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1.
The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg2+ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.  相似文献   

2.
Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca2+ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca2+ release from mitochondria when respiration is supported either by the NAD+-linked substrate β-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca2+ much better with succinate than with β-hydroxybutyrate, as respiratory substrate. When Ca2+ release is induced by t-butylhydroperoxide succinate-supported Ca2+ retention is impeded by rotenone. Ca2+ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca2+ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca2+ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.  相似文献   

3.
Mitochondria isolated from pea leaves (Pisum sativum L. var Massey Gem) and purified on a linear sucrose density gradient were substantially free of contamination by Chl and peroxisomes. They showed high respiratory rates and good respiratory control and ADP/O ratios. Malate, glutamate, succinate, glycine, pyruvate, α-ketoglutarate, NADH, and NADPH were oxidized but little or no oxidation of citrate, isocitrate, or proline was detected. The oxidation of NADPH by the purified mitochondria did not occur via a transhydrogenase or phosphatase converting it to NADH. NADPH oxidation had an absolute requirement for added Ca2+, whereas NADH oxidation proceeded in its absence. In addition, oxidation of the two substrates showed different sensitivities to chelators and sulfhydryl reagents, and faster rates of O2 uptake were observed with both substrates than with either alone. This indicates that the NADPH dehydrogenase is distinct from the exogenous NADH dehydrogenase.  相似文献   

4.
The oxidation of reduced nicotinamide adenine dinucleotide, malate-pyruvate, and succinate by corn mitochondria in buffered 0.2 m KCl was determined as a function of divalent cations. Ni2+, Mg2+, Co2+, Ca2+, Mn2+, Sr2+, and Ba2+ stimulated reduced nicotinamide adenine dinucleotide oxidation in the absence of inorganic phosphate, with Ca2+ and Sr2+ having the greatest effect. Malate-pyruvate and succinate oxidation was stimulated by Ca2+, Ba2+, and Sr2+, but only in the presence of inorganic phosphate. Ca2+, Sr2+, and Ba2+ produced a simulated state 4 to state 3 transition with all three substrates, but only with malate-pyruvate and succinate was there a return to state 4. The order of divalent cation effectiveness suggests that the rate of water substitution from the cation inner coordination hydration sphere may be a rate-limiting step in certain mitochondrial reactions involving electron transport and phosphorylation.  相似文献   

5.
Isolated liver mitochondria oxidized acetaldehyde in the following order: State 4< state 3< valinomycin. Ca2+, in concentrations greater than 0.10 mM, inhibited the oxidation of acetaldehyde by isolated liver mitochondria under all conditions. Valinomycin-stimulated oxidation of acetaldehyde was more sensitive to inhibition by Ca2+ than were the state 3 or 4 rates of acetaldehyde oxidation. Acetaldehyde could support an energy-dependent uptake of Ca2+ at rates about 20 percent that found with succinate. Ruthenium red, an inhibitor of Ca2+ translocation, almost completely prevented the inhibition by Ca2+, under all conditions. The addition of externally added NAD+ or NADH provided complete relief against the inhibitions by Ca2+ of the state 4 and 3 rates of acetaldehyde oxidation. Although some relief was also observed with the valinomycin-stimulated system, significant inhibition persisted. Cations such as Zn2+, Cu2+, or Hg2+ also inhibited acetaldehyde oxidation, whereas Mg2+ and Mn2+ were without effect. These cations also blocked glutamate oxidation and presumably inhibit acetaldehyde oxidation by preventing reoxidation of NADH. The greater sensitivity of the ionophore-stimulated oxidation of acetaldehyde to inhibition by Ca2+ may reflect release of intramitochondria K+, which is known to occur in the presence of Ca2+, suggesting that acetaldehyde oxidation is influenced by the cation environment within the mitochondria.  相似文献   

6.
A comparative study was made of the in vitro respiratory capacity of mitochondria isolated from Saccharomyces cerevisiae and Candida utilis grown in glucose-limited chemostat cultures. An electron-microscopic analysis of whole cells revealed that the volume density of mitochondria was the same in both yeasts. Mitochondria from both organisms exhibited respiratory control with NADH, pyruvate + malate, 2-oxoglutarate + acetate or malate, and ethanol. The rate of oxidation of these compounds by isolated mitochondria was the same in both yeasts. The rate of oxidation of NADPH by mitochondria from S. cerevisiae was 10 times lower than by those from C. utilis. However, this low rate probably has no influence on the overall in vivo respiratory capacity of S. cerevisiae. The results are discussed in relation to the differences in metabolic behaviour between S. cerevisiae and C. utilis upon transition of cultures from glucose limitation to glucose excess. It is concluded that the occurrence of alcoholic fermentation in S. cerevisiae under these conditions does not result from a bottleneck in the respiratory capacity of the mitochondria.  相似文献   

7.
The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na+ and 10 mM K+; these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca2+ or Mg2+. However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K+ and Na+, and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K+/Na+-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na+ or K+. In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a ΔΨ due to a low AOX activity. In D. hansenii mitochondria K+ and Na+ optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.  相似文献   

