Succinoxidase Activity Research Articles

Influence of the energetic state of mitochondria on the inhibition of oxidative phosphorylation by N-ethylmaleimide

N-Ethylmaleimide inhibitory effect on oxidative phosphorylation, adenylic nucleotide translocation, succinate dehydrogenase and succinoxidase activities was studied as a function of the energetic state of mitochondria. 1. 1. Using a reversible thiol reagent (mersalyl), in order to protect the phosphate carrier against irreversible action of N-ethylmaleimide, it was found that: (a) when mersalyl-pretreated mitochondria were in a ‘non-energized’ state, i.e. preincubated without a substrate and in the presence of rotenone, only a slight inhibition of succinate oxidation coupled to ATP synthesis by N-ethylmaleimide was observed. (b) when mersalyl-pretreated mitochondria were in an ‘energized’ state, i.e. preincubated in the presence of an oxidizable substrate, N-ethylmaleimide strongly inhibited the coupled oxidation of succinate. 2. 2. Mitochondrial energization was also shown to enhance the inhibitory effect of N-ethylmaleimide on adenylic nucleotide translocation and succinoxidase activity. However, other sulphydryl groups seem to be involved in the inhibition mechanism, but their function is unknown. 3. 3. As N-ethylmaleimide inhibitory effect increased, an enhancement of N-[ 14 C]ethylmaleimide binding to mitochondrial sulphydryl groups was obtained.

Inhibition of mitochondrial respiration by model phenolic compounds

A variety of model phenolic compounds were tested for their ability to inhibit the beef heart mitochondrial NADH-oxidase and succinoxidase enzyme systems in vitro. Specifically, we determined the hydroxy and methoxy configurations of the model phenolic compounds that were mandatory for inhibition of mitochondrial respiration. Data are presented that supported the conclusion that the relative potency of inhibition of the beef heart mitochondrial succinoxidase enzyme system was methyl hydroquinone > hydroquinone > 4-methyl catechol > 3-methyl catechol > catechol, whereas o-cresol, p-cresol, resorcinol, 2- methyl resorcinol and orcinol were non-inhibitory. None of the model phenolic compounds tested were inhibitory toward the beef heart mitochondrial NADH-oxidase enzyme system. These findings indicate that the site of inhibition for the catechol and hydroquinone derivatives is in complex II. Furthermore, it is proposed that a capacity for ortho- or para-quinone formation is mandatory for inhibition of mitochondrial succinoxidase activity by model phenolic compounds.

Selection of succinic dehydrogenase mutants of Neurospora crassa

A method is described which permits the selection of mutants of Neurospora crassa that are deficient in succinic dehydrogenase activity. The method relies on the observation that succinic dehydrogenase-deficient strains fail to reduce the dye nitrotetrazolium blue when overlaid with the dye in the presence of succinate and phenazine methosulfate. Wild-type colonies reduced the dye and turned blue, whereas mutant colonies remained colorless. In this communication we present studies of a mutant, SDH-1, isolated by this method. The mutant had 18% of the succinic dehydrogenase activity of the parent strain used in the mutation experiments as determined from the ratio of Vmax activities obtained from Lineweaver-Burk plots. The SDH-1 mutant segregated in a Mendelian manner when back-crossed to its parent strain. Succinate oxidase activity in SDH-1 was low and was markedly inhibited by adenosine 5'-diphosphate. The succinate oxidase activity of the parent strain was high and was not affected by the presence of adenosine 5'-diphosphate.

Seasonal Changes in the Structure and Function of Mitochondrial Membranes of Artichoke Tubers

The temperature limits of the order-disorder transition, and the Arrhenius activation energy of succinate oxidase activity for mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers were determined from the initiation to the termination of dormancy. The temperature limits for the transition at the initiation of dormancy were 25 and 3 C. These changed to 9 and -5 C at mid-dormancy and returned to 25 and 2 C at the termination of dormancy. The Arrhenius activation energy measured in the temperature range above the transition was 35 kilojoules per mole at middormancy and decreased to 17 kilojoules per mole at the termination of dormancy when sprouting was evident. The coincidence of the changes in membrane structure and function with dormancy suggests that artichokes possess a mechanism for regulating membrane lipid structure so that cellular integrity of tuber tissue is maintained even when the tubers are exposed to low temperatures.

Neuroanastomosis of orthotopically transplanted palmaris longus muscles.

