Photophosphorylation by Chloroplasts: Effects of Low Concentrations of Ammonia and Methylamine Christoph Giersch Botanisches Institut der Universität Düsseldorf, Universitätsstr. 1, D-4000 Düsseldorf, Bundesrepublik Deutschland Z. Naturforsch. 37 c, 242-250 (1982); received December 23, 1981 Photophosphorylation, Uncoupling, Amines, Proton Motive Force, Chloroplasts, Chemiosmotic Theory Intact chloroplasts exposed to hypotonic assay conditions are capable of photophosphorylating exogenous ADP. The rate of phosphorylation by these unbroken plastids is increased by 10—50% upon the addition of low concentrations (< mM) of N H 3 or CH3NH 2. Stimulation of phosphory­ lation is abolished by washing chloroplasts with MgCl2. Evidence is presented that washing re­ moves a factor responsible for amine-induced increase of A TP production and that this factor is associated with the thylakoid membrane. Addition of CH3NH 2 increased the proton permeability of the thylakoid membrane of unbroken and washed chloroplasts during the light/dark transition. Hence, differences of the membrane permeability for protons between the two preparations seem not to be responsible for an increase of ATP production upon the addition of amines. Stimulation of photophosphorylation by methylamine is observed even at light itensities which do not saturate the proton motive force, which in turn is reduced upon the addition of the uncoupler. Apparently, phosphorylation can be stimulated, although the limiting driving force is diminished. It is con­ cluded that phosphorylation by unbroken chloroplasts under low light illumination is limited kinetically, not energetically. Consequences of these findings for observation made with intact chloroplasts are discussed. Introduction creased pmf in the presence of low amine * concen­ trations although the rate of steady-state light-de­ The chemiosmotic concept holds the trans­ pendent ATP production is increased [4]. This ob­ membrane electrochemical proton gradient to be the servation is not readily accomodated within the driving force of photosynthetic energy conversion framework of the chemiosmotic concept of energy [1]. However, the relationship between the rate of conversion [1]. Additional work with isolated intact ATP production and the magnitude of the driving chloroplasts provides further, although indirect, evi­ force is not predicted by this concept. Quantitative dence for increased phosphorylation in the presence description of the relationship can be formulated by of a diminished proton gradient [5]. Sorgato etal. irreversible thermodynamics, provided the force- showed that the rate of ATP synthesis in sub- flow relations of energy conversion have been exper­ mitochondrial particles can decrease at a constant imentally characterized [2, 3]. Therefore, ob­ pmf [6], At the moment it seems unclear whether servations concerning the dependence of the rate of these observations demonstrate that ATP production phosphorylation on the pmf should aid not only an is at least partially independent of the pmf or understanding of the molecular mechanism of whether they can be explained as having resulted energy conversion but will also be relevant for the from artificial, non-physiological conditions. Thus, formulation of force-flow relations. it is the aim of this contribution (i) to report ob­ Recently, it has been shown that intact chloro­ servations which are relevant for understanding the plasts in hypotonic assay conditions exhibit a de- mechanism of stimulated ATP production at a de­ creased pmf, (ii) to show that phosphorylation by unbroken chloroplasts is limited kinetically, not Abbreviations: Ap5A, P\P5-di(adenosine-5'-)pentaphos- energetically, not only under saturating illumination phate; BSA, bovine serum albumin; Chi, chlorophyll; DCCD, dicyclohexyl-carbodiimide; HEPES, N-2-hy- [4] but also under limiting light, and (iii) to propose droxyethylpiperazine-N'-2-ethanesulphonic acid; pmf, that such a kinetic limitation should be considered proton motive force; 9-AA, 9-aminoacridine. Reprint requests to Dr. Christoph Giersch. * In this article, “ amine” is frequently understood to 0341-0382/82/0300-0242 $01.30/0 include ammonia and its cation, N H 4+. Ch. Giersch • Effect of Low Amine Concentrations on Photophosphorylation 243 by thermodynamic descriptions of energy conver­ Results sion. 1. Electron transport and phosphorylation by unbroken chloroplasts Materials and Methods Intact chloroplasts were isolated from spinach Unbroken chloroplasts (type B, ref. 13) were ob­ leaves of greenhouse plants by a modification [7] of tained by suspending isolated intact chloroplasts in Jensen and Bassham’s procedure [8]. BSA (0.3%, an assay medium containing 0.1 m sorbitol as os- w/v) was added to the grinding