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1.
The effects of fusaric acid, a phytotoxin produced byFusarium pathogens, on the metabolism of isolated maize root mitochondria and on maize seed germination and seedling growth were investigated. The phytotoxin inhibited basal and coupled respiration when succinate and α-ketoglutarate were the substrates. Coupled respiration dependent on NADH was inhibited, but basal respiration was not. Consistently, succinate cytochromec oxidoreductase activity was decreased whereas NADH cytochromec oxidoreductase was not affected. The ATPase activities of carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone stimulated mitochondria and of freeze-thawing disrupted mitochondria were inhibited. These results indicate that the phytotoxin impairs the respiratory activity of maize mitochondria by at least three mechanisms: (1) it inhibits the flow of electrons between succinate dehydrogenase and coenzyme Q, (2) it inhibits ATPase/ATP-synthase activity and (3) it possibly inhibits α-ketoglutarate dehydrogenase. Seed germination and seedling growth were also affected by fusaric acid with the most pronounced effect on root development. These effects can possibly contribute to the diseases ofFusarium- infected plants  相似文献   

2.
Anion transporters in plant mitochondria   总被引:11,自引:9,他引:2       下载免费PDF全文
The swelling of potato (Solanum tuberosum L.) mitochondria in isosmotic ammonium salts of phosphate, chloride, malate, succinate, and citrate was investigated by measuring light scattering. Potato mitochondria swell spontaneously in ammonium phosphate, and this swelling can be inhibited in N-ethylmaleimide. They swell in ammonium malate or succinate only after the addition of inorganic phosphate and in ammonium citrate only after the addition of both phosphate and a dicarboxylic acid. Pentylmalonate inhibits swelling in ammonium citrate solutions by competing for dicarboxylate entry. The results indicate that potato mitochondria possess a phosphate-hydroxyl carrier, a dicarboxylate carrier, and a tricarboxylate carrier.  相似文献   

3.
Day DA  Hanson JB 《Plant physiology》1977,59(2):139-144
A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O2 uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K+ permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low Vmax, probably reflecting a diffusion-limited process.  相似文献   

4.
The effect of inhibitors of carboxylic acid anion transport on the oxidation of substrates by mung bean (Phaseolus aureus) mitochondria was investigated. The oxidation of malate in the presence of either glutamate or cysteine sulfinate was inhibited by 2-butylmalonate, 2-phenylsuccinate, benzylmalonate, and p-iodobenzylmalonate in both intact and broken mitochondria. The oxidation of succinate, on the other hand, was inhibited in intact but not in broken mitochondria. The oxidation of reduced nicotinamide adenine dinucleotide was inhibited only by p-iodobenzylmalonate. This inhibition occurred only in coupled mitochondria and could be reversed by the addition of adenosine diphosphate.  相似文献   

5.
The chloroplastic and cytosolic forms of spinach (Spinacia oleracea cv Long Standing Bloomsdale) leaf NADH:dihydroxyacetone phosphate (DHAP) reductase were separated and partially purified. The chloroplastic form was stimulated by dithiothreitol, reduced thioredoxin, dihydrolipoic acid, 6-phosphogluconate, and phosphate; the cytosolic isozyme was stimulated by fructose 2,6-bisphosphate but not by reduced thioredoxin. End product components that severely inhibited both forms of the reductase included lipids and free fatty acids, membranes, and glycerol phosphate. In addition, two groups of inhibitory peptides were obtained from the fraction precipitated by 70 to 90% saturation with (NH4)2SO4. Chromatography of this fraction on Sephadex G-50 revealed a peptide peak of about 5 kilodaltons which inhibited the chloroplastic DHAP reductase and a second peak containing peptides of about 2 kilodaltons which inhibited the cytosolic form of the enzyme. Regulation of the reduction of dihydroxyacetone phosphate from the C3 photosynthetic carbon cycle or from glycolysis is a complex process involving activators such as thioredoxin or fructose 2,6-bisphosphate, peptide and lipid inhibitors, and intermediary metabolites. It is possible that fructose 2,6-bisphosphate increases lipid production by stimulating DHAP reductase for glycerol phosphate production as well as inhibiting fructose 1,6-bisphosphatase to stimulate glycolysis.  相似文献   

