Uncouplers of Spinach Chloroplast Photosynthetic Phosphorylation.

Abstract

reduction in the absence of simultaneous phosphorylation will be stimulated by the reagent, at least as much as by phosphorylating reagents. With these criteria nowv available, we have investigated two compounds that are effective as un-couplers of oxidative phosphorylation by mitochon-dria: dinitrophenol (11) and pentachlorophenol (15). Treatment of chloroplasts by dilution in sodium chloride at pH 6.0, which was previously found to activate them to a faster rate of ferricyanide reduction (12) is shown here to fulfill the criteria for uncoup-ling of the plastids. Arsenate was previously shown to replace phosphate in the stimulation of the Hill reaction (5) ; in the present work we show that arse-nate fulfills one of the criteria for an uncoupling re-agent. Finally we have found that ammonium ions are very effective uncouplers of chloroplast phosphory-lation. AIATERIALS AND METHODS Chloroplasts were prepared from grocery spinach as described previously (6). Leaves were ground in 0.4 Ml sucrose, 0.01 MI NaCl buffered at pH 7.8 with 0.05 WI TRIS. The chloroplasts were washed once in the same medium. Chlorophyll was determined by the method of Arnon (1). Incorporation of P32 into ATP was determined by adsorbing the ATP32 on charcoal, washing the charcoal, then removing the labelled phosphate by hydrolysis (10). Two kinds of reaction mixtures were employed in the present study. The first (control) contained 40 micromoles (MMAI) of TRIS buffer at pH 7.8, 70 ,uM of NaCl, 10 MMI of MIgCl,, 15 juAl of phosphate at pH 7.8, 2.0 j24 of potassium ferricyanicle, 0.1 MuM of ATP and chloroplasts containing 0.030 mg of chlorophyll in a total volume of 3.0 ml. The optical density of the entire reaction mixture w,as determined at 400 m,u in a Beckman spectrophotometer; the cuvette was exposed to 5000 ft-c of white light from a tungsten lamp for 2 minutes, and then the (lecreased optical density wvas measured again. A 10 cm path of water was used as a heat shield, and control experiments had shown no changes with boiled chloroplasts. The reaction mixture proper contained the same components as the control, and in addition 2.0 MAM of ADP and approximately 2 X 105 cpm of P32. Thus the only difference from the control mixture was the presence of ADP, which permitted net phosphoryla-tion to occur. The rate of reduction in the complex control mixture is approxinmately 90 % of that seen in a much simpler control containing only chloro-plasts, ferricyanide, buffer and NaCl (7), and the response to inhibitors was in all cases the same as in the simpler control. The complex control mixture was used in order to approximate as closely as possible the conditions in the phosphorylating reaction, except for the absence of net ATP formation. After the amount of ferricyanide reduction had been measured, the reaction mixtures in the phos-phorylation cuvettes were denatured by adding 0.3 ml of 20 % trichloroacetic acid, the mixture was centrifuged , and an aliquot was removed for ATP32 determination. In all experiments a zero time control was included in which all reagents were added directly to trichloroacetic acid; this control value for ATP32 was subtracted from all of the experimental measurements. The values shown in the tables represent the average of duplicate or triplicate determinations of both the rate of ferricyanide reduction and of phosphorylation. Duplicate values generally agreed within 10 %. Each experiment shown was repeated at least 4 times. From the measurements obtained in the phos-phorylating cuvettes it is possible to determine a P/2e ratio. This is defined here as micromiioles of A'TP formed per 112 X no. of / equivalents of Hill oxidant reduced, and is listed in the tables as the observed P/2e ratio. If the amount of ferricyanide reduced in the control cuvette is subtracted from that reduced in the phosphorylation cuvette, one cletermines the extra ferricyanide reduction due to simultaneous phos-phorylation. Using this net value for electron flow a calculated P/2e ratio is obtained, defined as micro-moles of ATP formed per 1/2 X net ,u e(quivalents of Hill oxidant reduced in the phosphorylating reaction. These calculated values are also listed in the tables. With a variable source of leaves, the extent of stimulation of the Hill reaction dlue to simultaneous phosphorylation has varied between 2 and 3.5-fold. The experiments chosen for the tables were ones in which the degree of stimulation wras in the upper range, since presumably these would be the better chloroplast preparations. In these experiments where the net stimulation of electron flowv is relatively high, the calculated P/2e ratio is minimal. The values shown in the tables are Mu equivalents of ferricvanide reduced or micromoles of ATP formed in the 3 ml of reaction mixture. MIultiplying anv of these numbers by 1000 will give the , equivalents or micromoles formed per mg chlorophyll per hour. RESULTS Table I shows the effect of dinitrophenol on ferri-cyanide reduction in the presence or absence of simultaneous phosphorylation, and on the amount of ATP formed in the phosphorylation. It can be seen that dinitrophenol at 3.3 X 10-' M, and even more at 1 X 10-3 M, inhibits ATP formation. These same concentrations also inhibit ferricyanide reduction either in the presence or absence of phosphorylation. The extent of the inhibitions of electron flow and of ATP formation are approximately equal in the phos-phorylating Hill reaction. Also shown in this table is the observed P/2e ratio which is close to 1.0. Note that in this experiment the net stimulation is 2.5 times as large as the control rate, and the total stimulated rate is 3.5 times as great as the control. The calculated P/2e ratios range 273