8.
Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca2+ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca2+ accumulation 6.7-fold. Hypotonic downshift-induced Ca2+ accumulation and steady-state Ca2+ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg2+. The Ca2+-influx pathway responsible for hypotonic-induced Ca2+ influx may account for about 10–35% of Ca2+ accumulation by cells growing in YPD. Ca2+ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd2+ suggesting that mutants resistant to acute Cd2+ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx. Received: 14 January 1997/Revised: 20 June 1997  相似文献   

9.
Summary Experiments were performed to obtain information on: (i) the specific properties of Ca2+ binding and transport in yeast (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast.The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca2+ in yeast. These were obtained mainly by considering actual concentrations of Ca2+ in the medium after substracting the amounts bound to the cell. Ak m of 1.9 m and aV max of 1.2 nmol (100 mg·3 min)–1 were calculated.The effects of some inhibitors and other cations on Ca2+ uptake allow one to postulate some independence between binding and transport for this divalent cation.Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca2+ uptake in yeast.The effects of Mg2+ on the uptake of Ca2+ agree with the existence of a single transport system for both divalent cations.The actions of Na+ and K+ on the transport of Ca2+ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.  相似文献   

10.
The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.  相似文献   

11.
Beffa, T., Pezet, R. and Turian, G. 1987. Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola. Mitochondria from young dormant α spores of Phomopsis viticola Sacc. (ATCC 44940) were isolated by grinding and differential centrifugation. They presented a good integrity of their inner and outer membranes as measured by biochemical assays. Electron microscopic analysis revealed an homogenous population. The highest respiratory activities were observed with NADH and ascorbate + tetra-methyl-p-phenylenediamine (TMPD). Malate stimulated the oxidation of pyruvate, citrate or α-ketoglutarate. The coupling of respiration to oxidative phosphorylation appeared at the time of spore germination. The respiratory activities of mitochondria isolated from young dormant α spores of P. viticola were strongly inhibited by S°. The sensitivity of mitochondrial oxidation of different substrates (NADH, pyruvate + malate, succinate and ascorbate + TMPD) to S° was heterogenous and indicated multiple-site action. Thus preincubation of mitochondria with 30 μM S° before addition of substrates fully prevented NADH oxidation (>98%), and strongly inhibited oxidation of pyruvate + malate (85%), succinate (60%) and ascorbate + TMPD (74%). S° inhibited more rapidly the oxidation of succinate than that of other substrates. In the presence of dithiothreitol (DTT), S°-inhibited oxidation of all substrates (except ascorbate + TMPD) could only be transiently and weakly reestablished. The inhibitory action of S° on the oxidation of NADH, pyruvate + malate and succinate was higher than that observed with sulfhydryl group reagents such as mersalyl, Hg-acetate or p - chloromercuribenzoate. In contrast to S° these SH-group reagents could not inhibit oxidation of ascorbate + TMPD. S°, by its dual capacity to oxidize the SH-groups and to self-reduce, probably at the level of cytochrome c oxidase, could produce a modification of the oxidation state of the respiratory complexes thereby disturbing the electron flux.  相似文献   

12.
R.P. Holmes  P.R. Stewart 《BBA》1979,545(1):94-105
A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other micro-organisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD.Mitochondria actively take up low concentrations of Ca2+ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca2+ concentrations in excess of 200 μM in the presence or absence of phosphate.Exogenous NADH is unique in supporting the uptake of large amounts of Ca2+ in the presence of phosphate and in showing an unusual ‘uncoupled’ response in the absence of phosphate.A sigmoidal relationship occurs between initial velocity of Ca2+ uptake and Ca2+ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 μM Ca2+.  相似文献   

13.
The respiratory rate of rat liver mitochondria in the presence of NADH as exogenous substrate is enhanced by the addition of CaCl2 (> 50 μM) when inorganic phosphate is present in the medium. The Ca-induced oxidation of NADH is inhibited by rotenone but is not affected by uncoupling agents. EDTA, which does not reverse the swelling of mitochondria which occurs in the presence of Ca2+ and phosphate, is able to inhibit reversibly the Ca-stimulated NADH oxidation. A stimulation of the rate of oxidation of NADH by Ca2+ is also observed in mitochondria partially swollen in a hypotonic medium.  相似文献   

14.
Whole cell respiration rates were measured polarographically for Naegleria gruberi during growth in agitated cultures. Log growth phase amebae consumed 80 ng atoms O/min/mg cell protein. At stationary phase, respiration rate decreased 4–fold. Intact mitochondria were isolated from N. gruberi and their oxidative and phosphorylative capacities were studied polarographically. As with the mammalian system, the mitochondria oxidized succinate and NAD-linked substrates, but unlike rat liver mitochondria, those from the protozoan rapidly oxidized citrate and NADH. The rates of substrate oxidation were ADP-dependent, with ADP:O ratios equalling ? 2.8 for NAD-linked substrates and ? 2.2 for succinate. The respiratory control ratios. 2 to 4 for 11 substrates, were dependent on Pi, Mg2+, and serum albumin. Potassium cyanide, azide, malonale, and rotenone inhibited electron transport the same way as that of the mammalian system: however, amytal inhibited both glutamate and succinate respiration. Pentachlorophenol, DNP, and bilirubin uncoupled oxidation from phosphorylation. Difference spectra of oxidized and dithionite-reduced mitochondria had distinct absorption bands of flavins and of c-, b-, and α-type cytochromes.  相似文献   