6.
1. Rat-liver mitochondria showed a decrease in amino acid production after preparation in 0·25m-sucrose containing EDTA (1mm), but an increase in water content. When EDTA was replaced by Mn2+ (1mm) or succinate (1mm), both amino acid production and water content were lowered, whereas preparation in 0·9% potassium chloride caused an increase in both. 2. Amino acid production by rat-liver homogenates prepared in 0·9% potassium chloride or 0·25m-sucrose was similar (qamino acid 0·047 and 0·042 respectively aerobically). After freezing-and-thawing qamino acid values were approximately doubled, and approached that of a homogenate prepared in water. 3. All cations tested inhibited amino acid production by mitochondria, Hg2+ and Zn2+ being the most effective in tris–hydrochloric acid buffer. In phosphate buffer Mg2+ and Mn2+ had no effect. Of the anions tested only pyrophosphate and arsenate had any inhibitory effect at final concn. 1mm. 4. Iodosobenzoate (1mm) and p-chloromercuribenzenesulphonate (1mm) inhibited mitochondrial amino acid production by 70–80%, whereas soya-bean trypsin inhibitor, EDTA and di-isopropyl phosphorofluoridate inhibited by a maximum of 30%. Respiratory inhibitors had no effect. 5. Rat-liver homogenate and subcellular fractions each showed an individual pattern of inhibition when a series of inhibitors was tested. 6. Amino acid production by mitochondria was decreased by up to 50% in the presence of oxidizable substrate, apart from α-glycerophosphate and palmitate, which had no effect. CoA stimulated amino acid production in tris–hydrochloric acid but not in phosphate buffer, α-oxoglutarate abolishing the stimulation. 7. Cysteine and glutathione stimulated amino acid production by whole mitochondria by 30%, but only reduced glutathione stimulated production in broken mitochondria. 8. Adrenocorticotrophic hormone and growth hormone stimulated mitochondrial amino acid production by 21–24%, whereas insulin inhibited production by 25%. 9. Coupled oxidative phosphorylation increased amino acid production by up to 154% at 25° and 40°. The increase was abolished by 2,4-dinitrophenol. 10. Amino acid incorporation in mitochondria was accompanied by an increase in amino acid production, both being decreased by chloramphenicol. 11. Mitochondrial production of ninhydrin-positive material was increased in the presence of albumin. The biggest increase was noted for the soluble fraction of broken mitochondria. No increase was found in the presence of 14C-labelled algal protein or denatured mitochondrial protein.  相似文献   

7.
Oxaloacetate transport into plant mitochondria   总被引:4,自引:1,他引:3       下载免费PDF全文
The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.  相似文献   

8.
Calcium ions are accumulated by intact mitochondria isolated from Ehrlich ascites tumour cells in a buffered system supplemented with ATP or succinate. In the ATP-supplemented system, the tumour mitochondria, in contrast to rat liver mitochondria, retain the accumulated Ca2+, do not exhibit a marked “irreversible” ATPase and do not swell. In the succinate-supplemented system, added Ca2+ stimulates respiration in either the absence or presence of added inorganic phosphate. Whereas respiration by rat liver mitochondria, measured in the presence of added phosphate, remains continuously activated after the addition of only a small amount of Ca2+, that by the tumour mitochondria can be stimulated by several successive additions of 100 μM Ca2+ and at all times exhibit appreciable activation ratios.  相似文献   

9.
Mitochondria from some plant tissues possess the ability to take up Ca2+ by a phosphate-dependent mechanism associated with a decrease in membrane potential, H+ extrusion, and increase in the rate of respiration (AE Vercesi, L Pereira da Silva, IS Martins, CF Bernardes, EGS Carnieri, MM Fagian [1989] In G Fiskum, ed, Cell Calcium Metabolism. Plenum Press, New York, pp 103-111). The present study reexamined the nature of the phosphate requirement in this process. The main observations are: (a) Respiration-coupled Ca2+ uptake by isolated corn (Zea mays var Maya Normal) mitochondria or carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced efflux of the cation from such mitochondria are sensitive to mersalyl and cannot be dissociated from the silmultaneous movement of phosphate in the same direction. (b) Ruthenium red-induced efflux is not affected by mersalyl and can occur in the absence of phosphate movement. (c) In Ca2+-loaded corn mitochondria, mersalyl causes net Ca2+ release unrelated to a decrease in membrane potential, probably due to an inhibition of Ca2+ cycling at the level of the influx pathway. It is concluded that corn mitochondria (and probably other plant mitochondria) do possess an electrophoretic influx pathway that appears to be a mersalyl-sensitive Ca2+/inorganic phosphate-symporter and a phosphate-independent efflux pathway possibly similar to the Na2+-independent Ca2+ efflux mechanism of vertebrate mitochondria, because it is not stimulated by Na+.  相似文献   

