共查询到20条相似文献,搜索用时 31 毫秒
1.
D. A. Aliverdieva D. V. Mamaev D. I. Bondarenko 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2008,2(4):354-364
Succinate and citrate transport into yeast (Saccharomyces cerevisiae) cells was studied by measuring substrate oxidation rates in the presence and in the absence of effective impermeable oxidation inhibitors O-palmitoyl-L-malate and 2-undecyl malonate. Linearity of the Dixon plot for 2-undecyl malonate suggests that this inhibitor blocked the rate-limiting step upon oxidation of both substrates, which was, most probably, transport of these substrates across the plasma membrane (due to inability of the inhibitor to penetrate into the membrane). This approach allowed fast (within 30–40 min) measurement of kinetic parameters of the transporter in individual samples without losing control over limiting conditions. In case of succinate transport, the limiting rate of succinate oxidation (V max) depended on pH and increased monotonously from near-zero at pH 4.5 to the maximum level at pH 7.5. At pH 5.5, succinate and citrate transport was insensitive to the protonophore FCCP, being activated by Na+ ions and competitively inhibited by 2-undecyl malonate and K+ ions. Values of K i for 2-undecyl malonate were similar for both substrates. These data suggest that citrate and succinate influx is mediated by a common plasma membrane transporter. This is not typical of fungi. At pH 6.5, Tris+, K+ and Na+ had no effect on succinate oxidation. In monosodium media pH increase was accompanied by a decrease of succinate K m due to higher proportion of the dianionic form of the substrate. Atypical substrate specificity and mechanisms of functional activity of the dicarboxylate transporter in plasma membrane of S. cerevisiae are discussed. 相似文献
2.
The active transport of 2-keto-D-gluconate in vesicles prepared from Pseudomonas purida. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The transport of 2-keto-D-gluconate (alpha-D-arabino-2-hexulopyranosonic acid; 2KGA) in vesicles prepared from glucose-grown Pseudomonas putida occurs by a saturable process with a Km of 110.0 +/- 2.9 microM and a Vmax. of 0.55 +/- 0.04 nmol X min-1 X (mg of protein)-1. The provision of phenazine methosulphate/ascorbate or L-malate leads to an accumulation of intravescular 2KGA, a decrease in the Km value to 50 +/- 2.1 microM and 35 +/- 2.9 microM respectively and no change in the Vmax. In the presence of electron donors the transport of 2KGA is inhibited by the respiratory poisons antimycin A, rotenone and the uncoupler 2,4-dinitrophenol. 2KGA transport is also competitively inhibited by 4-deoxy-4-fluoro-2-keto- or 3-deoxy-3-fluoro-2-keto-D-gluconate with Ki values of 50 microM and 160 microM respectively. The carrier system for 2KGA is repressed in vesicles from cells grown on succinate. Such vesicles transport 2KGA by non-specific physical diffusion with a Km value of infinity in the absence or presence of electron donors. Vesicles from glucose or succinate grown cells, in the presence of phenazine methosulphate/ascorbate at pH 6.6, generate a proton-motive force (delta p) of approx. 140 mV. The delta p, composed of proton gradient (delta pH) and a membrane potential (delta psi), is collapsed in the presence of dinitrophenol. Based on the results obtained with valinomycin, nigericin and carbonyl cyanide m-chlorophenylhydrazone, the active transport of 2KGA at pH 6.6 is coupled predominately to the delta pH component of delta p. 相似文献
3.
