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Y de Kouchkovsky

Publications and source records attributed to Y de Kouchkovsky.

14 recordsLinked to original sources
PubMed2000

Complex I impairment, respiratory compensations, and photosynthetic decrease in nuclear and mitochondrial male sterile mutants of Nicotiana sylvestris.

We have previously shown that in Nicotiana sylvestris cytoplasmic male-sterile (CMS) mutants where the mtDNA lacks the nad7 gene coding for a subunit of respiratory Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3), glycine (Gly) oxidation was lower than in the wild type and insensitive to rotenone, suggesting Complex I dysfunction. In contrast, the oxidation rate of exogenous NADH and the capacity of the cyanide-resistant respiration (AOX) were enhanced. Here we report that, in contrast to Gly, the rate of malate oxidation was not affected, but proceeded totally in a rotenone-insensitive pathway, strongly suggesting that survival of CMS plants depends on the activation of internal and external alternative NAD(P) H dehydrogenases and that Gly decarboxylase activity depends on Complex I functioning. A similar defect in Complex I activity and Gly oxidation was found in the NMS1 nuclear mutant, defective in the processing of the nad4 transcript, but alternative NAD(P) H dehydrogenases were less activated. In CMS and NMS1, the fraction of the AOX pathway was increased, as compared to wild type, associated with higher amounts of aox transcripts, AOX protein, and plant resistance to cyanide. Non-phosphorylating respiratory enzymes maintained normal in vivo respiration levels in both mutants, but photosynthesis was decreased, in correlation with lower leaf conductance, emphasizing mitochondrial control on photosynthesis.

Cell Nucleus↗
PubMed1997

In vivo interference of paromomycin with mitochondrial activity of Leishmania.

Paromomycin is an aminocyclitol aminoglycoside antibiotic used for the treatment of leishmaniasis. In view of the central role of mitochondria in cellular energetics and metabolism, its effect on in vivo mitochondrial activities of Leishmania donovani promastigotes-the parasite flagellate form-was investigated. The approach used flow cytometry, amperometric measure of O2 consumption, and, as a global estimate of mitochondrial dehydrogenases, thiazolyl blue reduction (MTT test); some in vitro controls were also made. When added to promastigote cultures for 24-72 h at 150-200 microM (= LC50), paromomycin doubled the generation time, inhibited respiration, and lowered its associated electric potential difference across mitochondrial membranes, as measured by rhodamine 123 fluorescence. The chemical analogue neomycin was ineffective. Furthermore, the in vivo mitochondrial dehydrogenase activities were lower, seemingly because of the shortage of respiratory substrates. Indeed, succinate addition to paromomycin-treated cultures partly restored mitochondrial membrane potential. However, no immediate effect of paromomycin on respiration was observed, neither inhibition of redox chain nor increase of membrane permeability (uncoupling). It is proposed that paromomycin acts at a metabolic level upstream of the respiratory chain itself. This would have the observed delayed consequence because the cell energy supply would progressively decline since it depends upon the proton gradient-viz., membrane potential-generated by respiration. In conclusion, paromomycin is an antibiotic affecting the cell's energetic metabolism; the respiratory dysfunction it induces may be a crucial aspect of its action against Leishmania and possibly other cells.

Animals↗
PubMed1997

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants.

We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection.

Amino Acid Sequence↗
PubMed1993

Flow-force relationships in lettuce thylakoids. 1. Strict control of electron flow by internal pH.

The regulation by the proton gradient of the electron flow from water to ferricyanide was investigated in thylakoids extracted from lettuce leaves. When the transmembrane proton current was varied by an uncoupler or by the ATP synthase activity, a unique relationship was found between the rate of ferricyanide reduction and the proton gradient, restricted here to its delta pH component. This behavior was conserved in CF1-depleted thylakoids where the passive proton flow was varied by the concentration of an Fo inhibitor or by the concentration of an uncoupler after 100% inhibition of Fo. This shows that under our experimental conditions no direct proton transfer exists in steady state between the site of regulation of the redox chain and the ATPase. Studies at two different pH's indicate that the internal pH, and not the transmembrane pH difference, controls the electron transfer between PS2 and PS1. Modeling the data suggests that a single deprotonation step is kinetically limiting.