10.
The trinuclear cyanine dye, tri-S-C7(5), at about 10 μM stimulated State 4 respiration of rat liver mitochondria more than 6-fold and released oligomycin-inhibited respiration completely. Thus, the dye is concluded to be a very effective cationic uncoupler of oxidative phosphorylation in mitochondria. However, for exhibition of its uncoupling action, the presence of Pi (or arsenate) was necessary, and a phosphate-transport inhibitor, N-ethylmaleimide or mersalyl, inhibited its action. The stimulation of phosphate transport via the Pi carrier by the dye is suggested to be directly related to the uncoupling action.  相似文献   

11.
Pyruvate and malate transport and oxidation in corn mitochondria   总被引:14,自引:13,他引:1       下载免费PDF全文
Day DA  Hanson JB 《Plant physiology》1977,59(4):630-635
Pyruvate oxidation and swelling in pyruvate solutions by corn (Zea mays) mitochondria were inhibited by α-cyano-4-hydroxy-cinnamic acid, an inhibitor of pyruvate transport in animal mitochondria; however, there was no inhibition of pyruvate dehydrogenase activity, and malate and NADH oxidation were not affected. These results suggest the presence of a pyruvate-OH exchange transporter which supplies the mitochondrion with oxidizable substrate. Lactate appears to be transported also, but not dicarboxylate anions or inorganic phosphate. The rate of pyruvate transport was much slower than that of malate, however, and valinomycin was required to elicit appreciable swelling in potassium pyruvate.  相似文献   

12.
Some effects of decenylsuccinic Acid on isolated corn mitochondria   总被引:2,自引:2,他引:0       下载免费PDF全文
The effects of decenylsuccinic acid on the swelling and respiratory capacities of mitochondria isolated from etiolated corn (Zea mays L., Wf9 × M14) shoots were studied. Decenylsuccinic acid (0.1 mM to 1.0 mM) inhibited the oxidation of succinate and malate-pyruvate, stimulated the oxidation of reduced nicotinamide adenine dinucleotide, and uncoupled phosphorylation. The swelling of isolated corn mitochondria, as determined by percentage of transmittance changes, was stimulated by decenylsuccinic acid in potassium chloride reaction media and in sucrose reaction media without bovine serum albumin. In a diaphorase (2, 6-dichlorophenolindophenol as acceptor) reaction with intact mitochondria, only the dehydrogenation rate of malate was reduced by the addition of decenylsuccinic acid. The dehydrogenation of reduced nicotinamide adenine dinucleotide or of succinate was either not affected or was stimulated depending on the diaphorase reaction medium. The oxygen uptake of mitochondria oxidizing N, N, N′, N′-tetramethyl-p-phenylenediamine diHCl and ascorbate was inhibited at decenylsuccinic acid concentrations greater than 0.5 mM.  相似文献   

13.
Malonyl-CoA decarboxylase (EC 4.1.1.9) was found to be localized in the mitochondria in rat liver. Low ionic strength (10 mm Na phosphate) buffer extracted the bulk (>85%) of the enzyme from the mitochondria. From this extract the enzyme was purified over 2,000-fold using a combination of (NH4)2SO4 precipitation, gel filtration with Sepharose 4B and Sephadex G-150, ion exchange chromatography with QAE-Sephadex and CM-Sephadex, and finally chromatography on NADP-agarose. The purified enzyme, which had a specific activity of about 16 μmol/min/mg, appeared to be electrophoretically homogeneous and had a molecular weight of 160,000. The decarboxylase had a broad pH optimum between 8.5 and 10.0 and showed a typical Michaelis-Menten substrate saturation pattern from which Km and V were calculated to be 54 μm and 18.8 μmol/min/mg, respectively. This enzyme decarboxylated neither malonic acid nor methylmalonyl-CoA and was severely inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide but not by iodoacetamide. Acetyl-CoA, propionyl-CoA, and methylmalonyl-CoA also inhibited the enzyme. The purified decarboxylase was immunogenic in rabbits and Ouchterlony double diffusion analysis revealed a single precipitant line with the purified enzyme. The IgG fraction isolated from the antiserum inhibited the enzyme from not only liver mitochondria but also the mammary gland, heart, and kidney of the rat. However, malonyl-CoA decarboxylase from rat brain mitochondria was not inhibited by the antibody. Malonyl-CoA decarboxylase purified from the uropygial gland of a domestic goose neither cross reacted nor was it inhibited by the antiserum prepared against the rat liver mitochondrial enzyme and the antibody against the goose enzyme neither cross-reacted nor inhibited the enzyme from the rat. It is proposed that a role for mitochondrial malonyl-CoA decarboxylase is to decarboxylate malonyl-CoA generated by propionyl-CoA carboxylase and thus protect mitochondrial enzymes susceptible to inhibition by malonyl-CoA.  相似文献   

