Nigericin-induced stimulation of photophosphorylation in chloroplasts

Amines have been shown recently to stimulate at low concentrations the steady-state rate of photophosphorylation by unbroken chloroplasts (Giersch, C. (1982) Z. Naturforsch. 37c, 242–250). In the present contribution it is demonstrated that not only amines but also the carboxylic ionophores nigericin and monensin at concentrations of 10 and 150 nM, respectively, stimulate the phosphorylation rate. The $\text{ATP}\text{2}\text{e}$ ratio is not decreased upon the addition of nigericin at concentrations that stimulate phosphorylation. Nigericin-induced stimulation is observed only in the presence of sufficient external potassium, indicating that the observed stimulation is unlikely to be a side-effect of the uncoupler but is related to H⁺-K⁺ exchange. The proton permeability of the thylakoid membrane is increased and the proton gradient decreased by amounts of nigericin that stimulate phosphorylation. The membrane potential is not affected in the steady state, indicating that the proton-motive force is slightly reduced upon addition of the ionophore. Data on the proton-motive force were related to maximum values of the phosphorylation potential, which was 45 000–50 000 M^?1 in the absence and 30 000–35 000 M^?1 in the presence of 10 nM nigericin. The observation that the $\text{ATP}\text{2}\text{e}$ ratio is not decreased in the presence of uncoupler-induced proton leakage is suggested to indicate that the thylakoid lumen does not represent a homogeneous phase of constant proton electrochemical potential. The results presented here are in agreement with the chemiosmotic concept as far as energetic aspects are concerned but seem to be at variance with the postulated free mobility of protons inside the thylakoids. A tentative model of uncoupler-induced stimulation of phosphorylation is presented.     

Flash-induced photophosphorylation in chloroplasts with activated ATPase

Chloroplasts which were rapidly isolated from illuminated leaves showed activity of ATP hydrolysis at a level much higher than that of the dark control. Under the high-intensity illumination or under repetitive flash excitation, the activated chloroplasts synthesized more ATP than those with a low ATP hydrolysis activity. formed under repetitive flashes was smaller in the activated chloroplasts than in the inactive chloroplasts. The inhibition of ATP yield per flash by valinomycin or nigericin in the presence of K⁺ was stronger in the inactive chloroplasts than in the activated chloroplast. ATP synthesis in the activated chloroplasts seems to have a lower threshold.     

Glycolate synthesis by intact chloroplasts: Studies with inhibitors of photophosphorylation

Intact spinach chloroplasts, capable of evolving O₂ in response to CO₂ at rates greater than 70 μmol/h · mg of chlorophyll, synthesize glycolate from added dihydroxyacetone phosphate, ribose 5-phosphate, or xylulose 5-phosphate, when illuminated in the presence of O₂. The synthesis of glycolate from these compounds is dependent upon photophosphorylation and is inhibited by each of the three classes of photophosphorylation inhibitors [Izawa, S., and Good, N. E. (1972) in Methods in Enzymology, Vol. 24, Part B, pp. 355–377)]: an uncoupler, carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP), an energy transfer inhibitor, Dio-9, and a phosphate analog, arsenate. Neither arsenate nor Dio-9 causes the collapse of the light-generated proton gradient between thylakoid and stroma compartments of the chloroplasts, so that the inhibition of glycolate synthesis by these compounds cannot be ascribed to an inactivation of Calvin cycle enzymes thought to be associated with the collapse of such a proton gradient. The dependency of glycolate synthesis upon photophosphorylation indicates that an ATP-consuming reaction(s) is involved in the conversion of the substrates (triose and pentose monophosphates) to glycolate. The formation of dihydroxyethylthiamine pyrophosphate, the “active glycolaldehyde” intermediate of the transketolase reaction, from triose and pentose monophosphates has no known requirements for ATP. On the other hand, the conversion of both triose and pentose monophosphates to ribulose 1,5-bisphosphate, the substrate for the ribulose 1,5-bisphosphate oxygenase reaction, requires ATP. It is concluded that glycolate synthesis by intact isolated chloroplasts is primarily the result of ribulose 1,5-bisphosphate oxygenase activity. No substantial role in glycolate synthesis can be attributed to the oxidation of dihydroxyethylthiamine pyrophosphate, the intermediate of the transketolase reaction.     

