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PubMed1980

Dependence of Delayed Luminescence upon Adenosine Triphosphatase Activity in Chlorella.

Delayed luminescence and fluorescence yield after illumination by a short flash were measured in Chlorella pyrenoidosa in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Addition of tri-N-butyl-tin (TNBT), a specific inhibitor of ATPase, drastically increases the life-time of the reduced photosystem II primary acceptor Q(-) and decreases the intensity of delayed luminescence. This indicates a slowing of the charge recombination between the oxidized donor and reduced acceptor of photosystem II centers. No inhibition is observed in isolated chloroplasts when the membrane is permeable to ions, i.e. in the presence of Gramicidin D and KCl.It is suggested that there exists in dark-adapted algae a permanent proton gradient which stimulates the charge recombination process. This proton gradient results from the hydrolysis of a pool of ATP by membrane-bound ATPases and collapses after the addition of TNBT. The long lifetime of this proton gradient (several hours) indicates that the ATP probably comes from the mitochondria.The rate of the back reaction occurring from state S(3) (as defined by Kok, Forbush, and McGloin 1970 Photochem Photobiol 11: 457-475) is more dependent upon the pH gradient than for state S(2).

Journal Article↗

PubMed1983

The electrochemical H+ gradient of platelet secretory alpha-granules. Contribution of a H+ pump and a Donnan potential.

Experiments were performed to determine the internal pH and membrane potential of platelet alpha-granules. Fluorescence microscopy showed accumulation of weak bases, indicative of an acidic interior, inside secretory vesicles in intact platelets and in isolated alpha-granules. Weak base uptake was pH-dependent and NH+4-sensitive. In isolated alpha-granules suspended in medium buffered at pH 7.2, a delta pH, the difference between internal and external pH, of 1.2 (inside acidic) was measured by [14C]methylamine distribution. Uptake of isotopic or fluorescent amines was reduced by H+/cation exchange via ionophores and by addition of NH+4, but also by increasing the ionic strength suggesting that delta pH is partly due to a Donnan potential. Transmembrane potential measurements by fluorescent or radioactive ion distribution indicated that in the absence of ATP, granules are internally negative. When measured with 86Rb+, this potential could be entirely collapsed by increasing the ionic strength. Addition of ATP X Mg in the absence of permeating anions made the intragranular space more positive, as expected from inward electrogenic H+ pumping. The results are compatible with the coexistence of sealed and leaky subpopulations of alpha-granules. Internal acidity was generated in sealed granules in vivo by a H+-pumping ATPase, whereas in leaky granules acidity is a consequence of an internally negative Donnan potential.

Ammonia↗

PubMed1975

The functional unit of electrical events and phosphorylation in chromatophores from Rhodopseudomonas sphaeroides.

1. From electron micrographs of chromatophores from Rhodopseudomonas spaeroides and from the estimated bacteriochlorophyll per chromatophore was estimated. The mean diameter of the chromatophore vesicles was 600 A. 2. The decay of the flash-induced electric potential across the chromatophore membrane measured by the carotenoid band shift was 20% accelerated by about one valinomycin molecule per 4700 bacteriochlorophyll, i.e. by one ionophore molecule per chromatophore. 3. The inhibition of the flash-induced ATP formation by valinomycin followed a similar pattern to the accelerated decay of the electric potential. 4. The single turnover flash yield of ATP synthesis gave a mean value of one ATP per 1470 bacteriochlorophyll or about 3 ATP per vesicle. 5. With regard to the partitioning of the ionophore between the membrane (85%) and aqueous phase (15%) we conclude that one molecule of valinomycin per chromatophore is sufficient to begin to collapse the electrical potential and inhibit ATP synthesis. It is therefore suggested that the membrane potential is an essential component of the energized state which is used for phosphorylation. The results correspond to those obtained for the 100-fold larger vesicles in chloroplasts (thylakoids) where one molecule of ioophore is also sufficient to quench both events.

Adenosine Triphosphate↗

PubMed1994

Role of Mg-ATP in norepinephrine biosynthesis in intact chromaffin granules.

