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.     

AMP photophosphorylation and binding by chloroplasts

Stoichiometric amounts of chloroplast thylakoids photophosphorylate free AMP to tightly bound ADP. Free ADP is a poor competitor for this AMP photoreaction, which saturates below 16 micronAMP. The inhibitor, diadenosine pentaphosphate, abolishes AMP photophosphorylation, and inhibits dark ADP binding. Taken together, these data imply that this photoreaction involves the high affinity nucleotide binding site(s) of chloroplast coupling factor CF1, and that little mixing with free nucleotides occurs.     

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.     

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.     

Further studies on the bicarbonate stimulation of photophosphorylation in isolated chloroplasts

The bicarbonate effect in stimulating the rate of photophosphorylation by isolated spinach (Spinacia oleracea var. Virginia blight-resistant savoy) chloroplasts at a pH below the optimum has been re-examined. Its seasonal nature may be related to the hormonal status of the plants. Bicarbonate anions stimulate adenosine 5′-triphosphate synthesis if added in the final, adenosine 5′-triphosphate-forming stage of either a postillumination or an acid-base experiment. They also stimulate the membrane-bound, Mg²⁺-dependent adenosine 5′-triphosphatase of chloroplasts, and the Ca²⁺-dependent adenosine 5′-triphosphatase of detached coupling factor. These and other data point to the interaction between energized thylakoid membranes and the coupling factor as the probable site of action of bicarbonate anions when they stimulate photophosphorylation.     

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; )     

Etude de la photophosphorylation endogene des chloroplastes isoles d'epinardStudy on endogenous photophosphorylation of isolated spinach chloroplasts

Whole spinach chloroplasts were able to perform photophosphorylation under nitrogen without the addition of any redox cofactor. This “endogenous” phosphorylation was totally insensitive to 3-(p-chlorophenyl)-1,1-dimethylurea. After osmotic shock endogenous ATP formation decreased but the addition of 3-(p-chlorophenyl)-1,1-dimethylurea stimulated it.

Under a stream of nitrogen, whole chloroplasts reduced NADP⁺ after an osmotic shock, in the absence of added ferredoxin. The resulting ATP/NADPH ratios were high (approx. 2 or 3). They decreased to 1 in the presence of either exogenous ferredoxin, 3-(p-chlorophenyl)-1,1-dimethylurea or limiting light: i.e. high ATP/NADPH ratios were observed only when the terminal step of NADP⁺ reduction was limiting.

The endogenous anaerobic phosphorylation was inhibited by antimycin A to the same extent as the O₂-dependent endogenous non-cyclic phosphorylation.

A direct inhibition of electron transport by antimycin A has never been observed.     

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.