15.
Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca2+/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg2+-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD+ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD+ in the case of NAD+-linked substrates.  相似文献   

16.
Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca2+. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca2+. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca2+. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn2+ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca2+ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca2+-chelators is not reversible or is enhanced by the addition of excess Ca2+. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca2+. It is suggested that a neutral 1:2 complex is formed between Ca2+ and chlorotetracycline which can substitute for Ca2+ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.  相似文献   

17.
Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti each have an active C4-dicarboxylic acid transport system dependent on an energized membrane. Free thiol groups are probably involved at the active site. Since EDTA inhibited succinate transport in R. leguminosarum bv phaseoli and R. loti, divalent cations may participate in the process; the activity was reconstituted by the addition of Ca2+ or Mg2+. However, EDTA had no effect on succinate transport in R. tropici, R. meliloti or R. trifolii strains. Ca2+ or Mg2+ had a similar effect on the growth rates of R. tropici and R. leguminosarum bv phaseoli; R. tropici did not require Ca2+ to grow on minimal medium supplemented with succinate but R. leguminosarum bv phaseoli required either or both of the divalent cations Ca2+ and Mg2+. A R. tropici Mu-dI (lacZ) mutant defective in dicarboxylic acid transport, was isolated and found unable to form effective bean nodules.The authors are with the Division of Biochemistry, Instituto de Investigaciones Biológicas Clemente Estable, Avda, Italia 3318, 11.600 Montevideo, Uruguay  相似文献   

18.
Tightly coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts were used in this study. The two yeasts are aerobes containing the fully competent respiratory chain with three energy conservation sites. Interaction of the yeast mitochondria with prooxidants (diamide, menadione, oxaloacetate, phenylarsine oxide, hydrogen peroxide, t-butyl peroxide, and ascorbate plus Fe2+) was studied. The prooxidants, depending on their chemical nature, either caused uncoupling (e.g., activated state 4 respiration) or inhibited oxidation of respiratory substrates. All of the agents dissipated the membrane potential without megachannel formation (no large-scale swelling of mitochondria was observed). Except for combined application of ascorbate and Fe2+, the prooxidant-induced decrease in the membrane potential was specifically prevented by ATP, even in the cases when classic antioxidants, e.g., N-acetylcysteine, were ineffective. No permeabilization of yeast mitochondria was observed under concerted action of prooxidants and Ca2+, suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with Ca2+ uptake.  相似文献   

19.
Some features of the Ca2+-transport system in mitochondria of the yeast Endomyces magnusii are considered. The Ca2+ uniporter was shown to be specifically activated by low concentrations of physiological modulators such as ADP, NADH, spermine, and Ca2+ itself. The Na+-independent system responsible for Ca2+ release from Ca2+-preloaded yeast mitochondria was characterized. The rate of the Ca2+ release was proportional to the Ca2+ load, insensitive to cyclosporin A and to Na+, inhibited by La3+, TPP+, Pi, and nigericin, while being activated by spermine. We conclude that Ca2+ release from preloaded E. magnusii yeast mitochondria is mediated by a Na+-independent pathway, very similar to that in mitochondria from nonexcitable mammalian tissues. A scheme describing an arrangement of the Ca2+ transport system of yeast mitochondria is proposed.  相似文献   

20.
Isolated mitochondria of wheat shoots oxidize α- ketoglutarate, DL-malate succinate and NADH with good relative respiration control and ADP: O ratio. They have high affinity for α-ketoglutarate and NADH as substrates and utilize malate and succinate with a respiration ratio of about one-half of α-ketoglutarate. The average ADP : O ratios approach the expected theoretical values, i.e., 3.6 ± 0.2 for α-ketoglutarate, 1.8 ± 0.2 for succinate, and 2.8 ± 0.2 for malate. The ADP: O ratio with NADH is 1.8 ± 0.2. The maximum coupling of oxidation and phosphorylation is obtained at concentrations of 10 mM, 2 mM, 10 mM and 8 mM for α-ketoglutarate, NADH, malate and succinate, respectively. — Wheat mitochondria have little or no dependence on added cofactors. Mitochondria prepared by our procedure apparently retain sufficient amounts of endogenous cofactors required for NAD-linked systems. FAD+ is found to improve succinate oxidation. Cytochrome c does not have any significant effect on respiratory parameters of wheat mitochondria. — Wheat mitochondria are some -what resistant to DNP at 1.7 × 10-5M. Malonate seems to improve coupling of α-ketoglutarate oxidation. Other Krebs cycle intermediates have been tested on three major substrates of TCA cycle, i.e., α-ketoglutarate, malate and succinate.  相似文献   

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