Calcium uptake into filipin-treated bovine spermatozoa is completely inhibited by the uncoupler CCCP or by ruthenium red. Both Pi and mitochondrial substrates are required to obtain the maximal rate of calcium uptake into the sperm mitochondria. Bicarbonate and other anions such as lactate, acetate or beta-hydroxybutyrate do not support a high rate of calcium uptake. There are significant differences among various mitochondrial substrates in supporting calcium uptake. The best substrates are durohydroquinone, alpha-glycerophosphate and lactate. Pyruvate is a relatively poor substrate, and its rate can be greatly enhanced by malate or succinate but not by oxalacetate or lactate. This stimulation is blocked by the dicarboxylate translocase inhibitor, butylmalonate and can be mimiced by the non-metabolized substrate D-malate. The Ka for pyruvate was found to be 17 microM and 67 microM in the presence and absence of L-malate, respectively. The Ka for L-malate is 0.12 mM. It is suggested that in addition to the known pyruvate/lactate translocase there is a second translocase for pyruvate which is malate/succinate-dependent and does not transport lactate. In the presence of succinate, glutamate stimulates calcium uptake 3-fold, and this effect is not inhibited by rotenone. In the presence of glutamate plus malate or oxalacetate there is only an additive effect. It is suggested that glutamate stimulates succinate transport and/or oxidation in bovine sperm mitochondria. The alpha-hydroxybutyrate is almost as good as lactate in supporting calcium uptake. Since the alpha-keto product is not further metabolized in the citric acid cycle, it is suggested that lactate can supply the mitochondrial needs for NADH from its oxidation to pyruvate by the sperm lactate dehydrogenase x. Thus, when there is sufficient lactate in the sperm mitochondria, pyruvate need not be further metabolized in the citric acid cycle in order to supply more NADH. 相似文献
4.
Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria 总被引:9,自引:3,他引:6
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme. 相似文献
5.
It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the Ki value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [14C] succinate and [14C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/Pi, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited Pi transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [14C]succinate/anion (Pi, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and Pi translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and Pi translocation across the mitochondrial membrane. 相似文献
6.
W W Kay 《Canadian journal of microbiology》1981,27(8):795-800
Membrane vesicles of Bacillus subtilis W23 actively transport the C4 and C5 dicarboxylates of the tricarboxylate cycle by system(s) of relatively high affinity for their requisite substrates (Km 4-53 microM). Glutamate and succinate binding activities were readily solubilized from membrane vesicles by nonionic detergents, particularly by Lubrol WX. From this extract, glutamate binding activity was highly enriched by affinity chromatography on phloroglucinol-expanded Sepharose-6B to which L-aspartate was coupled via divinylsulfone. Another protein (41000 molecular weight), which bound both L-glutamate and L-malate, was purified from affinity columns to which either L-glutamate or L-malate had been coupled via bisdiglycidyl ether. This protein bound labelled L-malate as well as L-glutamate with affinities similar to those seen with membrane vesicles (Kd's 8 microM L-malate and 52 microM L-glutamate). 相似文献
7.
The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15+/-6 nmol min-1 mg protein-1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 microM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8-17 nmol min-1 mg protein-1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface. 相似文献
8.
2-Alkylmalonates and O-acyl-L-malates have been found to competitively inhibit the dicarboxylate transporter of Saccharomyces cerevisiae cells, and the substrate derivatives chosen did not penetrate across the plasmalemma under the experiment conditions. Probing of the active site of this transporter has revealed a large lipophilic area stretching between the 0.72 to 2.5 nm from the substrate-binding site. Itaconate inhibited the transport fivefold more effectively than L-malate. This suggests the existence of a hydrophobic region immediately near the dicarboxylate-binding site (to 0.72 nm). The yeast plasmalemmal transporter was different from the rat liver mitochondrial dicarboxylate transporter. An area with variable lipophilicity adjoining the substrate-binding site has been revealed in the latter by a similar method. This area is mainly hydrophobic at distances up to 1.76 nm from the binding site and is separated by a hydrophilic region from 0.38 to 0.88 nm. Fumarate but not maleate competitively inhibited succinate transport into the S. cerevisiae cells. It is suggested that the plasmalemmal transporter binds the substrate in the trans-conformation. The prospects of the proposed approach for scanning lipophilic profiles of channels of different transporters are discussed. 相似文献
9.
