[Effects of Medium Viscosity Increasing Agents on ATP Synthesis in Chloroplast Thylakoids].

The effect of an increase in the medium viscosity on cyclic photophosphorylation in chloroplast thylakoids and on Ca2+ -dependent ATP hydrolysis by the chloroplast coupling factor CF, was studied. With 0.1-0.2 mM ADP used it was found that the rate of ATP synthesis decreases after addition of various agents that increase the medium viscosity (sucrose, dextran 40 or polyethylene glycol 6000 provided that these agents cause neither uncoupling nor electron transport inhibition in the absence of ADP. Dextran and polyethylene glycol inhibited ATP synthesis by 50% when their concentrations were much lower (6-10%) than that of sucrose (30-40%), while 50% inhibition of Ca2+ -dependent ATP hydrolysis by CFI-ATPase was observed at higher concentrations of dextran and polyethylene glycol (9-13%) and lower concentrations of sucrose (about 20%). For ADP, the effective Michaelis constant (KM) was shown to increase 2-3-fold with the increasing viscosity; meanwhile the maximal rate of cyclic photophosphorylation remained virtually unchanged. The dependence of K(M) on the medium viscosity can serve as a criterion for the process of diffusion-controlled photophosphorylation. Possible mechanisms of ADP and ATP diffusion are discussed.

Two types of ammonium uncoupling in pea chloroplasts.

The effect of ammonium on ATP synthesis, electron transfer, and light-induced uptake of hydrogen ions in pea chloroplasts was studied. It is shown that the dependence of these reactions on ammonium concentration could be due to effects of two different uncoupling processes. The first process is induced by low ammonium concentrations (<0.2 mM); the second one is observed in the NH(4)Cl concentration interval of 0.5-5.0 mM. The first type of uncoupling is stimulated by palmitic acid or by N,N'-dicyclohexylcarbodiimide, while the second is stimulated by chloroplast thylakoid swelling caused by energy-dependent osmotic gradients. In the presence of the fluorescent dye sulforhodamine B, which does not penetrate through the cell membrane, this swelling causes the dye to enter the lumens. It is supposed that ammonium activates two different routes of cation leakage from the lumen. The first route involves channel proteins, while the second is a mechanosensitive pore that opens in response to osmotic gradients.

Synergism of ammonium and palmitic acid in uncoupling of electron transfer and ATP synthesis in chloroplasts.

Uncoupling by ammonium of electron transfer and ATP synthesis during linear transfer of electrons from water to photosystem 1 acceptors was studied in pea chloroplasts. It was shown that 40 microM palmitic acid decreased several-fold the ammonium concentrations necessary for 50% inhibition of ATP synthesis. The protonophore carbonyl cyanide m-chlorophenylhydrazone has no such property. The enhancement by palmitate of ammonium-induced uncoupling is accompanied by acceleration of basal electron transfer and decrease in the photoinduced uptake of hydrogen ions (H+). In the absence of ammonium, palmitate has no effect on basal transport and stimulates uptake of hydrogen ions. This means that in the case of combined action of palmitate and ammonium an additional leakage of H+ takes place, resulting in dissipation of the pH gradient. Synergic action of two metabolites, free fatty acid and ammonium, is supposed to provide for functioning of a system of mild regulation of energy coupling processes in native plant cell chloroplasts. Possible mechanisms of synergism are discussed.

[Ammonium factor in the life of plant cells].

In the present review the role of ammonium component not connected with the synthesis of nitrogen-containing substances in plant cells is discussed. In particular, it is devoted to the influence of NH4+ ions on the morphological changes of surface root cells, heterotrophic CO2 fixation by cells, dark and light-dependent reduction of nitrate in plant cells, formation and functioning of cytoplasmic ribosomes in plant cells and function of photosynthesizing organelle membrane systems. Our experimental data together with the results of the other researchers allow us to look at ammonium participation in the life of plant cells more widely and not to consider NH4+ ions only as a substrate for ketoacids amination processes in plant cells.

Light-induced reversible local fusions of thylakoid membranes in the presence of dibucaine or tetracaine.

The dynamics of structural changes in pea chloroplasts in the presence of 25-50 microM dibucaine or tetracaine has been examined using electron microscopy. The light-induced uptake of anesthetic cations by thylakoids is attended by the appearance of local fusions of stroma-exposed thylakoid membranes. The first membrane protrusions and interthylakoid contacts are observed after 4 s illumination and they become numerous by 10 s. As a result, a network of anastomoses is formed which is maintained during at least 10 min. These effects are reversible in the dark and can be reproduced several times. The formation of membrane fusions is inhibited by the addition of protonophore. It is supposed that the energy-dependent uptake of protonated anesthetics by thylakoids leads to an increase in positive surface charge and thus a lateral pressure on the inner side of the thylakoid membrane. The appearance of membrane protrusions (crinkles) having the positive curvature of their inner surface may be considered as a way of compensating for lateral pressure. Presumably, anastomoses result from the fusion of crinkles to adjacent thylakoids.

Induction of an electrogenic transfer of monovalent cations (K+, NH4+) in thylakoid membranes by N,N'-dicyclohexylcarbodiimide.