Alkaloids as inhibitors of photophosphorylation in spinach chloroplasts

A group of 12 alkaloids were tested as inhibitors of photophosphorylation in spinach chloroplasts. Ajmaline, a dihydroindole alkaloid, was found to be the strongest inhibitor of both cyclic and non-cyclic photophosphorylation. Low concentrations of ajmaline also inhibited the dark and light ATPases, and the coupled electron flow from water to ferricyanide, measured either as ferrocyanide formed or as oxygen evolved, but not the uncoupled electron transport or the pH rise of illuminated unbuffered suspensions of chloroplasts. Higher concentrations of ajmaline stimulated, instead of inhibiting, photosynthetic electron transport or oxygen evolution and decreased the pH rise, thus behaving as an uncoupler, such as ammonia.Photophosphorylation was partially inhibited by 100 μM dihydrosanguinarine, 100 μM dihydrochelerythrine (benzophenanthridine alkaloids); 500 μM O,O'-dimethylmagnoflorine, 500 μM N-methylcorydine (aporphine alkaloids) and 1 mM julocrotine. They also inhibited coupled oxygen evolution and only partially (dihydrosanguinarine and dihydrochelerythrine) or not at all (the other alkaloids) uncoupled oxygen evolution.Spegazzinine (dihydroindole alkaloid), magnoflorine, N-methylisocorydine, coryneine (aporphine alkaloids), candicine and ribalinium chloride were without effect on photophosphorylation at 500 μM.     

Efficiency of photophosphorylation in chloroplasts with steady and pulsed illumination

The quantum yield of noncyclic photophosphorylation in chloroplasts excited by a series of 8 mus flashes of the saturating intensity displays a two-fold decrease when the flash-frequency is reduced from about 1.1 to about 0.8 s-1, whereas further decrease of flash frequency does not affect the average ATP yield per flash. Under excitation by two-flashes series the ATP yield is also about half-maximal. These observations are inconsistent with the concept postulating accumulation of energy contributions from several parallel or consecutive one-electron transfers as a prerequisite for ATP formation. The two-state model of a thylakoid membrane and of a coupling site is put forward according to which only one of these states ensures ATP formation in response to one electron transfer through one coupling site, whereas the other state is nonphosphorylating.     

Linolenic acid binding by chloroplasts

The binding of linolenic acid with chloroplasts was investigatedwith ¹⁴C-labelled linolenic acid. The effect of the fatty acidon the activity of the electron transport system was also studied. The amount of linolenic acid bound to chloroplasts increasedwith increasing concentration of the fatty acid added in a mannersuggesting a cooperative mode of binding. At the highest concentrationof linolenic acid added (100 µM), the molar ratio of boundfatty acid to chlorophyll was four. The bound fatty acid to chlorophyll ratios were inversely proportionalto the amounts of chloroplasts added. The bound fatty acid wasreleased by addition of bovine serum albumin or by washing ofthe chloroplasts. The mode of release during repeated washingindicates that binding of linolenic acid to chloroplast membraneoccurred through partition of the fatty acid between the membraneand the aqueous medium. Time courses and temperature dependency of the development oflinolenic acid-induced inhibition of the Hill reaction weremarkedly different from those of the fatty acid binding. Theinhibition was at least partially reversible. The results indicatethat inactivation of electron transport is due to disorganizationof the functional integrity of the membrane caused by penetrationof the fatty acid molecules into the hydrophobic region of membrane. ¹ Present address: Biological Laboratory, Nippon Medical School,Kosugi, Kawasaki, Japan. (Received December 16, 1976; )     

Peptide alkaloids as inhibitors of photophosphorylation in spinach chloroplasts

Photophosphorylation in isolated spinach chloroplasts was inhibitedby all 21 peptide alkaloids tested. Zizyphine A and B, adounetineZ, amphibine B, C and D and scutianine A inhibited the coupledbut not the uncoupled electron transport. The other peptidealkaloids stimulated nonphosphorylating electron flow behavinglike uncouplers. Aralionine A, lasiodine A and mucronine B werethe strongest inhibitors and uncouplers. Lasiodine A and homalinestimulated by several times the light-induced proton uptakeby chloroplasts. (Received January 20, 1977; )