medium instead of moticum. These type B plastids are reported to be ascorbate. The same concentration of BSA was ad­ morphologically intact [13], but posses an envelope ded to the storage medium. Thylakoids were obtain­ which is permeable to smaller molecules like fer­ ed by washing intact chloroplasts twice in MgCl2. ricyanide or adenine nucleotides. They are capable The washing procedure was as follows: intact plas- of phosphorylating exogenous ADP at rates up to tids suspended in the storage medium were diluted 180 nmol/mg Chi • h. Recently, it has been demon­ 20 fold with 5 mM MgCl2; after centrifugation for strated that the rate of phosphorylation by these 5 min at 3900 x g, the pellet was resuspended in the chloroplasts in increased (rather than decreased) storage medium. Chlorophyll was determined upon the addition of low (< 1 mM) concentrations of according to Amon [9]. Chlorophyll concentra­ amines [4]. Fig. la shows the dependence on the tion in the assay was 25 — 33 ^g/ml. pH changes concentrations of NH 4C1 and methylamine of elec­ of weakly buffered chloroplast suspensions were re­ tron transport and phosphorylation. corded by a glass electrode (M3, LOT-405, Ingold) As reported before [4], methylamine is more ef­ connected to a Knick-53 pH meter. Oxygen evolu­ fective in stimulating phosphorylation. The concen­ tion was measured using a Clark-type electrode; the tration of uncoupler causing maximum stimulation is electron acceptor was 1 mM ferricyanide if not in­ higher for methylamine than for NH 4C1. The rate of dicated otherwise. Quenching of 9-AA fluorescence electron transport from H20 to ferricyanide is slight­ and absorption changes of chloroplast suspensions ly stimulated upon the addition of methylamine, were measured as decribed previously [5], Actinic whereas concentrations of N H 4C1 exceeding 0.6 mM light was provided by passing a beam of white light drastically increase the rate, indicating that ammo­ through a heat protection filter, 1 mm Calflex C nia is a more potent uncoupler than methylamine. (Balzer), and a 2 mm RG 630 (Schott) glass filter. Cyclohexylamine, another uncoupling amine, causes Light intensities were determined by a radiometer practically no stimulation of ATP production (Yellow Springs Instruments Co., model 65A). (maximum rate in the presence of 0.1 mM cyclo­ Photophosphorylation was measured by enzymic de­ hexylamine was 103% of the control) and drastically termination of ATP, by determination of Pi5 or in­ increases electron transport. Since cyclohexylamine corporation of 32Pi. For the enzymic ATP-test [10], has about the same pK-value as methylamine corrections were made for activity of adenylate ki­ (pK = 10.6) it is clear that the differences observed nase in the assay (E.C. 2.7.4.3), or activity of this en­ between the three uncouplers cannot be due to dif­ zyme was inhibited by addition of 0.15 mM Ap5A ferent pK-values. Results comparable to those [11] which does not affect the rate of phosphory­ shown in Fig. 1 were obtained when ferricyanide lation as measured by incorporation of 32Pj. Unla­ was replaced by methylviologen in the presence of belled phosphate was determined according to Fiske KCN (data not shown). and Subbarow [12]. For measurement of incorpor­ As expected, the ATP/e2 ratio is decreased in the ation of labelled phosphate, Pi was replaced by presence of cyclohexylamine (not shown) or N H 4C1 KH 232P04 (5 mCi/mmol). The label recovered in (Fig. 1 b). It is not affected or even slightly increased phosphate and in ATP was determined by high (Fig. lb ) with low concentrations of methylamine; performance liquid chromatography as described in higher concentrations lead to a decrease which, how­ [4]. The rate of phosphorylation was calculated from ever, is less dramatic than with ammonia. Un­ the percentage of total label recovered in the ATP couplers like simple amines or ammonia are known peak, the amount of added Pi5 and the chlorophyll to stimulate electron transport, probably by increas­ concentration. ing the permeability of the thylakoid membrane for 244 Ch. Giersch • Effect of Low Amine Concentrations on Photophosphorylation 2. Effects o f chloroplast pretreatment The envelope of intact isolated chloroplasts expos­ ed to 0.1 M sorbitol is likely not to be disrupted [13, 15]. Fig. 2 shows the effect of methylamine on phos­ phorylation with chloroplasts exposed to isotonic conditions after brief osmotic shock in 5 mM MgCl2. This procedure is known to disrupt the envelope to­ tally and to produce type D chloroplasts [13]. Stimu­ lation upon the addition of methylamine is observed also under this assay condition and may even exceed that measured under permanent osmotic stress in 0.1 m sorbitol (Fig. 2, H). Intact chloroplasts were washed twice in