The effect of disulfiram on the aldehyde dehydrogenases of sheep liver.

1. The effect of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. 2. Disulfiram causes an immediate inhibition of the enzyme reaction. The effect on the cytoplasmic enzyme is much greater than on the mitochondrial enzyme. 3. In both cases, the initial partial inhibition is followed by a gradual irreversible loss of activity. 4. The pH-rate profile of the inactivation of the mitochondrial enzyme by disulfiram and the pH-dependence of the maximum velocity of the enzyme-catalysed reaction are both consistent with the involvement of a thiol group. 5. Excess of 2-mercaptoethanol or GSH abolishes the effect of disulfiram. However, equimolar amounts of either of these reagents and disulfiram cause an effect greater than does disulfiram alone. It was shown that the mixed disulphide, Et2N-CS-SS-CH2-CH2OH, strongly inhibits aldehyde dehydrogenase. 6. The inhibitory effect of diethyldithiocarbamate in vitro is due mainly to contamination by disulfiram.     

Effects of Phosphonic Acid Analogues of Glycerol-3-Phosphate on the Growth of Escherichia coli

The four-carbon phosphonate, 3,4-dihydroxybutyl-1-phosphonate, is similar to glycerol-3-phosphate in its ability to inhibit cell growth of Escherichia coli strain 8 cultured in low-phosphate synthetic medium supplemented with either succinate or casein hydrolysate as the sole carbon source. The three-carbon phosphonate, 2,3-dihydroxypropyl-1-phosphonate, does not appear to exhibit a similar effect. The inhibition caused by the four-carbon phosphonate differs from that caused by glycerol-3-phosphate in at least three ways. (i) Its inhibitory effect is not offset by the presence of glucose in the culture medium. (ii) It is capable of exerting its inhibitory effect on cells containing an active aerobic glycerol-3-phosphate dehydrogenase. (iii) Its inhibitory effect is maintained in synthetic medium containing high concentrations of inorganic phosphate. The four-carbon phosphonate appears to be bacteriostatic and inhibits the uptake of labeled glycerol-3-phosphate by E. coli strain 8.     

Effect of disulfiram and its reduced metabolite, diethyl-dithiocarbamate on aldehyde dehydrogenase of human erythrocytes

Inhibition of human erythrocyte aldehyde dehydrogenase (ALDH) activity was studied using disulfiram and its reduced metabolite, diethyldithiocarbamate (DDC). The enzyme was rapidly inactivated by disulfiram and the inhibition was protected by reduced glutathione (GSH), in a concentration dependent manner when the enzyme premixed with GSH was reacted with disulfiram. Higher reactivity of the thiol group of the enzyme than that of GSH to disulfiram was suggested from the observation that half of the enzyme activity was inhibited when the ratio of disulfiram to GSH was 1:10. Although DDC alone showed no inhibitory effect on the enzyme, inactivation was mediated by a low concentration of heme-containing peroxidases, but not by methemoglobin. Under this condition, the inhibition potential was not protected, even with a high concentration of GSH. The constant reoxidation system of DDC is probably directly related to the enzyme inactivation.     

The participation of primary amino groups of succinate dehydrogenase in the formation of succinate-Q reductase

(1) Purified succinate dehydrogenase contains about 49 mol of lysine residues per mol enzyme. Titration of succinate dehydrogenase with fluorescamine indicates that half the lysyl groups are located on the surface of the protein and the other half are buried inside. (2) The reconstitutive activity and the low Km ferricyanide reductase activity of succinate dehydrogenase decreased as the extent of alkylation of amino groups by fluorescamine increased. (3) The inhibitory effects of fluorescamine on both activities are parallel and are succinate concentration dependent. (4) Alkylation of the native succinate-Q reductase by fluorescamine does not affect the enzymatic activity or alter the enzyme kinetic parameters. This indicates that the inhibitory effect of fluorescamine on succinate dehydrogenase is due to the modification of a specific amino group(s) on succinate dehydrogenase which is essential in the interaction with QPs to form succinate-Q reductase. The participation of an ionic group in the formation of succinate-Q reductase supports the idea of the involvement of ionic interaction between succinate dehydrogenase and QPs.     