G C Brown 《The Journal of biological chemistry》1989,264(25):14704-14709
Several different proton pumps were used to generate a proton motive force (delta p, proton motive force across the mitochondrial inner membrane) in isolated rat liver mitochondria, and the relationship between delta p and pump rate was investigated by titrating with various inhibitors of the pumps. It was found that this relationship was the same for mitochondria respiring on succinate irrespective of whether respiration was inhibited with malonate, antimycin or cyanide, indicating that the relationship was independent of the redox state of the respiratory chain. When delta p was generated by either the cytochrome bc1 complex, cytochrome oxidase, both together, or ATP hydrolysis (and transport), the reaction rates (in moles of electrons or ATP) were in the ratio of close to 3:1.5:1:1, respectively, at all accessible values of delta p. This suggests that the proton stoichiometries (H+/e and H+/ATP, where H+/e is the number of protons translocated vectorially across the inner membrane per electron transferred by the respiratory chain and H+/ATP is the number of protons translocated vectorially per ATP molecule hydrolyzed externally) were in the ratio of close to 1:2:3:3, respectively, at all values of delta p. Possible reasons for previous contradictory results are suggested. 相似文献
10.
Treatment of rats with thiamphenicol in a dose of 125 mg/kg per day for 60–64 h causes specific inhibition of mitochondrial protein synthesis, leading to a drastic decrease of the cytochrome c oxidase activity in intestinal epithelium. At the same time the mitochondrial ATPase activity becomes resistant to inhibition by oligomycin. Experiments with isolated intestinal mitochondria revealed that respiration in state 3 is diminished by 55% with succinate (5 mM) and by 40% with pyruvate (10 mM) plus L-malate (2 mM) as the substrates, both as compared to intestinal mitochondria isolated from control rats. P : O ratios as well as respiratory control indices are comparable in the two groups of animals. Uncoupled respiration is inhibited by 35% with succinate as the substrate, while the succinate cytochrome c reductase activity remains unaltered. No inhibition of uncoupled respiration with pyruvate plus L-malate as the substrates was observed. The ATP-Pi exchange activity in the mitochondria from the treated animals is diminished by about 75%. It is concluded that in the mitochondria of the treated animals the inhibition of the coupled respiration (state 3) is caused by the limitation of the ATP-generating capacity and that electron transport is rate limiting only with the rapidly oxidized substrates such as succinate, if respiration is uncoupled. 相似文献
11.
The relationship between the rate of substrate oxidation and the protonmotive force (electrochemical proton gradient) generated by bovine heart submitochondrial particles has been examined. Unexpectedly, oxidation of succinate generated a higher protonmotive force than the oxidation of NADH, although the rate of proton translocation across the membrane was inferred to be considerably lower with succinate as substrate. The data suggest that the flow of electrons through site 1 of the respiratory chain may increase the conductance of the mitochondrial membrane for protons. Upon reduction of the rate of succinate oxidation by titration with malonate, the protonmotive force remained essentially constant until the extent of inhibition was greater than 75%. The general conclusion from this work is that a constant passive membrane conductance for protons cannot be assumed. 相似文献
12.
Isolated plasma membrane vesicles and the plasma membrane NADH oxidase partially purified from soybean plasma membrane vesicles exhibited a cyanide-insensitive vitamin K(1) hydroquinone oxidase activity with isolated plasma membrane vesicles. Reduced vitamin K(1) (phylloquinol) was oxidized at a rate of about 10 nmol/min/mg protein as determined by reduced vitamin K(1) reduction or oxygen consumption. The K(m) for reduced K(1) was 350 microM. With the partially purified enzyme, reduced vitamin K(1) was oxidized at a rate of about 600 nmol/min/mg protein and the K(m) was 400 microM. When assayed in the presence of 1 mM KCN, activities of both plasma membrane vesicles and of the purified protein were stimulated (0.1 microM) or inhibited (0.1 mM) by the synthetic auxin growth factor 2, 4-dichlorophenoxyacetic acid. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via reduced vitamin K(1) to acceptors (molecular oxygen or protein disulfides) at the cell surface. 相似文献
13.