The effect of N,N'-dicyclohexylcarbodiimide (DCCD) on photosynthetic electron transport and light-induced NH4+ and K+ uptake in the presence of ammonium or nigericin was studied. DCCD alone had no effect on either the electron transport or the uptake of protons. The simultaneous action of DCCD and low concentrations of ammonium or nigericin was shown to lead to a significant increase in the electron transport rate and a decrease in the steady-state uptake value of H+ and NH4+ or K+. The effect of DCCD on these processes was compared with the effect of the ionophore, valinomycin, which transports potassium and ammonium cations through membranes. The conclusion was made that 1.0-1.5 mol DCCD per mol chlorophyll activated the transfer system of monovalent cations (K+ and NH4+) in thylakoid membranes.

[Study of DNA-interaction with protons of the medium by means of buffer capacity method]. / Izuchenie vzaimodeistviia DNK s protonami sredy metodom bufernoi emkosti.

pH-dependences of buffer capacity of native and denaturated DNA have been obtained. These dependences were expanded into basic functions each corresponding to ionised groups of equal pK. The pH dependence of buffer capacity of native DNA have two narrow g- and s-peaks and are shifted (relatively to nucleosides mixture) to acidic pH values, whereas those of heat denaturated DNA have only one wide assymmetric peak shifted to neutral pH. It was shown that s-and g-peaks of pH dependences of buffer capacity correspond to polymorphic transformation of the double helix (h leads to s transition) and to its disordering (s leads to g transition) respectively. From our data it follows that h leads to s transition at mu = 0.01 M NaCl is observed at temperatures T less than 50 degrees C. The independence of DNA protonation degrees, at which h leads to s leads to g transitions occur, on ionic strength of the solution is, apparently, related to linear dependence of the positions of g- and s-peaks maxima on (lg mu). This points to an important role of protonated bases in double helix destabilisation upon salt elimination. High sensitivity of the buffer capacity method allows us to reveal some differences between states of heat and acid denaturated DNA. Analysing pH dependences of buffer capacity of denaturated DNA it was shown that at neutral pH it contained some double or triple helix regions formed by H-linked bases holding unshared proton. Simms' method was shown to be inapplicable to determine base ionization constants both in native and denaturated DNA.

[Amino acid composition and proton capacity of pea chloroplast thylakoid membranes]. / Aminokislotnyi sostav i protonnaia emkost' tilakoidnykh membran khloroplastov gorokha.

The amino acid composition and pH dependence of proton capacity of pea chloroplast thylakoid membranes have been studied. The thylakoid membranes possess a high content of amino acids, whose functional groups can either bind or split off protons depending on pH of the reaction mixture. The number of amino acids with proton-accepting functional groups per mg of chlorophyll significantly exceeds the proton capacity of the native membranes. It is concluded that the buffer capacity of thylakoid membranes is predetermined by the proton-accepting groups of protein components which are largely blocked within the native structure of the membranes. The protein/chlorophyll weight ratio for pea chloroplast thylakoid membranes is 3.22 +/- 0.05.

[Evaluation of changes in free energy of proteins and biomembranes under pH-induced conformational transitions. Coupling membranes of peak chloroplasts]. / Metod otsenki velichiny izmeneniia svobodnoi energii belkov i biomembran pri pH-indutsirovannykh konformatsionnykh perekhodakh. Sopiagaiushchie membrany khloroplastov gorokha.

It was shown that changes in the free energy during pH-induced conformational transitions can be calculated from the buffer spectrum of the biopolymer. These changes are similar to those of the chemical potential of cooperatively protonating groups (delta Go = 2,3 RT (pKt--pKi)Nt) and can be determined independently for each transition of the complex object. Consequently, in case of biomembranes with the ionic strength greater than or equal to 0,1 it is possible to determine not only the maximal efficiency of each transition according to the formula magnitude of delta Go congruent to 2,3 RTNt, but also that of the membrane component which initiates the given transition. Thus, a study of the photo-dependent buffer spectrum of the coupling membranes of chloroplasts showed that in the membrane the ATPase complex performs a reversible conformational transition within the pH region of photophosphorylation with the energy of 100--130 kcal/mol for the coupling factor or 190--260 cal/mol for the chlorophyll.

[Buffer capacity of polyproton substances]. / Bufernaia emkost' poliprotonnykh obdektov.

Mathematical analysis of protolytic properties of a polyelectrolite with arbitrary set of stepwise dissotiation constants is carried out, based on disintegration of summary titration curves and buffer capacity for basic functions. It is demonstrated, that in some cases (for example, investigation of biomembranes and their components) simplified disintegration variants can be used. Buffer titration curve of biopolymers and biomembranes is a "buffer spectrum", the position of bands on the pH scale, their intensity and half-width being determined by the nature of the object (the quantity and quality of hydrophilic groups and the set of its conformation states). Sharp peaks of the buffer spectrum correspond to the conformation transition of the object, and their half-width on the pH scale determines the cooperative degree of each conformation transition. The mathematical analysis described is not specific to the formation of polyprotonic complex, and it can be used in investigation of the complex formation with any monodentant ligand. It is concluded that the method described can be used in the investigation of complex biomembranes and different processes, in which these membranes participate.

[Structure of electrochemically reduced adenine nucleotides. Their interaction with inorganic phosphate]. / Osobennosti struktury élektrokhimicheski vosstanovlennykh adeninnukleotidov. Ikh vzaimodeistvie s neorganicheskim fosfatom

The stable reduced form of adenosine phosphates has probably cyclic or dimeric structure of the type: adenine "head"--phosphate "end". Inorganic phosphate produce a stabilyzing effect on reduced adenosine due to a strong ionic interaction with the amino group. In the case of reduced ADP, inorganic phosphate can incorporate into adenine nucleotide, which oxidation results in the formation of small amounts of ATP. A scheme of possible mechanism of this process is given.