Proapoptotic action of alpha-tocopheryl succinate in rat thymocytes is conditioned by the inhibition of mitochondrial succinate dehydrogenase

It has been established that alpha-tocopheryl succinate in concenrations 10-100 microM inhibits in a dose-dependent manner the viability of primary culture rats thymocytes and causes the DNA internucleosomal degradation that testifies to apoptotic way of thymocytes destruction. These effects were accompanied by an enhanced production of intracellular superoxide. This is the first report demonstrating that apoptosis induced by alpha-tocopheryl succinate was accompanied by a dose-dependent inhibition of mitochondrial succinate dehydrogenase. Known apoptosis inducers--actinomicin D, staurosporin and hydrogen peroxide decreased a cell survival but neither induced any significant changes in succinate dehydrogenase activity which means that this effect is characteristic only of alpha-tocopheryl succinate and seems to be an important event triggering the apoptotic response by it. It was supposed that alpha-tocopheryl succinate might appear as a pseudosubstrate for mitochondrial succinate dehydrogenase leading to its inhibition, dysfunction of the mitochondrial electron transport chain, generation of reactive oxygen species and iduction of apoptosis.     

双硫仑抗癌作用研究进展

双硫仑作为一种治疗慢性酒精中毒的药物在临床中广泛使用。近几十年研究发现它除了戒酒作用还在治疗癌症中具有巨大潜力,针对它在体外和体内模型的研究结论已有部分在临床治疗中得到证实。双硫仑通过其代谢产物抑制乙醛脱氢酶活性导致体内乙醛含量积累,增加细胞毒性从而抑制肿瘤干细胞增殖分化;提高细胞内活性氧的浓度诱导细胞凋亡;抑制蛋白酶体活性,积累大量废弃蛋白质诱导细胞凋亡;通过抑制NF-κB下调来抑制上皮间质转化等。此外双硫仑与抗癌药物联合使用可提升抗癌药物药效。由于具有低毒、低成本且对肿瘤组织有趋向性等特点,双硫仑重新应用于临床作为抗癌药物具有广阔前景。简要回顾了双硫仑最新研究中阐明的双硫仑抗癌作用分子机制,展望了未来双硫仑用作新临床抗癌药物的前景,以期为双硫仑在抗癌药物中的应用研究提供参考。     

Further studies of the action of disulfiram and 2,2''-dithiodipyridine on the dehydrogenase and esterase activities of sheep liver cytoplasmic aldehyde dehydrogenase.

1. Pre-modification of cytoplasmic aldehyde dehydrogenase by disulfiram results in the same extent of inactivation when the enzyme is subsequently assayed as a dehydrogenase or as an esterase. 2. 4-Nitrophenyl acetate protects the enzyme against inactivation by disulfiram, particularly well in the absence of NAD+. Some protection is also provided by chloral hydrate and indol-3-ylacetaldehyde (in the absence of NAD+). 3. When disulfiram is prevented from reacting at its usual site by the presence of 4-nitrophenyl acetate, it reacts elsewhere on the enzyme molecule without causing inactivation. 4. Enzyme in the presence of aldehyde and NAD+ is not at all protected against disulfiram. It is proposed that, under these circumstances, disulfiram reacts with the enzyme-NADH complex formed in the enzyme-catalysed reaction. 5. Modification by disulfiram results in a decrease in the amplitude of the burst of NADH formation during the dehydrogenase reaction, as well as a decrease in the steady-state rate. 6. 2,2'-Dithiodipyridine reacts with the enzyme both in the absence and presence of NAD+. Under the former circumstances the activity of the enzyme is little affected, but when the reaction is conducted in the presence of NAD+ the enzyme is activated by approximately 2-fold and is then relatively insensitive to the inactivatory effect of disulfiram. 7. Enzyme activated by 2,2'-dithiodipyridine loses most of its activity when stored over a period of a few days at 4 degrees C, or within 30 min when treated with sodium diethyldithiocarbamate. 8. Points for and against the proposal that the disulfiram-sensitive groups are catalytically essential are discussed.     

Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase.

The effect of disulfiram, [1-14C]disulfiram and some other thiol reagents on the activity of cytoplasmic aldehyde dehydrogenase from sheep liver was studied. The results are consistent with a rapid covalent interaction between disulfiram and the enzyme, and inconsistent with the notion that disulfiram is a reversible competitive inhibitor of cytoplasmic aldehyde dehydrogenase. There is a non-linear relationship between loss of about 90% of the enzyme activity and amount of disulfiram added; possible reasons for this are discussed. The remaining approx. 10% of activity is relatively insensitive to disulfiram. It is found that modification of only a small number of groups (one to two) per tetrameric enzyme molecule is responsible for the observed loss of activity. The dehydrogenase activity of the enzyme is affected more severely by disulfiram than is the esterase activity. Negatively charged thiol reagents have little or no effect on cytoplasmic aldehyde dehydrogenase. 2,2'-Dithiodipyridine is an activator of the enzyme.     

An antimycin-insensitive succinate-cytochrome c reductase activity in pure reconstitutively active succinate dehydrogenase

Antimycin-insensitive succinate-cytochrome c reductase activity has been detected in pure, reconstitutively active succinate dehydrogenase. The enzyme catalyzes electron transfer from succinate to cytochrome c at a rate of 0.7 mumole succinate oxidized per min per mg protein, in the presence of 100 microM cytochrome c. This activity, which is about 2% of that of reconstitutive (the ability of succinate dehydrogenase to reconstitute with coenzyme ubiquinone-binding proteins (QPs) to form succinate-ubiquinone reductase) or succinate-phenazine methosulfate activity in the preparation, differs from antimycin-insensitive succinate-cytochrome c reductase activity detected in submitochondrial particles or isolated succinate-cytochrome c reductase. The Km for cytochrome c for the former is too high to be measured. The Km for the latter is about 4.4 microM, similar to that of antimycin-sensitive succinate-cytochrome c activity in isolated succinate-cytochrome c reductase, suggesting that antimycin-insensitive succinate-cytochrome c activity of succinate-cytochrome c reductase probably results from incomplete inhibition by antimycin. Like reconstitutive activity of succinate dehydrogenase, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase is sensitive to oxygen; the half-life is about 20 min at 0 degrees C at a protein concentration of 23 mg/ml. In the presence of QPs, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase disappears and at the same time a thenoyltrifluoroacetone-sensitive succinate-ubiquinone reductase activity appears. This suggests that antimycin-insensitive succinate-cytochrome c reductase activity of succinate dehydrogenase appears when succinate dehydrogenase is detached from the membrane or from QPs. Reconstitutively active succinate dehydrogenase oxidizes succinate using succinylated cytochrome c as electron acceptor, suggesting that a low potential intermediate (radical) may be involved. This suggestion is confirmed by the detection of an unknown radical by spin trapping techniques. When a spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), is added to a succinate oxidizing system containing reconstitutively active succinate dehydrogenase, a PBN spin adduct is generated. Although this PBN spin adduct is identical to that generated by xanthine oxidase, indicating that a perhydroxy radical might be involved, the insensitivity of this antimycin-insensitive succinate-cytochrome c reductase activity to superoxide dismutase and oxygen questions the nature of this observed radical.     

Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition

Lipid peroxidation in rat brain mitochondria was induced by NADH in the presence of ADP and FeCl3. CV-2619 inhibited the lipid peroxidation in a concentration-dependent manner; the concentration giving 50% inhibition (IC50) was 84 microM. In addition, the inhibitory effect of CV-2619 was strongly enhanced by adding substrates of mitochondrial respiration; when succinate, glutamate, or succinate plus glutamate was added, the IC50 of CV-2619 was changed to 1.1, 10, or 0.5 microM, respectively. Metabolites of CV-2619 also inhibited the lipid peroxidation. The inhibitory effect of CV-2619 on mitochondrial lipid peroxidation disappeared when TTFA, an inhibitor of complex II in mitochondrial respiratory chain, was added. The results indicate that in mitochondria CV-2619 is changed to its reduced form which inhibits lipid peroxidation.     