E Wodtke 《Biochimica et biophysica acta》1981,640(3):710-720
The acclimation temperature of carp does not affect the amount of cytochrome c oxidase per mg mitochondrial protein as revealed from the reduced-minus-oxidized difference spectra of red muscle mitochondria from cold- and warm-acclimated carp. There are no differences between cold- and warm-acclimated fish in the substrate binding properties of the enzyme as judged from the Km values for cytochrome c at 30 degrees C (3.34 +/- 0.ee microM, acclimation temperature 10 degrees C and 3.55 +/- 0.31 microM, acclimation temperature 30 degrees C). The molar activities of the enzyme, however, differ for both acclimation temperatures: when intercalated in the 10 degrees C-acclimated mitochondrial membrane, the enzyme can catalyze the oxidation of 117.6 +/- 17.2 mol ferrocytochrome c/s per mol heme a as compared with 85.6 +/- 17.2 in the 30 degrees C-acclimated membrane (experimental temperature 30 degrees C). Correspondingly, higher specific activities of the succinate oxidase system are observed in mitochondria from cold-acclimated carp as compared with those obtained from warm-acclimated carp. The results indicate that cold acclimation of the eurythermic carp is accompanied by a partial compensation of the acute effect of decreasing temperature on the activity of cytochrome c oxidase in red muscle mitochondria. Based on the temperature-induced lipid adaptation reported for carp red muscle mitochondria (Wodtke, E. (1980) Biochim. Biophys. Acta 640, 698--709), it is concluded that during thermal acclimation the molar activity of cytochrome c oxidase is controlled by viscotropic regulation. The results fit to the conception that cardiolipin constitutes a lipid shell (annulus) surrounding the oxidase within the native membrane, but that it is the bilayer fluidity and not the annular fluidity which determines the activity of cytochrome c oxidase. 相似文献
14.
Wu JL Wu QP Peng YP Zhang JM 《Physiological research / Academia Scientiarum Bohemoslovaca》2011,60(2):329-336
Accumulation of oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the aging process. The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and aging. L-malate, a tricarboxylic acid cycle intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production. Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. In the present study we focused on the effect of L-malate on the activities of electron transport chain in young and aged rats. We found that mitochondrial membrane potential (MMP) and the activities of succinate dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats were significantly decreased when compared to young control rats. Supplementation of L-malate to aged rats for 30 days slightly increased MMP and improved the activities of NADH-dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats when compared with aged control rats. In young rats, L-malate administration increased only the activity of NADH-dehydrogenase. Our result suggested that L-malate could improve the activities of electron transport chain enzymes in aged rats. 相似文献
15.
The kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius with N-acetylimidazole was studied by a newly modified histochemical technique. Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely. Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme. The value of the inhibition constant (Ki = 34 microM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with the Ki value (33 microM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group. 相似文献
16.
Oxygraphic measurements of the rates of mitochondrial respiration in the presence of varying amounts of chalepin, imperatorin and marmesin, three naturally occurring furanocoumarins, revealed that the oxidation of NAD(+)-linked substrates was inhibited by chalepin and imperatorin and less significantly by marmesin. The order of potency being rotenone much greater than chalepin imperatorin greater than marmesin. There was no effect whatsoever on succinate oxidation by the furanocoumarins tested (up to 60 microM). State 3 respiration was also inhibited by these furanocoumarins; by at least 80% by 10 microM chalepin and by 48 and 29% with 60 microM imperatorin and 60 microM marmesin, respectively. Consequently, ADP control of respiration was diminished by those concentrations of furanocoumarins that inhibited respiration. At 60 microM, respiratory control ratio was reduced by about 88, 49 and 28% with chalepin, imperatorin and marmesin, respectively. A measurement of the rate of proton and Ca2(+)-movements across the mitochondrial coupling membrane demonstrated that succinate-supported transport was not affected by these furanocoumarins. On the other hand, pyruvate/malate-supported proton ejection was significantly inhibited by chalepin, imperatorin and marmesin. The order of the degree of inhibition of proton flux is rotenone much greater than chalepin greater than imperatorin greater than marmesin. The pattern of the inhibition of pyruvate/malate-supported Ca2(+)-transport was identical to that seen during proton transport. A comparison of the effects of chalepin to that of rotenone suggests that chalepin might be about 10 times less potent than rotenone. 相似文献
17.
