was performed as described before [17]. Membranes were isolated and purified essentially according to [21]. EF1 was extracted by treatment with 1 mM EDTA in the presence of 0.5 mM DTT and applied to an anion-exchange column (Tosoh Fractogel TSKDEAE 650(S), Toyo Soda, Darmstadt, Germany) equilibrated with buffer A (50 mM Tris/H2SO4, 10 (v/v) methanol, pH 7.4). EF1 was eluted from the column using a stepwise gradient of 0.5 M Na2SO4 in buffer A. Enzyme-containing fractions (eluted at about 150 mM Na2SO4) were combined, stabilized with 0.1 mM Mg2+-ATP, and reduced with 1 mM DTT. Aliquots were further purified by size exclusion chromatography, using PD-10 columns (Amersham Pharmacia Biotech), which were equilibrated with buffer B (20 mM MOPS, 5 mM MgCl2, 50 mM KCl, 10 (v/v) glycerol, pH 7.5). For the rotation assay an equimolar amount of biotin-PEAC5-maleimide was used for the biotinylation of mutants (20 min incubation at room temperature) at residue cC109 and cA213C. The reaction was stopped by adding 2 mM of N-acetylcysteine. Then samples were applied to a Ni-NTA affinity chromatography column (5 mg of protein/ml Ni-NTAagarose) equilibrated with the same buffer. After washing with buffer B (containing 20 mM imidazole), pure EF1 was eluted with 200 mM imidazole in buffer B.ATPase hydrolysis activityATPase activity was determined colorimetrically as described previously [17,23]. Proteins (10 mg/ml) in 50 mM Tris-HCl, pH 8.0, 3 mM MgCl2, and 10 mM sodium-ATP were incubated for 5 min at 37uC, before the reaction was stopped by the addition of trichloroacetic acid, and the released Pi was determined colorimetrically. The activity of reduced and oxidized samples was determined after exposure overnight at room temperature to 20 mM DTT and 200 mM DTNB, respectively. For control experiments re-reduction was achieved by incubating the oxidized samples with 20 mM DTT for 2 hours at room temperature.Rotation Assay and.