Dopamine beta-monooxygenase converts dopamine to norepinephrine in intact chromaffin granules using intragranular ascorbic acid as a cosubstrate. Mg-ATP with external ascorbic acid is required for maximal norepinephrine biosynthesis. Mechanisms to explain these requirements were investigated specifically using intact granules. The effect of Mg-ATP was independent of membrane potential (delta psi) because norepinephrine biosynthesis was unchanged whether delta psi was positive or collapsed. Furthermore, the effect of Mg-ATP was independent of absolute intragranular and extragranular pH as well as the pH difference across the chromaffin granule membrane (delta pH). Nevertheless, norepinephrine biosynthesis was inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzofurazane, and N,N-dicyclohexylcarbodiimide, specific inhibitors of the secretory vesicle ATPase that may directly affect proton pumping. Biosynthesis occurred normally with other ATPase inhibitors that do not inhibit the ATPase in secretory vesicles. The data indicate that the effect of Mg-ATP with ascorbic acid is mediated by the granule membrane ATPase but independent of maintaining delta psi and delta pH. An explanation of these findings is that Mg-ATP, via the granule ATPase, may change the rate at which protons or dopamine are made available to dopamine beta-monooxygenase.

Adenosine Triphosphatases↗

PubMed2000

Dynamic in vivo (31)P nuclear magnetic resonance study of Saccharomyces cerevisiae in glucose-limited chemostat culture during the aerobic-anaerobic shift.

The purpose of this work was to analyse in vivo the influence of sudden oxygen depletion on Saccharomyces cerevisiae, grown in glucose-limited chemostat culture, using a recently developed cyclone reactor coupled with (31)P NMR spectroscopy. Before, during and after the transition, intracellular and extracellular phosphorylated metabolites as well as the pHs in the different cellular compartments were monitored with a time resolution of 2.5 min. The employed integrated NMR bioreactor system allowed the defined glucose-limited continuous cultivation of yeast at a density of 75 g DW/l and a p(O(2)) of 30% air saturation. A purely oxidative metabolism was maintained at all times. In vivo (31)P NMR spectra obtained were of excellent quality and even allowed the detection of phosphoenolpyruvate (PEP). During the switch from aerobic to anaerobic conditions, a rapid, significant decrease of intracellular ATP and PEP levels was observed and the cytoplasmic pH decreased from 7.5 to 6.8. This change, which was accompanied by a transient influx of extracellular inorganic phosphate (P(i)), appeared to correlate linearly with the decrease of the ATP concentration, suggesting that the cause of the partial collapse of the plasma membrane pH gradient was a reduced availability of ATP. The complete phosphorous balance established from our measurement data showed that polyphosphate was not the source of the increased intracellular P(i). The derived intracellular P(i), ATP and ADP concentration data confirmed that the glycolytic flux at the level of glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and enolase enzymes is mainly controlled by thermodynamic constraints.

Adenosine Diphosphate↗

PubMed1998

Protein phosphatase inhibitors and heat preconditioning prevent Hsp27 dephosphorylation, F-actin disruption and deterioration of morphology in ATP-depleted endothelial cells.

The vascular endothelium response to ischemic depletion of ATP was studied in vitro. Endothelial cells (EC) cultured from human aorta or umbilical vein were incubated in a glucose-free medium containing CCCP or rotenone. Such blockade of energy metabolism caused a drop in ATP, destruction of actin filaments, morphological changes, and eventually disintegration of EC monolayer within 2-2.5 h. While ATP fell and F-actin collapsed, the 27-kDa heat shock protein (Hsp27) lost basal phosphorylation and became Triton X-100-insoluble forming granules inside the cell nuclei. Protein phosphatase (PP) inhibitors (okadaic acid, cantharidin, sodium orthovanadate) did not delay the ATP decrease in energy-deprived EC but arrested both the alterations in the Hsp27 status and the changes for the worse in F-actin and cell morphology. Similarly, the Hsp27 dephosphorylation/insolubilization/granulation and the cytoskeletal and morphological disturbances resulting from lack of ATP were suppressed in heat-preconditioned (thermotolerant) cultures, this effect being sensitive to quercetin, a blocker of Hsp induction. The longer preservation of the cytosolic pool of phosphorylated Hsp27 during ATP depletion in the PP inhibitor-treated or thermotolerant EC correlated with the acquired resistance of F-actin and morphology. These data suggest that PP inhibitors as well as heat-inducible Hsp(s) can protect ischemia-stressed cells by preventing the ATP loss-provoked protein dephosphorylation and breakdown of the actin cytoskeleton.