Guinea pig heart mitochondria loaded with [-14C]citrate show exchanges of radioactivity at 30 degrees C with added citrate, L-malate and phosphoenolpyruvate. These exchanges are inhibited by benzene-1,2,3-tricarboxylate. Measurements of rates of citrate transport indicate that the activity of this transporting system is low in heart mitochondria compared to that observed in liver mitochondria. The K(m) values obtained indicate a similarity to those obtained in liver. Citrate oxidation by coupled mitochondria was also found to be slow at 30 degrees C but was inhibited by benzene-1,2,3-tricarboxylate. The role of mitochondrial citrate transport in control of glycolytic flux in the heart is discussed. 相似文献
18.
Heinen A Aldakkak M Stowe DF Rhodes SS Riess ML Varadarajan SG Camara AK 《American journal of physiology. Heart and circulatory physiology》2007,293(3):H1400-H1407
Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O(2) concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH(2)-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O(2)(*-)); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate + rotenone-dependent H(2)O(2) production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca(2+)-sensitive K(+) channel (mtBK(Ca)) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O(2) consumption, membrane potential (DeltaPsi(m)), H(2)O(2) release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 microM) increased state 2 and state 4 respiration by 26 +/- 4% and 14 +/- 4%, respectively; this increase was abolished by the BK(Ca) channel blocker paxilline (5 microM). Paxilline alone had no effect on respiration. NS-1619 did not alter DeltaPsi(m) or redox state but decreased H(2)O(2) production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K(+) influx through mtBK(Ca) channels reduces mitochondrial H(2)O(2) production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H(2)O(2) production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K(+) influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics. 相似文献
19.
DeSantis Aurelio; Arrigoni Oreste; Palmieri Ferdinando 《Plant & cell physiology》1976,17(6):1221-1233
- The mechanism of transport of Krebs cycle intermediates, phosphateand sulfurcontaining compounds across the membrane of purifiedbean mitochondria was investigated by directly measuring dieexchange between intramitochondrial labelled substrates andexternal anions and by testing die inhibitor sensitivity ofdiese transport processes.
- The exchange between intramitochondrialphosphate and externalphosphate or sulfite is insensitive toN-ediylmaleimide or butylmalonatewhen either is added alone,but is completely inhibited by N-ethylmaleimideplus butylmalonateor by mersalyl. Internal phosphate is exchangedwith malate,succinate, oxaloacetate, sulfate and thiosulfate;these reactionsare inhibited by butylmalonate but not affectedby N-ethylmaleimide.
- Internal sulfate is exchanged with malate, malonate, succinate,phosphate and sulfite in a butylmalonate- and mersalyl-sensitivereaction. Also the exchanges of malonate with phosphate, sulfateand sulfite are inhibited by butylmalonate and mersalyl. Onthe other hand, the exchange between intra- and extramitochondrialmalonate is completely inhibited only by the combination ofbutylmalonate and 1,2,3-benzenetricarboxylate.
- Citrate isexchanged with some di- and tricarboxylates and phosphoenolpyruvate(but not with phosphate, sulfate, oxoglutarate, trans-aconitateand benzenetricarboxylates). These exchanges are inhibited by1,2,3-benzenetricarboxylate, but not by 1,2,4-benzenetricarboxylateor 1,3,5-pentanetricarboxylate.
- Oxoglutarate is exchangedwith succinate, malate, malonate andoxaloacetate (but not withphosphate, citrate or phosphoenolpyruvate)in a mersalyl-insensitive,butylmalonate- and phenylsuccinate-sensitivereaction.
- Weconcluded that bean mitochondria contain the following transportsystems: a phosphate carrier inhibited by N-ethylmaleimide ormersalyl, a dicarboxylate carrier inhibited by butylmalonateor mersalyl, a citrate carrier inhibited by 1,2,3-benzenetricarboxylateand an oxoglutarate carrier inhibited by phenylsuccinate orbutylmalonate but insensitive to mersalyl.
20.
1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues. 相似文献