Calcium↗
PubMed1993

Flow-force relationships in lettuce thylakoids. 2. Effect of the uncoupler FCCP on local proton resistances at the ATPase level.

The relationship between the steady-state proton gradient (delta pH) and the rate of phosphorylation was investigated in thylakoids under various conditions. Under partial uncoupling by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), the rate of ATP synthesis was reduced by less than expected from the decrease of delta pH. This was observed in the case of the pyocyanine-mediated cyclic electron flow around photosystem 1, but not with the H2O-->photosystem 2-->cytochrome b6f-->photosystem 1-->methyl viologen system. In state 4, a unique relation was found between delta pH and the "phosphate potential", delta Gp, regardless of whether the energy level was controlled by light input or FCCP. The anomalous effect of FCCP on the rate of ATP synthesis disappeared when the ATPase was partially blocked by the reversible inhibitor venturicidin, but not in the presence of tentoxin, an irreversible inhibitor. These results are consistent with the existence of a small kinetic barrier for protons, limiting their access to the ATPase. This resistance would be collapsed by FCCP.

Adenosine Triphosphate↗
PubMed1992

ATPase state and activity in thylakoids from normal and water-stressed lupin.

To compare with the case of phosphorylation, ATP hydrolysis was investigated in thylakoids from plants submitted to drought. For medium stresses the relationship between hydrolysis rate and deactivation constant of the de-energized enzyme when delta pH varies is unchanged, hence the basic mechanisms are preserved; (PS1-driven) energization than is neither affected, at any light intensity. As drought intensifies, hydrolysis and monogalactolipids fall somewhat in parallel, whereas the deactivation constant changes with digalactolipids and phosphatidic acid content. This alteration of the F0 lipid environment must be transduced to F1, the subunits of which, however, are preserved, as shown by LDS-PAGE. ATP synthesis (thiol-oxidized enzyme) and hydrolysis (thiol-reduced enzyme) show an identical decrease with stress. Thus the latter impairs a common early step, probably the activation of the oxidized enzyme.

Adenosine Triphosphatases↗
PubMed1992

An attempt to discriminate catalytic and regulatory proton binding sites in membrane-bound, thiol-reduced chloroplast ATPase.

The question of the possible identity of catalytic and regulatory proton pathways in the chloroplast FoF1 ATPase has been studied using different energy-transfer inhibitors. Venturicidin, a reversible inhibitor of Fo, affects neither the delta mu H(+)-dependent thiol reduction of the membrane-bound chloroplast ATPase nor its ability to be activated by the proton gradient. It seems therefore to block only the proton flow required by the catalytic function of the enzymes. Venturicidin, however, also slows down the deactivation of the thiol-reduced ATPases during uncoupled ATP hydrolysis, following a delta mu H+ activation, but phloridzin, a reversible F1 inhibitor, has the same effect. Tentoxin, an irreversible F1 inhibitor, decreases the rate of ATP hydrolysis but does not affect the rate of deactivation. These findings suggest that catalytic and regulatory H(+)-binding sites are different. No distinction can be made, if any, between protons involved in unmasking the thiol-sensitive groups of F1 and in activating the enzyme. The effect of venturicidin and phloridzin on the deactivation is consistent with an inhibitory effect of newly formed--by ATP hydrolysis--ADP molecules, which might affect the enzyme without passing through the medium. Phosphate at millimolar concentration has an effect similar to low concentrations of phloridzin and venturicidin, probably by a simple back-reaction effect.

Adenosine Triphosphate↗
PubMed1991

Dependence of kinetic parameters of chloroplast ATP synthase on external pH, internal pH, and delta pH.

ATP synthesis by the membrane-bound chloroplast ATPase in the oxidized state of its gamma disulfide bridge was studied as a function of the ADP concentration, delta pH, and external pH values, under conditions where delta pH was clamped and delocalized. At a given pH, the rate of phosphorylation at saturating ADP concentration (Vmax) and the Michaelis constant Km (ADP) depend strictly on delta pH, irrespective of the way the delta pH is generated: there evidently is no specific interaction between the redox carriers and the ATPase. It was also shown that both Km (ADP) and Vmax depend on delta pH, not on the external or internal pH. This suggests that internal proton binding and external proton release are concerted, so that net proton translocation is an elementary step of the phosphorylation process. These results appear to be consistent with a modified "proton substrate" model, provided the delta G0 of the condensation reaction within the catalytic site is low. At least one additional assumption, such as a shift in the pK of bound phosphate or the existence of an additional group transferring protons from or to reactants, is nevertheless required to account for the strict delta pH dependence of the rate of ATP synthesis. A purely "conformational" model, chemically less explicit, only requires constraints on the pK's of the groups involved in proton translocation.