Inhibition of membrane-bound succinate dehydrogenase by fluorescamine

Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.     

Purification of aldehyde dehydrogenase reconstitutively active in fatty alcohol oxidation from rabbit intestinal microsomes

This work presents the purification and further characterization of the aldehyde dehydrogenase reconstitutively active in fatty alcohol oxidation, from rabbit intestinal microsomes. Microsomal aldehyde dehydrogenase was solubilized with cholate and purified by using chromatography on 6-amino-n-hexyl-Sepharose and 5'-AMP-Sepharose. The purified enzyme migrated as a single polypeptide band with molecular weight of 60,000 on SDS-polyacrylamide gel. By gel filtration in the presence of detergent, its apparent molecular weight was estimated to be 370,000. In the detergent-free solution, in contrast, it had a much higher molecular weight, indicating its association in forming large aggregates. The pH optimum was 9.0 when pyrophosphate buffer was used. The enzyme was active toward various aliphatic aldehydes with more than three carbons. The Km value for substrate seemed to decrease with increase in the chain length. The microsomal aldehyde dehydrogenase was not affected by disulfiram and MgCl2, which were, in contrast, highly inhibitory towards the activity of the cytosolic aldehyde dehydrogenase separated from intestinal mucosa.     

Involvement of a carboxyl group in the interaction between succinate dehydrogenase and its membrane-anchoring protein (QPs) fraction

The involvement of the carboxyl groups in the membrane-anchoring protein (QPs) in reconstitution of succinate dehydrogenase to form succinate-ubiquinone reductase is studied by using a carboxyl group modifying reagent, dicyclohexylcarbodiimide (DCCD). Inactivation of QPs by DCCD is found to be dependent on the temperature, pH, detergent, and DCCD concentration used. When QPs is treated with 300 molar excess DCCD at room temperature for 10 min, about 90% of the original reconstitutive activity is lost. When intact or reconstituted succinate-ubiquinone reductase formed from reconstitutively active succinate dehydrogenase and QPs is treated with DCCD under the same conditions, no loss of succinate-ubiquinone reductase activity is observed. However, when a mixture of reconstitutively inactive succinate dehydrogenase and QPs is treated with DCCD before being reconstituted with active succinate dehydrogenase, an inactivation behavior similar to that with QPs alone is observed. These results indicate that DCCD modifies the carboxyl groups of QPs which are essential for the interaction with succinate dehydrogenase to form succinate-ubiquinone reductase. Inactivation of QPs by DCCD parallels the incorporation of DCCD into QPs. About two carboxyl groups per molecule of QPs are essential for the interaction with succinate dehydrogenase. These essential carboxyl groups are located in the smaller subunit (Mr 13,000) of QPs. Modification of QPs by DCCD also alters the heme environment of cytochrome b560.     

Pentachlorophenol inhibition of succinate oxidation by the respiratory chain in submitochondrial particles from the bovine heart

Study on the effect of pentachlorophenol on the succinate oxidase activity of submitochondrial particles and on the reduction level of cytochromes b revealed that the Ki value for PCP is equal to 2-4 microM. The succinate-DCPIP-reductase activity is noncompetitively inhibited with PCP (by 75-85%) (Ki = 3.6 microM). In the case of the succinate-PMS-reductase activity PCP at micromolar concentrations decreases the value of V only by 40% (C50 = 2 microM) with a simultaneous increase of the Km value for PMS. The identity of Ki values for PCP under these conditions suggests that the effect of PCP is due to the inhibitor interaction with the same component of the succinate dehydrogenase complex. The type of action of PCP on the succinate-acceptor-reductase activities indicates that the inhibiting effect of PCP on succinate oxidations is similar to that exerted by traditional inhibitors of succinate dehydrogenase--tenoyltrifluoroacetone and carboxins. Since PCP inhibits succinate dehydrogenase at low concentrations, it seems likely that the biological (pesticidal) effect of PCP is provided for not only by its uncoupling action but also by the inhibition of succinate oxidation in the respiratory chain.     