Actins↗

PubMed2002

The carboxyl terminus of the epsilon subunit of the chloroplast ATP synthase is exposed during illumination.

The epsilon subunit of the chloroplast ATP synthase is an inhibitor of activity of the enzyme. Recombinant forms of the epsilon subunit from spinach chloroplasts lacking the last 10, 32, or 45 amino acids were immobilized onto activated Sepharose. A polyclonal antiserum raised against the epsilon subunit was passed over these immobilized protein columns, and the purified antibodies which were not bound recognized the portions of the epsilon subunit missing from the recombinant form present on the column. The full polyclonal antiserum can strip the epsilon subunit from the ATP synthase in illuminated thylakoid membranes [Richter, M. L., and McCarty, R. E. (1987) J. Biol. Chem. 262, 15037-15040]. Exposure of illuminated thylakoid membranes to antibodies recognizing the last 32 amino acids of the epsilon subunit collapses the proton gradient and hinders ATP synthesis with similar efficiency as the full polyclonal preparation. These results indicate that antibodies against the last 32 amino acids of the epsilon subunit are capable of stripping the subunit from the ATP synthase in illuminated membranes. Neither of these effects was seen when the membranes were exposed to the antibodies in the dark. This is direct evidence that the chloroplast ATP synthase undergoes a conformational shift during its activation by the electrochemical proton gradient which specifically alters the conformation of the carboxyl-terminal domain of the epsilon subunit from protected to solvent-exposed. The relation between this shift and activation of the enzyme by the electrochemical proton gradient is discussed.

Antibodies↗

PubMed1990

Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial F1F0-ATPase and construction of mutant yeast lacking the protein.

A gene coding for yeast 15-kDa protein, a regulatory factor of mitochondrial F1F0-ATPase, was isolated. The cloned gene was disrupted in vitro and mutant strains that did not contain the 15-kDa protein were constructed by transformation of yeast cells with the disrupted gene. The ATP-synthesizing activity of the mutant mitochondria was the same as that of wild-type cells, suggesting that the 15-kDa protein is not required for mitochondrial oxidative phosphorylation. Collapse of the membrane potential induced ATP-hydrolyzing activity of F1F0-ATPase of the mutant mitochondria but not of normal mitochondria. Activation of the enzyme was also observed during incubation of submitochondrial particles from mutant cells, but not of those from wild-type cells. Thus, it is inferred that the 15-kDa protein supports the action of an intrinsic ATPase inhibitor of the ATP-hydrolyzing activity of the enzyme upon de-energization of mitochondrial membranes.

Amino Acid Sequence↗

PubMed1996

ATP-dependent release of glucocorticoid receptors from the nuclear matrix.

Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNA-containing nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a subpopulation of simian virus 40 large tumor antigen with the nuclear matrix is not quantitatively altered in ATP-depleted Cos-1 cells. Nuclear GRs which are not bound to the nuclear matrix of metabolically active cells (i.e., a DNA-binding domain deletion mutant and a beta-galactosidase chimera possessing the GR nuclear localization signal sequence) are not recruited to the matrix upon depletion of cellular ATP. Thus, it appears that ATP depletion does not expose the GR to nuclear matrix interactions which are not normally encountered in cells but merely alters the dynamics of such interactions. The dynamic association of steroid receptors with the nuclear matrix may provide a mechanism which is utilized by these regulable transcription factors to facilitate their efficient scanning of the genome.

