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Effect of inhibitors on the EFP reaction

Table 1 Effect of inhibitors on the EFP reaction

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Fig.l. Time course of ATP formation in the EFP reaction in presence and absence of tentoxin. Abscissa: time after application of the electric field. Ordinate: amount of ATP formed. 0.5 ml of a reaction mixture containing 200 mM sucrose, 0.5 mM MgCh, 2.5 mM 32P;, 0.5 mM ADP and 10 mM Na—HEPES and chloroplasts (75 pg (4) or 166 pg (¢) at pH 8.0 and 21°C were placed in a cuvette between two stainless steel electrodes 0.3 cm apart. The mixture was exposed to 1200 V/cm AC field for 50 ms and automatically quenched by rapid injection of 0.5 ml of 2 M perchloric acid. When indicated 2.2 »M tentoxin (co) was present in the assay medium and 2 min incubation time allowed before the exposure to the.electric pulse. 1 ymol of cold ATP was then added to the quenched reaction mixture and the sample analyzed as described in [11]. Every point represents the mean value of three independent measurements on the same chloroplasts. Insert: 0.8 m of a reaction mixture containing 200 mM sucrose, 0.5 mM MgCh, 2.5 mM P;, 0.5 mM ADP (purified of residual ATP by pretreatment with glucose and hexokinase), 0.42 mM D-luciferin, 50 A (0.5 mg protein/ml) luciferase and 150 pg of chlorophyll at pH 8.0 and 21°C were exposed to a 30 ms of 1200 V/cm AC pulse. (A) control, (B) in presence of 7.7 »M tentoxin. The initial spike is an electric field-induced artefact unrelated to ATP synthesis being much faster than the assay’s response time.
Fig.l. Time course of ATP formation in the EFP reaction in presence and absence of tentoxin. Abscissa: time after application of the electric field. Ordinate: amount of ATP formed. 0.5 ml of a reaction mixture containing 200 mM sucrose, 0.5 mM MgCh, 2.5 mM 32P;, 0.5 mM ADP and 10 mM Na—HEPES and chloroplasts (75 pg (4) or 166 pg (¢) at pH 8.0 and 21°C were placed in a cuvette between two stainless steel electrodes 0.3 cm apart. The mixture was exposed to 1200 V/cm AC field for 50 ms and automatically quenched by rapid injection of 0.5 ml of 2 M perchloric acid. When indicated 2.2 »M tentoxin (co) was present in the assay medium and 2 min incubation time allowed before the exposure to the.electric pulse. 1 ymol of cold ATP was then added to the quenched reaction mixture and the sample analyzed as described in [11]. Every point represents the mean value of three independent measurements on the same chloroplasts. Insert: 0.8 m of a reaction mixture containing 200 mM sucrose, 0.5 mM MgCh, 2.5 mM P;, 0.5 mM ADP (purified of residual ATP by pretreatment with glucose and hexokinase), 0.42 mM D-luciferin, 50 A (0.5 mg protein/ml) luciferase and 150 pg of chlorophyll at pH 8.0 and 21°C were exposed to a 30 ms of 1200 V/cm AC pulse. (A) control, (B) in presence of 7.7 »M tentoxin. The initial spike is an electric field-induced artefact unrelated to ATP synthesis being much faster than the assay’s response time.
Fig.2. The EFP reaction in NaBr treated particles. Chlo- roplasts (52 pg of chlorophyll/ml of either NaBr treated or non-treated particles) were added to a reaction mix- ture containing 200 mM sucrose, 0.5 mM MgCl», 5 mM P;, 50 mM KCl, 10 mM Na—HEPES, 0.225 mM ADP (purified of ATP by pretreatment with glucose and hexokinase), 0.05 mM phenazine methosulfate and 0.06 mM PI!,P5 diadenosine pentaphosphate, 0.5 mM _ lu- ciferin and 40 A luciferase (0.5 mg protein/ml) at pH 8.0 and 21°C. The mixture was placed in a cuvette between two gold electrodes as described in [8] and exposed to a 1800 V/cm DC field for 1.3 ms. An exposure to light for I s followed. NaBr treated particles were prepared as described in [13].
Fig.2. The EFP reaction in NaBr treated particles. Chlo- roplasts (52 pg of chlorophyll/ml of either NaBr treated or non-treated particles) were added to a reaction mix- ture containing 200 mM sucrose, 0.5 mM MgCl», 5 mM P;, 50 mM KCl, 10 mM Na—HEPES, 0.225 mM ADP (purified of ATP by pretreatment with glucose and hexokinase), 0.05 mM phenazine methosulfate and 0.06 mM PI!,P5 diadenosine pentaphosphate, 0.5 mM _ lu- ciferin and 40 A luciferase (0.5 mg protein/ml) at pH 8.0 and 21°C. The mixture was placed in a cuvette between two gold electrodes as described in [8] and exposed to a 1800 V/cm DC field for 1.3 ms. An exposure to light for I s followed. NaBr treated particles were prepared as described in [13].
Fig.3. The effect of tentoxin on the EFP reaction and on photophosphorylation. 0.8 ml of reaction mixture con- taining the same as in the insert to fig.1 but also with 0.06 mM phenazine methosulfate were exposed to a 2-s light pulse and then to a 1200 V/cm AC field for 30 ms. Two minutes preincubation time with 7.7 4M tentoxin were allowed in experiment B.
Fig.3. The effect of tentoxin on the EFP reaction and on photophosphorylation. 0.8 ml of reaction mixture con- taining the same as in the insert to fig.1 but also with 0.06 mM phenazine methosulfate were exposed to a 2-s light pulse and then to a 1200 V/cm AC field for 30 ms. Two minutes preincubation time with 7.7 4M tentoxin were allowed in experiment B.
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BiophysicsChemistryATP synthaseBiochemistry and cell biologyATP synthesis
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