Inhibition of mitochondrial function in isolated rat liver mitochondria by azole antifungals

Ketoconazole is an imidazole oral antifungal agent with a broad spectrum of activity. Ketoconazole has been reported to cause liver damage, but the mechanism is unknown. However, ketoconazole and a related drug, miconazole, have been shown to have inhibitory effects on oxidative phosphorylation in fungi. Fluconazole, another orally administered antifungal azole, has also been reported to cause liver damage despite its supposedly low toxicity profile. The primary objective of this study was to evaluate the metabolic integrity of adult rat liver mitochondria after exposure to ketoconazole, miconazole, fluconazole, and the deacetylated metabolite of ketoconazole by measuring ADP-dependent oxygen uptake polarographically and succinate dehydrogenase activity spectrophotometrically. Ketoconazole, N-deacetyl ketoconazole, and miconazole inhibited glutamate-malate oxidation in a dose-dependent manner such that the 50% inhibitory concentration (I₅₀ was 32, 300, and 110 μM, respectively. In addition, the effect of ketoconazole, miconazole, and fluconazole on phosphorylation coupled to the oxidation of pyruvate/malate, ornithine/malate, arginine/malate, and succinate was evaluated. The results demonstrated that ketoconazole and miconazole produced a dose-dependent inhibition of NADH oxidase in which ketoconazole was the most potent inhibitor. Fluconazole had minimal inhibitory effects on NADH oxidase and succinate dehydrogenase, whereas higher concentrations of ketoconazole were required to inhibit the activity of succinate dehydrogenase. N-deacetylated ketoconazole inhibited succinate dehydrogenase with an I₅₀ of 350 μM. In addition, the reduction of ferricyanide by succinate catalyzed by succinate dehydrogenase demonstrated that ketoconazole caused a dose-dependent inhibition of succinate activity (I₅₀ of 74 μM). In summary, ketoconazole appears to be the more potent mitochondrial inhibitor of the azoles studied; complex I of the respiratory chain is the apparent target of the drug's action. © 1997 John Wiley & Sons, Inc.     

Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.     

Effects of fusaric acid on respiration in maize root mitochondria

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     

Inactivation of horse liver mitochondrial aldehyde dehydrogenase by disulfiram. Evidence that disulfiram is not an active-site-directed reagent.

The inhibition of mitochondrial (pI 5) horse liver aldehyde dehydrogenase by disulfiram (tetraethylthiuram disulphide) was investigated to determine if the drug was an active-site-directed inhibitor. Stoichiometry of inhibition was determined by using an analogue, [35S]tetramethylthiuram disulphide. A 50% loss of the dehydrogenase activity was observed when only one site per tetrameric enzyme was modified, and complete inactivation was not obtained even after seven sites per tetramer were modified. Modification of only two sites accounted for a loss of 75% of the initial catalytic activity. The number of functioning active sites per tetrameric enzyme, as determined by the magnitude of the pre-steady-state burst of NADH formation, did not decrease until approx. 75% of the catalytic activity was lost. These data indicate that disulfiram does not modify the essential nucleophilic amino acid at the active site of the enzyme. The data support an inactivation mechanism involving the formation of a mixed disulphide with a non-essential cysteine residue, resulting in a lowered specific activity of the enzyme.     

甘蓝型油菜子油分的积累与某些生理变化关系的研究

油菜种子发育过程中,其内部的生理代谢过程发生了规律性的变化。伴随着种子的发育进程,６－磷酸葡萄糖脱氢酶、异柠檬酸裂解酶、异柠檬酸脱氢酶和琥珀酸脱氢酶的活性均有不同程度的增强。在油分旺盛合成期,６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均达到了最大值,而此时,异柠檬酸脱氢酶和琥珀酸脱氢酶的活属于匀增加较慢;在种子的不同发育时期,高含油量品系的６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均高于低含油量的     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.