Adenosine Triphosphate↗

PubMed1993

A new paradigm for biochemical energy coupling. Salmonella typhimurium nicotinate phosphoribosyltransferase.

The pncB gene of Salmonella typhimurium was used to develop an overexpression system for nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11), which forms nicotinate mononucleotide (NAMN) and PPi from nicotinate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAPRTase hydrolyzes ATP in 1:1 molar stoichiometry to NAMN synthesis. Hydrolysis of ATP alters the ratio of products/substrates for the reaction nicotinate + PRPP <--> NAMN + PPi from its equilibrium value of 0.67 to a steady-state value of 1100. The energy for the maintenance of this ratio must come from ATP hydrolysis. However, in contrast to other ATP-utilizing enzymes, when all ATP is hydrolyzed the unfavorable product/substrate ratio collapses. ATP/ADP exchange results suggest that the overall reaction involves a phosphoenzyme (E-P) arising from E.ATP. Km values for nicotinate and PRPP each decreased by 200-fold when ATP was present to phosphorylate the enzyme. PPi stimulated the ATPase activity of the enzyme to Vmax values, suggesting that PPi formation during catalysis provides a trigger for cleavage of the putative E-P in the overall reaction and regenerates the low affinity form of the enzyme. A model is presented in which alternation of high and low affinity forms of NAPRTase provides a "steady-state" coupling between ATP hydrolysis and NAMN formation.

Cloning, Molecular↗

PubMedDate not supplied

[The role of the mitochondria in the clearance of cytosolic calcium].

The magnitude and space-temporal profile of the intracellular Ca2+ transients are determined both by the mechanism that decrease and increase calcium levels in the cytoplasm. By the use of cocktails with different content of specific inhibitors of the extrusion and sequester mechanisms, the ability of mitochondrial Ca2+ transport to limit the elevation in free cytosolic Ca2+ concentration, following an imposed Ca2+ load was reexamined, indicating variable data with respect to various cells. In chromaffin cells, inhibition of mitochondrial Ca2+ accumulation with protonophore, dramatically modifies the shape of the [Ca2+]c response, indicating that mitochondrial Ca2+ uptake is an important mechanism for clearance of large Ca2+ loads. By contrast, using digital imaging in the presence of specific mitochondria inhibitors to investigate the [Ca2+]c responses of cerebellar granule cells in which ATP generation has been totally separated from mitochondrial Ca2+ transport, indicates surprising results: it was confirmed that mitochondria in these cells accumulate Ca2+ entering the cell in response to plasma membrane depolarization, but specific abolition of mitochondrial Ca2+ accumulation without ATP depletion significantly decreases the bulk cytoplasmic Ca2+ transients generated by elevated KCl levels, whereas the response in greatly increased when protonophore are present and ATP/ADP ratios are allowed to collapse. The results suggest that nonmitochondrial ATP-dependent transport pathways are primarily responsible for removing excess Ca2+ from the cytoplasm. Far from restricting the elevation in [Ca2+]c in response to a Ca2+ load, functional mitochondria may enhance the elevation in the bulk cytoplasm. The existent conflict of data, suggests the need for a new reevaluation of the role of mitochondria in Ca2+ clearance, and the possibility that mitochondria contribute to, rather than protect against, excitoxicity has to be investigated.

Animals↗

PubMed2004

Bacillus amyloliquefaciens strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells.

Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 microg dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (Delta Psi(m)) and the plasma membrane (Delta Psi(p)) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K(+):Na(+):Ca(2+) of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.

Air Microbiology↗

PubMed2002

Mechanochemical ATPases and transcriptional activation.

Transcriptional activator proteins that act upon the sigma54-containing form of the bacterial RNA polymerase belong to the extensive AAA+ superfamily of ATPases, members of which are found in all three kingdoms of life and function in diverse cellular processes, often via chaperone-like activities. Formation and collapse of the transition state of ATP for hydrolysis appears to engender the interaction of the activator proteins with sigma54 and leads to the protein structural transitions needed for RNA polymerase to isomerize and engage with the DNA template strand. The common oligomeric structures of AAA+ proteins and the creation of the active site for ATP hydrolysis between protomers suggest that the critical changes in protomer structure required for productive interactions with sigma54-holoenzyme occur as a consequence of sensing the state of the gamma-phosphate of ATP. Depending upon the form of nucleotide bound, different functional states of the activator are created that have distinct substrate and chaperone-like binding activities. In particular, interprotomer ATP interactions rely upon the use of an arginine finger, a situation reminiscent of GTPase-activating proteins.

