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.     

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; )     

Photosynthetic control and photophosphorylation in photosystem II of isolated spinach chloroplasts

Two cycles of photosynthetic control have been observed in isolated spinach chloroplasts in the presence of lipophilic class III electron acceptors, which may accept electrons at PS II. $\text{ADP}\text{O}$ ratios of 0.8 to 0.9 were recorded;rates of oxygen evolution were stimulated by phosphorylating reagents and uncouplers. Addition of the plastoquinone antagonist DBMIB decreased photosynthetic control, oxygen evolution and photophosphorylation. We believe that there is a coupling site associated with PSII which can be rate limiting. Comparison of the $\text{P}\text{2e}$ ratios observed with class I and class III electron acceptors leads us to propose that more than 0.6 and possibly approaching one molecule of ATP can be formed for every pair of electrons transported from water to PSII acceptors.     

Estimation of ^{3}J_{HN\hbox{-}H\alpha} and ^{3}J_{H\alpha\hbox{-}H\beta} coupling constants from heteronuclear TOCSY spectra

(3)J proton-proton coupling constants bear information on the intervening dihedral angles. Methods have been developed to derive this information from NMR spectra of proteins. Using series expansion of the time dependent density matrix, and exploiting the simple topology of amino acid spin-systems, formulae for estimation of (3)J(HN-Halpha) and (3)J(Halpha-Hbeta) from HSQC-TOCSY spectra are derived. The results obtained on a protein entailing both alpha-helix and beta-sheet secondary structure elements agree very well with J-coupling constants computed from the X-ray structure. The method compares well with existing methods and requires only 2D spectra which would be typically otherwise recorded for structural studies.     

Temperature dependence of chlorophyll {alpha} fluorescence in lettuce and spinach chloroplasts at sub-zero temperatures

The temperature dependence of chlorophyll fluorescence wasmeasured in spinach and lettuce chloroplasts at sub-zero temperaturesin the presence of 50% ethylene glycol. In the presence of 5mM Mg²⁺, a fluorescence maximum appeared at –31?C in boththe spinach and lettuce chloroplasts, while in the presenceof only 5 mM Na⁺ as cations the maximum shifted to –20?Cin the spinach chloroplasts and to –11?C in the lettucechloroplasts. Since the occurrence of a maximum in the temperatureversus fluorescence curve is an indication for the transitionof the physical phase of thylakoid membrane lipids between theliquid crystalline and the phase-separation state (16, 18),these findings suggest that the (major) phase transition ofmembrane lipids occurs at these low temperatures in chloroplastsof higher plants and also that the phase transition temperatureis markedly lowered by the presence of divalent cations. Ethylene glycol at a concentration of 50% had almost no effecton the temperature dependence of chlorophyll fluorescence ina lamellar membrane preparation of Anabaena variabilis. In awater suspension of dimyristoylphosphatidylcholine, the additionof ethylene glycol to 50% did not alter the characteristic featureof the temperature dependence of fluorescence of 1-anilinonaphthalene-8-sulfonate.These findings suggest that 50% ethylene glycol does not affectthe temperature of the transition of the physical phase of membranelipids. ¹ C.I.W.-D.P.B. Publication No. 592. ² Present Address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Hongo 113, Tokyo, Japan. (Received June 22, 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.     

Light-dependent cleavage of the gamma subunit of coupling factor 1 by trypsin causes activation of Mg2+-ATPase activity and uncoupling of photophosphorylation in spinach chloroplasts

Trypsin treatment of spinach chloroplast thylakoids in the light but not in the dark, results in a highly active Mg2+-ATPase and an uncoupling of photophosphorylation. These light-dependent effects are due to a modification of coupling factor 1 (CF1). CF1 purified from thylakoids treated with trypsin in the light contained a clipped beta subunit and a partially clipped gamma subunit, whereas that from thylakoids treated in the dark with trypsin contained only the clipped beta subunit. CF1 containing this modified gamma subunit also retained a high level of Ca2+-ATPase activity in solution. These results suggest that the gamma subunit becomes highly sensitive to trypsin only when the CF1 is in an active conformation. A similar hypersensitivity to proteases of the gamma subunit in highly purified CF1 is seen only after the enzyme is activated (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). The conversion of the enzyme to its active form, both on the membrane and in solution, therefore, seems to involve conformational changes that expose the gamma subunit to proteolysis.     

Suppression of lipid peroxidation by {beta}-carotene in illuminated chloroplast fragments: Evidence for {beta}-carotene as a quencher of singlet molecular oxygen in chloroplasts

The effect of ß-carotene on light-induced lipid peroxidationwas examined in heptane-extracted chloroplasts. Lipid peroxidationwas suppressed by 50% when the extracted chloroplasts were reconstitutedwith ß-carotene. However, ß-carotene addedto unextracted chloroplasts did not affect the lipid peroxidation.As the molal ratio of ß-carotene to chlorophyll duringthe reconstitution increased, the rate of the lipid peroxidationdecreased but became independent of the molal ratio above thevalue of 0.3. The results led to the conclusion that ß-carotenein thylakoid membranes functions as an efficient quencher ofsinglet molecular oxygen which induces the lipid peroxidation. (Received April 20, 1978; )     

Ca2+ and calmodulin antagonists inhibit the proton gradients associated with non-cyclic and cyclic photophosphorylation in spinach chloroplasts

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.     

Cooperativity among manganese-binding sites in the H+-ATPase of chloroplasts

Coupling factor, isolated from lettuce chloroplasts, contained several binding sites for Mn2+ ions. Three of these sites showed strong cooperative interactions having a Hill coefficient of 2.9 +/- 0.20 and a Kd of 14.7 +/- 0.44 microM. Three additional non-interacting Mn2+-binding sites were found with a Kd of 46.7 +/- 2.3 microM. Chemical modification with naphthylglyoxal of 1 arginyl residue/chloroplast coupling factor 1, which inhibited ATPase activity, inhibited the cooperativity among the sites but did not prevent Mn2+ binding to the enzyme. It is suggested that the cooperative interaction among the Mn2+-binding sites is an expression of the interaction among the active sites of the enzyme which is required for catalysis.     

Proton transport-coupled unisite catalysis by the H+-ATPase from chloroplasts

Proton transport-coupled unisite catalysis was measured with the H⁺-ATPase from chloroplasts. The reaction was measured in the ATP hydrolysis direction under deenergized conditions and in the ATP synthesis direction under energized conditions. The equilibrium constant of the enzyme does not change upon energization, whereas the dissociation constants of substrates and products change by orders of magnitude. This indicates that the Gibbs free enthalpy derived from proton translocation is used to change binding affinities of substrates and products, and this results in synthesis of free ATP.