Ter the cells had been cultured as described above, the cells were chilled on ice and then centrifuged at 10,0006g for 5 min at 4uC. The pellets were treated with a AZ876 solution containing 20 mM Tris-HCl, 50 mM MgSO4, 4 mM EDTA, and 50 methanol at pH 7.4 for 30 min at 70uC [19] and then 1379592 were centrifuged at 10,0006g for 5 min at 4uC. The ATP content of the supernatant was measured using a luminometer (Turner Designs, Inc.) as described previously [20]. Luciferase and standard ATP were purchased from Sigma Chemical Co. The measurement was repeated three times using separate culture, and the mean value and the standard deviation were calculated.Measurement of Intracellular pH (pHi)The pHi was determined by the distribution of salicylic acids between outside and inside the cells, as described previously [11,21]. After the cells had been adapted in EG medium at pH 5.5 for 4 h, the harvested cells were suspended in EG medium at pH 5.5 or 2.5 at approximately 16109 cells per ml, and [14C]salicylic acids (10 mM; 0.2 mCi/ml) was added. After incubation at 37uC for 15 min, 1 ml of the medium was centrifuged at 10,0006g for 5 min through an oil mixture (laurylbromide and liquid paraffin). The radioactivity of the supernatant and the pellet were measured to obtain the indicator concentrations outside and inside cells, respectively. The amount of protein in the pellet was measured, and the radioactivity of the pellet was divided by the internal water content calculated from the protein content of the pellet. The pHi was calculated by the following equation: pHi log ?? in = out 10pKa z10pHout {10pKa ,Measurement of ATPase ActivityThe ATP hydrolysis activity in the membranes was determined by the amount of inorganic phosphate (Pi) released from ATP, as previously described [24,25]. After 5 mg of the membranes had been added to the reaction 125-65-5 supplier buffer containing 300 ml of buffer solution (200 mM Tris-maleate and 5 mM MgCl2 at pH 9.0) and 270 ml of water, the mixture was incubated at 37uC for 5 min, and then 30 ml of 100 mM ATP was added. After incubation at 37uC for 20 min, the reaction was stopped by the addition 300 ml of cold 15 trichloroacetic acid and immediately chilled on an ice bath. The resulting mixture was centrifuged at 3,0006g for 10 min at 4uC, 24195657 and 800 ml of the supernatant was mixed with 1.87 ml of the reagent (10 ml of 5 N H2SO4, 10 ml of 2.5 ammonium molybdate, 10 ml of the solution containing 3 NaHSO3 and 1 p-methylaminophenol sulfate, and 40 ml of H2O). The mixture was incubated at 18uC for 10 min, and the absorbance at 660 nm was measured. K2HPO4 (10 mM) was used as aRespiration and F1Fo-ATPase Enhance AR in E. colistandard phosphate. One unit of ATPase activity was defined as the activity to release 1 mmol of Pi from ATP for 1 min. The measurement was repeated three times using separate culture, and the mean value and the standard deviation were calculated.Other MethodsTransduction with P1kc was performed as described previously [31]. Protein was measured as described previously [32], and bovine serum albumin was used as a standard.Measurement of Proton Pumping ActivityThe proton pumping activity of the membranes was determined using the quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) as described previously [26,27]. The membranes were suspended with the buffer containing 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) and 10 mM MgCl2 (pH 7.5) at 60 mg/ml of membrane protein. After 1 ml of 0.1 ACMA was added to the react.Ter the cells had been cultured as described above, the cells were chilled on ice and then centrifuged at 10,0006g for 5 min at 4uC. The pellets were treated with a solution containing 20 mM Tris-HCl, 50 mM MgSO4, 4 mM EDTA, and 50 methanol at pH 7.4 for 30 min at 70uC [19] and then 1379592 were centrifuged at 10,0006g for 5 min at 4uC. The ATP content of the supernatant was measured using a luminometer (Turner Designs, Inc.) as described previously [20]. Luciferase and standard ATP were purchased from Sigma Chemical Co. The measurement was repeated three times using separate culture, and the mean value and the standard deviation were calculated.Measurement of Intracellular pH (pHi)The pHi was determined by the distribution of salicylic acids between outside and inside the cells, as described previously [11,21]. After the cells had been adapted in EG medium at pH 5.5 for 4 h, the harvested cells were suspended in EG medium at pH 5.5 or 2.5 at approximately 16109 cells per ml, and [14C]salicylic acids (10 mM; 0.2 mCi/ml) was added. After incubation at 37uC for 15 min, 1 ml of the medium was centrifuged at 10,0006g for 5 min through an oil mixture (laurylbromide and liquid paraffin). The radioactivity of the supernatant and the pellet were measured to obtain the indicator concentrations outside and inside cells, respectively. The amount of protein in the pellet was measured, and the radioactivity of the pellet was divided by the internal water content calculated from the protein content of the pellet. The pHi was calculated by the following equation: pHi log ?? in = out 10pKa z10pHout {10pKa ,Measurement of ATPase ActivityThe ATP hydrolysis activity in the membranes was determined by the amount of inorganic phosphate (Pi) released from ATP, as previously described [24,25]. After 5 mg of the membranes had been added to the reaction buffer containing 300 ml of buffer solution (200 mM Tris-maleate and 5 mM MgCl2 at pH 9.0) and 270 ml of water, the mixture was incubated at 37uC for 5 min, and then 30 ml of 100 mM ATP was added. After incubation at 37uC for 20 min, the reaction was stopped by the addition 300 ml of cold 15 trichloroacetic acid and immediately chilled on an ice bath. The resulting mixture was centrifuged at 3,0006g for 10 min at 4uC, 24195657 and 800 ml of the supernatant was mixed with 1.87 ml of the reagent (10 ml of 5 N H2SO4, 10 ml of 2.5 ammonium molybdate, 10 ml of the solution containing 3 NaHSO3 and 1 p-methylaminophenol sulfate, and 40 ml of H2O). The mixture was incubated at 18uC for 10 min, and the absorbance at 660 nm was measured. K2HPO4 (10 mM) was used as aRespiration and F1Fo-ATPase Enhance AR in E. colistandard phosphate. One unit of ATPase activity was defined as the activity to release 1 mmol of Pi from ATP for 1 min. The measurement was repeated three times using separate culture, and the mean value and the standard deviation were calculated.Other MethodsTransduction with P1kc was performed as described previously [31]. Protein was measured as described previously [32], and bovine serum albumin was used as a standard.Measurement of Proton Pumping ActivityThe proton pumping activity of the membranes was determined using the quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) as described previously [26,27]. The membranes were suspended with the buffer containing 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) and 10 mM MgCl2 (pH 7.5) at 60 mg/ml of membrane protein. After 1 ml of 0.1 ACMA was added to the react.