Adenosine Triphosphatases↗

PubMed2004

Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA+ transcriptional activator protein.

Enhancer-dependent transcriptional activators that act upon the sigma54 bacterial RNA polymerase holoenzyme belong to the extensive AAA+ superfamily of mechanochemical ATPases. Formation and collapse of the transition state for ATP hydrolysis engenders direct interactions between AAA+ activators and the sigma54 factor, required for RNA polymerase isomerization. A DNA fork junction structure present within closed complexes serves as a nucleation point for the DNA melting seen in open promoter complexes and restricts spontaneous activator-independent RNA polymerase isomerization. We now provide physical evidence showing that the ADP.AlF(x) bound form of the AAA+ domain of the transcriptional activator protein PspF changes interactions between sigma54-RNA polymerase and a DNA fork junction structure present in the closed promoter complex. The results suggest that one functional state of the nucleotide-bound activator serves to alter DNA binding by sigma54 and sigma54-RNA polymerase and appears to drive events that precede DNA opening. Clear evidence for a DNA-interacting activity in the AAA+ domain of PspF was obtained, suggesting that PspF may make a direct contact to the DNA component of a basal promoter complex to promote changes in sigma54-RNA polymerase-DNA interactions that favour open complex formation. We also provide evidence for two distinct closed promoter complexes with differing stabilities.

Adenosine Diphosphate↗

PubMed1996

A reevaluation of the role of mitochondria in neuronal Ca2+ homeostasis.

The ability of mitochondrial Ca2+ transport to limit the elevation in free cytoplasmic Ca2+ concentration in neurones following an imposed Ca2+ load is reexamined. Cultured cerebellar granule cells were monitored by digital fura-2 imaging. Following KCl depolarization, addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) to depolarize mitochondria released a pool of Ca2+ into the cytoplasm in both somata and neurites. No CCCP-releasable pool was found in nondepolarized cells. Although the KCl-evoked somatic and neurite Ca2+ concentration elevations were enhanced when CCCP was present during KCl depolarization, this was associated with a collapsed ATP/ADP ratio. In the presence of the ATP synthase inhibitor oligomycin, glycolysis maintained high ATP/ADP ratios for at least 10 min. The further addition of the mitochondrial complex I inhibitor rotenone led to a collapse of the mitochondrial membrane potential, monitored by rhodamine-123, but had no effect on ATP/ADP ratios. In the presence of rotenone/oligomycin, no CCCP-releasable pool was found subsequent to KCl depolarization, consistent with the abolition of mitochondrial Ca2+ transport; however, paradoxically the KCl-evoked Ca2+ elevation is decreased. It is concluded that the CCCP-induced increase in cytoplasmic Ca2+ response to KCl is due to inhibition of nonmitochondrial ATP-dependent transport and that mitochondrial Ca2+ transport enhances entry of Ca2+, perhaps by removing the cation from cytoplasmic sites responsible for feedback inhibition of voltage-activated Ca2+ channel activity.

Adenosine Triphosphate↗

PubMed1988

Effect of the electrochemical proton gradient and anions on the ATPase activity of soybean submitochondrial particles.