Palmaris longus (PML) muscles of rhesus monkeys were transplanted, with or without anastomosis of the median nerve, to the nerve stump of the autograft. Because PML autografts revascularize spontaneously, vascular anastomoses were not performed. Muscle fibers regenerated in all autografts with neuroanastomosis, but in only three of eight autografts without neuroanastomosis. Five autografts without neuroanastomosis were replaced by noncontractile connective tissue. Growth and differentiation of muscle fibers into three fiber types and development of capillarity were analyzed histochemically, and succinate oxidase activity of whole-muscle homogenates was determined. None of these measures reached values for control PML muscles within 100 days of transplantation. In comparison to control muscles, autografts had slower times to peak tension and less absolute tension, but similar tension per square centimeter of muscle fiber cross-sectional area. Monkey PML autografts with neuroanastomosis were similar in structure and function to cat extensor digitorum longus autografts that had not had neuroanastomosis.

Structure — Inhibition relationships of various phenolic compounds towards mitochondrial respiration

Summary A variety of phenolic compounds were assessed for their ability to inhibit beef heart mitochondrial respiration in vitro . Specifically, pyrogallol and various methylated analogs of pyrogallol were tested for their inhibitory properties towards beef heart mitochondrial NADH- and succinoxidase enzyme systems. In addition, various benzaldehyde and benzoic acid analogs were similarly assessed. Our data show that most of the model phenolic compounds tested do not inhibit mitochondrial respiration; however, 3-methoxycatechol, gallic acid and pyrogallol inhibited beef heart mitochondrial succinoxidase activity as the I 50 values were 175, 1000, and >1000 nmoles/mg. mitochondrial protein respectively. The only compound tested that showed inhibition of the NADH- oxidase enzyme system was 2,4,6-trihydroxybenzaldehyde which was only a marginal inhibitor. We concluded that 3-methoxycatechol, gallic acid and pyrogallol inhibited the respiratory chain at a site in complex II. These findings are consistent with the view that the capacity for ortho quinone formation is essential for inhibition of the beef heart mitochondrial succinoxidase enzyme system by model phenolic compounds.

Temperature dependence of anesthetic effects on succinate oxidase activity in uncoupled submitochondrial particles

Temperature dependence of anesthetic effects on succinate oxidase activity in uncoupled submitochondrial particles

Seasonal acclimatization to temperature in cardueline finches

1. Seasonal variation in body constituents and utilization of lipid, protein, and carbohydrate during cold stress in American goldfinches were studied to determine relations of these functions to the pronounced seasonal shift in thermogenic capacity documented in a previous study (Dawson and Carey, 1976). 2. Mean body mass for adults increases from a low of 11.4 g in July to a high of 15.1 g in December and January. Seasonal variation in lipid content accounts for the major part of the observed changes in body mass, but such variation in water and protein content is also appreciable. 3. Linoleic acid (18∶2) is the predominant fatty acid in neutral lipids of liver, pectoralis muscle, and furcular depots at all seasons. Unsaturated fatty acids comprise a much greater proportion of total fatty acids in liver and pectoralis muscle during winter (71% and 73%, respectively) than in spring or fall. 4. Fasting winter goldfinches exposed to −10°C for 17 h overnight utilize significant amounts of body lipid. However, total body protein, liver and pectoralis muscle carbohydrate, and pectoralis muscle fatty acids do not differ significantly between control and cold-stressed individuals. 5. Glycogen stores in the pectoralis muscles are significantly higher in winter than in summer birds. Winter goldfinches exposed to −70°C utilize significant amounts of total body lipids and pectoralis glycogen. Birds tested in this manner in summer do not do so and quickly become hypothermic. 6. Histochemical characteristics and succinate oxidase activities of pectoralis muscles do not vary appreciably over the year. 7. Increased stores of body lipid and muscle carbohydrate and the ability to mobilize these substrates rapidly during cold stress seem to be key factors in the superior thermogenic capacities of winter goldfinches.

55] Extraction and reincorporation of ubiquinone in submitochondrial particles

Publisher Summary This chapter describes the extraction and reincorporation of ubiquinone in submitochondrial particles. Methods for extraction of ubiquinone from mitochondria and submitochondrial particles are based on the use of organic solvents. An early procedure is applied on iso-octane to extract ubiquinone from aqueous suspensions of beef-heart mitochondria or submitochondrial particles. Extraction of lyophilized beef or rat-liver mitochondria with 96% acetone has subsequently been shown to result in a preparation that required both ubiquinone and cytochrome c for the restoration of succinate oxidase activity. The dried, lyophilized particles, either before or after pentane extraction and reincorporation of ubiquinone, can be stored at -15 to -20 ° in vacuo for several weeks without any apparent loss of enzyme activities. Caution must be taken, however, that the preparations, which are highly hygroscopic, remain absolutely dry. Even a slight amount of water impairs both the extraction and the reincorporation of ubiquinone.