14.
Osmotic shock was found to be better than freezing and thawing, a French press, or sonic oscillation for the preparation of submitochondrial particles from mung bean (Phaseolus aureus) hypocotyl mitochondria. Particles prepared by osmotic shock rapidly oxidize reduced nicotinamide adenine dinucleotide and succinate, but they oxidize malate slowly. NADH oxidation was slightly stimulated by cytochrome c, ATP, and ADP; succinate oxidation was markedly increased by ATP, slightly by ADP and cytochrome c; and malate oxidation required the addition of NAD+ NADH oxidation is inhibited weakly by amytal, completely by antimycin A and KCN, but not by rotenone. Chlorsuccinate, malonate, antimycin A, and KCN inhibit succinate oxidation. The action of antimycin A and KCN is incomplete, while chlorsuccinate and malonate were competitive inhibitors. Antimycin A combined stoichiometrically with particle protein in the ratio of 0.23 millimicromole per milligram of protein.  相似文献   

15.
Previous investigators have shown that following doxorubicin treatment heart mitochondria appear swollen and contain intramitochondrial dense inclusion bodies identified as calcium phosphate. In vitro studies have shown that similar morphological changes occur in mitochondria previously loaded with excess calcium. The present studies were performed to determine the effects of doxorubicin and its aglycone metabolite on 45Ca2+ uptake by mitochondria isolated from the heart, liver, and kidney of the rabbit. Doxorubicin (100 μM) significantly inhibited the initial rate of 45Ca2+ accumulated by mitochondria isolated from the three tissues. In contrast, the aglycone metabolite (100 μM) induced the reverse effect. In preloaded mitochondria the aglycone stimulated the release of calcium while doxorubicin was without effect. Mitochondria from the heart were significantly more sensitive to the effects of these anthracyclines than were mitochondria from the other two tissues. If these in vitro effects also occur in vitro, then the aglycone metabolite would be a more likely candidate in explaining the morphological changes in heart mitochondria previously described.  相似文献   

16.
A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD+ at 25°C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl2, and 5 millimolar α-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD+ efflux was observed instead. Furthermore, the rate of NAD+ accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3′-NAD+. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD+ content. This led to a slow decrease of NAD+-linked substrates oxidation by isolated mitochondria The rate of NAD+ efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD+ transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.  相似文献   

17.
Storey BT 《Plant physiology》1971,48(6):694-701
Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (Pi) and ATP are present. No reduction occurs in the absence of Pi, even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.  相似文献   

18.
D.A. Day  B.L. Bertagnolli  J.B. Hanson 《BBA》1978,502(2):289-297
Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca2+ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage.  相似文献   

19.
Thymidine kinase is present in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis. Previous workers have been unable to find a specific thymidine kinase activity in this organism. The cytoplasm of Tetrahymena contained a thymidine phosphorylating activity which was ATP dependent, was stimulated by Mg2+, and was inhibited by dTTP. This activity was also partly inhibited by dCTP. Although the mitochondrial fraction also exhibited ATP-dependent phosphorylation, it is not stimulated by Mg2+ and not significantly inhibited by dTTP. Nucleoside phosphotransferase activity is detectable both in cytoplasmic and mitochondrial fractions, although it is not clear whether they represent separate enzymes. Nucleoside phosphotransferase activity is inhibited both by NaF and by ATP. Thymidine kinase and nucleoside phosphotransferase activities were separated by polyacrylamide gel electrophoresis, establishing the presence of both enzymes in this organism. Both crude mitochondrial lysate and postmitochondrial supernatant samples exhibited similar gel electrophoretic patterns for thymidine kinase and nucleoside phosphotransferase activities. The former, however, exhibited a relatively small peak of thymidine kinase migrating at the same rate as that of the postmitochondrial supernatant. A separate peak of thymidine kinase was not found in the mitochondria of Tetrahymena.  相似文献   

20.
The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase were assayed during embryogenesis of R. microplus. The influence of poly P3 and poly P15 were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P3. However, superoxide dismutase was not affected by poly P15, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos.  相似文献   

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