Adenosine Diphosphate↗
PubMed1980

Measurement of chloroplast internal protons with 9-aminoacridine. Probe binding, dark proton gradient, and salt effects.

A defined ratio, gamma, of the total proton uptake to the concentration change of free internal H+ is observed for illuminated envelope-free chloroplasts (Haraux, F. and de Kouchkovsky, Y. (1979) Biochim. Biophys. Acta, 546, 455-471). Proton uptake is measured by the external pH shift, free internal H+ by 9-aminoacridine fluorescence quenching. Extension of this work leads to the following conclusions, which, in the case of 9-aminoacridine behaviour, should apply to any kind of diffusible protonizable delta pH probe: 1. The gamma constancy is preserved when the internal volume (Vi) is modulated by chlorophyll and osmolarity changes: thus, 9-aminoacridine behaves as expected from the delta pH distribution of an amine of high pK; previous doubts on this point are attributed to the lack of control of the external proton uptake. 2. With variable 9-aminoacridine concentration, however, some variation of gamma confirms the existence of slight light-induced probe-membrane interactions. 3. According to the diffuse layer theory, salts decrease the negative potential at the 'plane of closest approach' of the thylakoids, thereby releasing the excess 9-aminoacridine in this diffuse layer, which increases its fluorescence. Although of equal valency, NH4+ is more potent than K+, suggesting competition between amines for specific anionic binding sites. 4. Two categories of membrane modifications are induced by salts: in addition to the above-mentioned electrical effect, mono- and divalent cations at high concentration increase the chloroplast proton binding capacity. La3+ is only able to release the excess dye in the diffuse layer and leaves gamma unchanged. Therefore the probe-membrane interactions should have limited importance for steady-state delta pH measurement. 5. A Donnan-type dark pH difference, which could seriously bias these delta pH estimates, is found experimentally to be less than 2 (no significant gamma change when Vi varies) and even theoretically less than 1 (on the basis of the concentration of the non-diffusible internal protonizable groups). Similarly, the predictable errors of Vi and its possible light-induced variations must have a small effect on delta pH under present experimental conditions.

Aminacrine↗
PubMed1975

Study of the chlorophyll fluorescence in chloroplasts and algae with the plastoquinone antagonist dibromothymoquinone.

The effect of the plastoquinone antagonist dibromothymoquinone on chlorophyll fluorescence in vitro and in vivo was investigated. 1. With chlorophyll a in solution quenching is observed, more efficient than that of p-benzoquinone (the Stern-Volmer constant K equals to 200 M-1); ascorbate removes this effect. 2. With isolated chloroplasts, a dramatic enhancement of quenching occurs (also abolished by ascorbate) in the following order of importance: thermal step, photochemical step (of variable fluorescence), and constant fluorescence; K for the total variable fluorescence approximately equal to 480 000 M-1, for constant approximately equal to 190 000 M-1. Parallel to the quenching effect, an enlargement of the complementary area of the variable fluorescence is observed. Addition of ascorbate suppresses the quenching and this enlargement. If this area in the presence of reduced dibromothymoquinone is similar to that of the control, this means that A, the pool of oxidant next to Q, remains connected to it, a result contradicted by the O2-burst measurement. However, when the excess of unbound dibromothymoquinone is washed out, which removes the quenching effect, a significant decrease of the area is seen, in agreement then with the burst results. Therefore, the meaning of the complementary area and the nature of the controlling factors may be questioned. 3. With whole cells (Chlorella), no quenching is observed; instead, an enhancement of the thermal step is noticed, together with an even larger increase of the complementary area. Dibromothymoquinone being in an oxidized form at the thylakoid level, it is proposed that in intact membranes, the chlorophyll molecules are not readily accessible to it: thus, no quenching is observable, and the predominant effect is on the redox chain. The specific effect of dibromothymoquinone on the thermal step supports the hypothesis that is normally controlled by a secondary quencher, related to plastoquinone.

Chlorella↗