Succinoxidase Activity of Avocado Fruit Mitochondria in Relation to Temperature and Chilling Injury throughout the Climacteric Cycle

Mitochondria were isolated from ;Fuerte' avocado fruit (Persea americana Mill.) at four different stages of the respiratory climacteric. Preclimacteric fruit had the highest rate of succinate oxidation and the postclimacteric mitochondria the lowest. Subsequently, successive additions of ADP increased the respiratory control ratio.Arrhenius plots of succinate oxidation of intact mitochondria from climacteric rise and climacteric peak fruit showed two transition temperatures, while only one was observed in preclimacteric fruit. The low temperature phase transition was at about 9 C, while the high one was at 20 C. In postclimacteric fruit, the low temperature transition decreased to between 5 and 2 C. The state 3 rate of succinate oxidation was highest for mitochondria from preclimacteric fruit and decreased for each later stage. The state 4 rates for preclimacteric and climacteric rise were the same, while both the climacteric peak and postclimacteric rates were about 40% lower than the preclimacteric O(2) uptake.The results indicate continuous changes in the mitochondrial membrane of the electron transport chain throughout the climacteric cycle. The change in the membrane influencing the phosphorylation system is greatest between climacteric rise and peak stages. Mitochondrial membranes of postclimacteric fruit are presumed to change from flexible disordered to solid ordered phase at a lower temperature than those of other climacteric stages.

Mutations affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli

A strain carrying a point mutation affecting the NADH dehydrogenase complex of Escherichia coli has been isolated and its properties examined. The gene carrying the mutation (designated ndh) was located on the E. coli chromosome at about minute 23 and was shown to be cotransducible with the pyrC gene. Strains carrying the ndh − allele were found to be unable to grow on mannitol and to grow very poorly on glucose unless the medium was supplemented with succinate, acetate or casamino acids. The following properties of strains carrying the ndh − allele were established which suggest that the mutation affects the NADH dehydrogenase complex but apparently not the primary dehydrogenase. Membrane preparations possess normal to elevated levels of d-lactate oxidase and succinate oxidase activities but NADH oxidase is absent. NADH is unable to reduce ubiquinone in the aerobic steady state and reduces cytochrome b very slowly when the membranes become anaerobic. NADH dehydrogenase, measured as NADH-dichlorophenolindophenol reductase is reduced but not absent. NADH oxidase is stimulated by menadione although not by Q-3 or MK-1 and in the presence of menadione, cytochrome b is reduced normally by NADH. Further mutants affected in NADH oxidase were isolated using a screening procedure based on the growth characteristics of the original ndh − strain. The mutations carried by these strains were all cotransducible with the pyrC gene and the biochemical properties of the additional mutants were similar to those of the original mutant. The properties of the group of ndh − mutants established so far suggest that they are affected in the transfer of reducing equivalents from the NADH dehydrogenase complex to ubiquinone.

Removal of "tightly bound" nucleotides from phosphorylating submitochondrial particles.

Phosphorylating submitochondrial particles from beef heart (ETPH) prepared here contained about 2.4 nmol of ATP and 1.9 nmol of ADP/mg of protein after repeated washing of the particles. Essentially all of the "tightly bound " ATP and ADP was removed by trypsin treatment. The trypsin-treated ETPH had increased ATPase activity, undiminished NADH oxidase and succinate oxidase activity, but energy-coupling activity (ATP-driven reversed electron transfer) was abolished. Removal of half the ATP and ADP occurred at low levels of trypsin and was associated with loss of half of the coupling activity. Gel filtration of ETPH in high ionic strength buffer also removed ADP and ATP from the particles, resulting in loss of energy-coupling activity, while ATPase activity was increased. The results support the contention that the tightly bound ADP is essential in energy coupling in mitochondria. Tightly bound ATP may also play an essential role.

Membrane-bound respiratory of Spirillum itersonii

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.

Carboxins: Powerful selective inhibitors of succinate oxidation in animal tissues

Carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) is a systemic fungicide, reported to inhibit succinate oxidation in certain fungi, particularly Ustilago maydis (corn smut). In the present study the action of carboxin and of other oxathiin derivatives on beef heart succinate dehydrogenase has been investigated. Carboxins inhibited the same activities to the same extent as thenoyltrifluoroacetone (TTF) but at much lower concentrations. For 14 carboxin derivatives the inhibition constants (concentration required to inhibit 50% of the carboxin-sensitive activity) ranged from 2 × 10 −8 to 2 × 10 −6 M . Like TTF, carboxin derivatives did not inhibit soluble succinate dehydrogenase but inhibited the reduction of coenzyme Q analogs, of 2,6-dichlorophenolindophenol, and of phenazine methosulfate (PMS) in Complex II preparations. The same reactions and succinoxidase activity were also inhibited in inner membranes (ETP). In ETP only ∼ 50% of the succinate-PMS activity was carboxin sensitive, the same fraction as is inhibited by TTF or is lost on extraction of coenzyme Q and on incubation with cyanide. While the inhibition of PMS reduction by carboxin was largely or entirely competitive in Complex II, it was predominantly non-competitive in ETP at low concentrations. Some other carboxin derivatives gave mixed inhibition patterns for PMS reduction in ETP even at low inhibitor concentrations. The complex inhibition pattern in the PMS assay seems more compatible with conformation changes affecting activity than with loss of a reaction site for PMS.

Changes in Respiratory Pathways of Potato Tuber (Solanum tuberosum L.) after Various Times of Storage of the Tuber. Comparison of Wounded and Non-Wounded Tissue

Summary Respiration of potato tuber discs increases during incubation for 1 day at 25 °C to a value which is roughly twice as high as the respiration immediately after slicing. During the storage of potato tubers the respiration level of non-incubated discs and that reached by incubated slices declines, mainly in the period from harvest to january. During the storage period the changes in activity of several parameters linked to respiration have been determined both in freshly cut slices and in incubated tissue. These parameters do not uniformly change parallel with the increase of respiration during incubation or with the decline of respiration levels during the storage period. The activity of mitochondrial malate dehydrogenase located in the matrix does not change much after wounding and incubation and decreases slowly during the storage period. Cytochrome oxidase and CN-sensitive succinate oxidase, located in the mitochondrial inner membrane slightly increase after wounding and incubation. The former rapidly decreases in activity in the first months of storage and more slowly afterwards. The latter decreases in the first months of storage and stabilizes afterwards. CN-resistant succinate oxidase activity (alternative oxidase) of the inner membrane is not present in mitochondria from non-incubated tissue. It is induced during incubation only when the potatoes have been stored for some time but is not induced immediately after harvest. Only a small activity of outer membrane located, antimycin-A resistant NADH-cytochrome c reductase could be found in freshly cut slices. During incubation it strongly increases immediately after harvest, but this increase disappears gradually during storage of the tubers. The protein content of the mitochondrial fraction roughly doubles during one day of incubation. The protein in this fraction both from fresh discs and incubated ones rises during storage of the tubers. The activity of the cytoplasmatic 6-phosphogluconate dehydrogenase doubles after incubation of discs but levels do not change throughout the storage period.

The branched respiratory system of photosynthetically grown Rhodopseudomonas capsulata

Various respiratory electron transport activities of Rhodopseudomonas capsulata were studied in membrane fragments prepared from photosynthetically grown cells of a parental strain and two terminal oxidase-defective mutant strains. The NADH and succinate oxidase activities of the mutant having a functional N,N,N 1,N 1- tetramethyl-p- phenylenediamine oxidase, M6, were considerably more sensitive to inhibition by either antimycin A or cyanide than the corresponding activities of the mutant lacking a functional N,N,N, 1N 1- tetramethyl-p- phenylenediamine oxidase, M7. The parental strain, Z-1, but not the mutants, showed biphasic inhibitory responses of NADH and succinate oxidase activities with either antimycin A or cyanide. In certain reactions no differences in inhibitor susceptibility were found among the strains tested, implying that the pathways involved were unaffected in the mutants. In this category were the actions of rotenone on NADH oxidase, antimycin A on cytochrome c reductase and, in M6 and Z-1, cyanide on N,N,N′,N′- tetramethyl-p- phenylenediamine oxidase. These results suggest that the respiratory chain of the parental strain branches at the ubiquinone-cytochrome b region into two pathways, each branch goes to a distinct terminal oxidase, and either may be blocked independently by genetic mutation.

Purification and properties of a low molecular weight protein factor of mitochondrial energy-linked functions