Submitochondrial particles from soybean (Glycine max L. cv Jupiter) hypocotyls with an ATPase activity of 0.3 to 1.0 micromole per minute per milligram were prepared by sonication with Mg-ATP. The particles catalyzed ATP synthesis with NADH and succinate; the ratios of ATP/O with these substrates were 1.0 and 0.1, respectively. As monitored by oxonol-VI, the particles built up and maintained a membrane potential that was higher with NADH than with succinate or Mg-ATP. The ATPase activity of the particles increased two to threefold by preincubation with 50 millimolar phosphate at a temperature of 38 degrees C. The increase in ATPase activity became higher (five to sixfold) when particles were preincubated with Mg-ATP plus phosphate. Under the latter conditions, collapse of DeltamuH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevented the activation. An increase in ATPase activity of the particles was also observed with NADH and succinate, although activation was lower with succinate. With these substrates, phosphate did not increase ATPase activation. When particles were preincubated with Mg-ATP, anions that stimulate ATP hydrolysis (malate, malonate, and bicarbonate) had an activating effect similar to that of phosphate. The data suggest that the soybean mitochondrial ATPase can be activated by DeltamuH but that this activation is increased by the binding of certain anions to a conformation of the enzyme that appears during hydrolytic cycles.

Journal Article↗

PubMed2001

A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons.

In order to investigate the relationship between nitric oxide-mediated regulation of mitochondrial function and excitotoxicity, the role of mitochondrial ATP synthesis and intracellular redox status on the mode of neuronal cell death was studied. Brief (5 min) glutamate (100 microM) receptor stimulation in primary cortical neurons collapsed the mitochondrial membrane potential (psi(m)) and transiently (30 min) inhibited mitochondrial ATP synthesis, causing early (1 h) necrosis or delayed (24 h) apoptosis. The transient inhibition of ATP synthesis was paralleled to a loss of NADH, which was fully recovered shortly after the insult. In contrast, NADPH and the GSH/GSSG ratio were maintained, but progressively decreased thereafter. Twenty-four hours after glutamate treatment, ATP was depleted, a phenomenon associated with a persistent inhibition of mitochondrial succinate-cytochrome c reductase activity and delayed necrosis. Blockade of either nitric oxide synthase (NOS) activity or the mitochondrial permeability transition (MPT) pore prevented psi(m) collapse, the transient inhibition of mitochondrial ATP synthesis, early necrosis and delayed apoptosis. However, blockade of NOS activity, but not the MPT pore, prevented the inhibition of succinate-cytochrome c reductase activity and delayed ATP depletion and necrosis. From these results, we suggest that glutamate receptor-mediated NOS activation would trigger MPT pore opening and transient inhibition of ATP synthesis leading to apoptosis in a neuronal subpopulation, whereas other groups of neurons would undergo oxidative stress and persistent inhibition of ATP synthesis leading to necrosis.

2-Amino-5-phosphonovalerate↗

PubMed2005

Inhibition of the mitochondrial ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene.

Polygodial is a naturally occurring sesquiterpene dialdehyde that exhibits several pharmacologically interesting activities. Among them, its antifungal properties have been more thoroughly studied. The mitochondrial ATPase has been suggested as one of the possible targets for polygodial action. However, its mechanism of action is not well defined yet. The effect of polygodial on the mitochondrial energy metabolism is described in this paper. Polygodial inhibited ATP synthesis coupled to succinate oxidation in beef-heart submitochondrial particles at concentrations (IC(50)=2.4+/-0.1 microM) which marginally affected electron transport and ATPase activity (IC(50)=97+/-4 microM). A transitory stimulation of the electron transport in intact rat liver mitochondria in state 4 was also obtained at low polygodial concentrations (EC(50)=20+/-4 microM). These results suggest that polygodial uncouples ATP synthesis from electron transport at low concentrations. Similar concentrations of polygodial partially abolished the ANS fluorescence enhancement (IC(50)=2.2+/-0.4 microM) induced by succinate oxidation in submitochondrial particles but did not collapse the DeltapH. We postulate that polygodial uncouples mitochondrial ATP synthesis by affecting the electrical properties of the membrane surface and consequently collapsing the membrane potential (Deltapsi) and/or the localized transmembrane pH difference (DeltapH(S)) without affecting the DeltapH between the two bulk aqueous phases (DeltapH(B)). The relevance of these findings for the understanding of the biochemical basis of the antifungal activity of polygodial and the evaluation of its potentiality as a therapeutic agent are discussed.

Adenosine Triphosphate↗