1. 1. A soluble protein with a molecular weight of 11–12 · 10 3 has been isolated from bovine-heart mitochondria, which stimulates the following ATP-dependent reactions of submitochondrial particles treated with 0.6 mM EDTA and 1 M NH 4 OH: reverse electron transfer from succinate to NAD, transhydrogenation from NADH to NADP, and ATP · P i exchange. The factor has no effect on the NADH oxidase, succinate oxidase and ATPase activities of the particles. 2. 2. The stimulatory effect of the factor in the ATP-dependent reduction of NAD by succinate is 12 μmol · min −1 · mg −1 of the factor protein. However, the NH 4OH · EDTA treated particles are saturated for maximal activation of the above reaction by very small amounts of the factor (about 20–40 μg factor per mg particle). 3. 3. Electrophoresis of the factor preparation on polyacrylamide gels showed a single protein band plus a nonprotein material which moved at the dye front and was weakly stained with Coomassie Blue. The protein was shown to be required for activation of the particles; whether the fast-moving, nonprotein material is also required is not known. 4. 4. The factor is inhibited by mercurials and N- ethylmaleimide . The former, but not the latter, inhibition is completely reversed by 1,4-dithiothreitol. 5. 5. The NH 4OH · EDTA treated particles are also stimulated by rutamycin up to about 0.1 nmol of rutamycin per mg particle; higher rutamycin concentrations inhibit. Depending on the particle preparation, the factor stimulates up to about 3 nmol per mg particle, but does not inhibit at higher concentrations. In addition, under certain conditions in which appropriate concentrations of rutamycin fail to stimulate the particles, the factor still does.

Discontinuities in the arrhenius plots of mitochondrial membrane-bound enzyme systems from a poikilotherm: Acclimation temperature of carp affects transition temperatures

1. Evidence is presented for ‘breaks’ in the Arrhenius plots of succinate and cytochrome oxidase systems of mitochondria from carp liver and red epaxial muscle. 2. Arrhenius activation energies (Ea) of succinate and cytochrome oxidase systems above and below the transition temperatures (T*) are about half those reported for homoiotherms (Table 3). Succinate oxidase and cytochrome oxidase obey Michaelis-Menten kinetics. The Km-values for succinate and tetramethyl-p-phenylenediamine (TMPD) (25°C) are similar to those in homoiotherms; they are not affected by acclimation temperature (Table 2). 3. The change inEa of carp liver mitochondria (C.L.M.) occurs at low (high) experimental temperatures if carps are acclimated to low (high) ambient temperatures for at least 4 (3) weeks prior to the experiments (Fig. 1). 4. TheT* shift in Arrhenius plots following a change in acclimation temperature has been studied for succinate oxidation by carp liver mitochondria (Fig. 2). The time constant ofT* change was calculated as taccl.1/2=4.3 days (Fig. 3). Based on the concept of lipid protein interaction the acclimation temperature dependency ofT* is discussed as a possible consequence of a lipid adaptation. 5. Carp acclimated to 26°C and 10°C do not differ significantly with respect to specific activity of liver succinate oxidase determined at 25°C (Table 1). The same is true for the liver-somatic index and the amount of mitochondrial protein per gram of liver (Table 1). Changes, however, are observed in these parameters if carp are in a transitory state of temperature acclimation (early after changing from 26°C to 10°C; Table 1). 6. A bimodal action of temperature change during the process of acclimation is discussed combining the results in a model of succinate oxidase system of carp liver (Fig. 4).

Different temperature dependences of oxidative phosphorylation in the inner and outer layers of tuna heart ventricle

1. The oxidative phosphorylation and the temperature dependence of mitochondria prepared from the outer compact layer and the inner spongy layer of adult tuna heart ventricle have been examined. 2. Mitochondria of the inner layer show higher succinoxidase and NADH-oxidase activities as compared with those of the outer layer. 3. Arrhenius plots for succinate oxidation by phosphorylating mitochondria show that the temperature dependence of the inner layer is higher than that of the outer layer. 4. Experiments performed with disrupted non-phosphorylating mitochondria demonstrate that this difference in temperature dependence of the two cardiac compartments depends on the integrity of the mitochondrial membranes. 5. These findings are discussed in relation to the physiology of the fish.

A comparative study of oxidative enzymatic potential of Sphermophilus lateralis and Spermophilus tereticaudus

1.1. Adult Spermophilus lateralis and Spermophilus tereticaudus were examined for possible differences in cellular metabolic potential as indicated by oxidative enzyme levels.2.2. The body weight of S. tereticaudus was 38% smaller than S. lateralis. The body to organ weight ratios of the two species were not significantly different except for the kidney which was higher in S. tereticaudus.3.3. The specific and total activity of succinoxidase in the heart, brown adipose tissue (BAT), muscle and kidney was greater (P ⩽ 0.05) in S. lateralis.4.4. The specific and total activity of glutamic acid oxidase was greater (P ⩽ 0.05) in kidney, muscle and liver of S. lateralis, but no differences were observed in BAT or heart.5.5. The total cytochrome oxidase activity of BAT was higher in S. lateralis; but there were no differences in the α-glycerophosphate dehydrogenase activity.6.6. These results indicate potential metabolic differences exist in two closely related but ecologically diversified scuirids.