An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.     

The intracellular localization of adrenal 3β-hydroxysteroiddehydrogenase/Δ5-isomerase by density gradient perturbation

The intracellular localization of 3β-hydroxysteroiddehydrogenase/Δ⁵-isomerase (Δ⁵-3β-OHD) in bovine adrenal cortex was determined by density perturbation with density gradient centrifugation and the use of marker enzymes. Mitochondrial rich fractions after incubation with succinate and iodonitrotetrazolium (INT) were loaded with insoluble reduced formazan and separated on a linear sucrose gradient. A shift in median density was observed in the INT-treated mitochondria for protein, succinic dehydrogenase activity, and for Δ⁵-3β-OHD activity, but not for 21-hydroxylase, a microsomal enzyme marker. Density perturbation and gradient fractionation reveals a dual location of Δ⁵-3β-OHD in both mitochondrial and microsomal fractions.     

A simplified dehydrogenase enzyme assay in contaminated sediment using 2-(p-Iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride

2-(p-Iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) accepts electrons from dehydrogenase enzymes and is reduced to a red-colored formazan (INTF), which can be quantified by colorimetric analysis. Use of previously published methods for this technique was unsuccessful due to background chemical reactions from high levels of polycyclic aromatic hydrocarbons (PAHs) and metals in the sediments. A modified method using acetonitrile extraction of the INTF was efficient and did not chemically reduce INT. This activity method is simple, quick, inexpensive and precise.     

An investigation of staining methods to determine total cell numbers and the number of respiring micro-organisms in samples obtained from the field and the laboratory

Abstract Dyes were evaluated in combination with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to enable total cell numbers and the numbers of respiring cells to be determined on the same preparation. Malachite green and 4',6-diamidino-2-phenylindole (DAPI) were unsuitable counter-stains. Cells which contained INT formazan crystals could be stained with ethidium bromide or auramine. At high concentrations of INT formazan, auramine fluorescence was reduced, although this effect was partially rectified by prior fixation with glutaraldehyde. Staining with ethidium bromide produced a strong fluorescence in cells containing crystals of INT formazan. This observation was developed into a procedure which allowed total cells to be determined and provided a useful estimate of the number of respiring cells in samples obtained from the laboratory and the field.     

Succinate oxidation in intact tissue sections: the effect of oxidation in the cytoplasm

Summary In intact tissue sections, neotetrazolium formazan production is greater when the mitochondrial succinate dehydrogenase and the cytoplasmic glucose-6-phosphate dehydrogenase activities are tested together than when measured individually. Studies with malonate have shown that this increase is due to an increase in the rate of oxidation of succinate. Such an increase does not occur however when the hydrogen transport pathway is by-passed with PMS. It is suggested that the cytoplasmic activity overcomes a rate-limiting step in the mitochondrial transport chain.     

A cytochemical staining procedure for succinate dehydrogenase activity in pre-ovulatory mouse oocytes embedded in low gelling temperature agarose.

We describe a cytochemical staining procedure for succinate dehydrogenase (SDH) activity in pre-ovulatory mouse oocytes. The oocytes were embedded in low gelling temperature agarose and treated with caffeine before cytochemical staining in the presence of nitro blue tetrazolium (NBT), phenazinemethosulfate (PMS), and succinate. This resulted in intense staining of the oocytes by formazan precipitate. The level of aspecific formazan production in the absence of succinate was very low. We applied the procedure to oocytes matured in vitro and found that the location of the formazan precipitate as a result of SDH activity correlated well with the location of mitochondria. The chromatin of the cytochemically stained oocytes could subsequently be analyzed by means of the DNA-specific fluorochrome DAPI. In pre-ovulatory oocytes, we found a correlation between chromatin organization and the location of mitochondria: in oocytes with an intact germinal vesicle the mitochondria were uniformly distributed in the cytoplasm, as shown by fine grains of formazan precipitate. In oocytes with condensed chromatin the mitochondria apparently had clustered, because the formazan precipitate was more coarse in these cells.     

Enzyme, electron microscopic and polarographic characteristics of isolated rat brain mitochondria. III. Quantitative assessment of their distribution in fractions of the homogenate]

Activities of succinate dehydrogenase, succinate- and NAD-H-cytochrome c--reductases, and cytochrome c--oxidase was compared in 1 g tissue homogenate and homogenate fractions made from 1 g brain tissue using various solutions. Fractionation resulted in the increased activities of NADH- and succinate cytochrome reductases, and in the loss of succinate dehydrogenase activity, cytochrome oxidase was less influenced. These phenomena are regarded as signs of the interrelation between mitochondria and other constituents of brain cell within homogenates. Maximal quantity of mitochondria isolated from homogenates is no more than 20% of all the mitochondrial homogenates (according enzyme data). The electronogram of the brain mitochondrial preparation isolated in the Krebs--Ringer solution without glucose pointed out to a high homogeneity of mitochondria in the residue.     

Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets

Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.     

A permeability test for the study of mitochondrial injury in in vitro cultured heart muscle and endothelioid cells.

A cytochemical permeability test for the detection of injury to in situ mitochondria of cultured heart cells is presented. The test is based on the increased rate at which injured mitochondria stain for succinate dehydrogenase activity. Whereas an intact inner mitochondrial membrane limits the rate at which Nitro Blue tetrazolium and phenazine methosulphate reach succinate dehydrogenase, injured mitochondria allow these reactants to reach the enzyme more rapidly to form microscopically-observable formazan granules. The extent of staining at fixed durations of incubation with the reactants was assessed on a blind basis with pseudo dark-field microscopy, using a standardized rating scale. Differences in the staining of control and treated cells were analysed statistically by a semi-quantitative method. Treatment of the cultures with either vitamin A or chlorpromazine, resulted in more rapid mitochondrial staining. Brief pre-fixation of the cells with cold acetone also labilized the mitochondria as did a delay in the change of culture medium.     

Evidence for Chloroplastic Succinate Dehydrogenase Participating in the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardtii

A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ([1983] Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K_m for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H₂ adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen [1986] Plant Physiol 82: 160-166).     

Effect of intermittent hypoxic hypoxia on energy supply of rat skeletal muscle during adaptation to physical load

The experiment, on Wistar male rats was carried out to investigate influence of endurance training (swimming with load 7.0 +/- 1.3% body weight, 30 min a day, during 4 weeks) and additional intermittent hypoxic training (12% O2 in N2 - 15 min, 21% O2 - 15 min, 5 sessions a day, during the first 2 weeks) on the following parameters: ADF-stimulated mitochondrial respiration, lactate/pyruvate ratio, succinate dehydrogenase activity, and lipid peroxidation in skeletal muscle. The next oxidation substrates were used: 1 mmol/l succinate and 1 mmol/l alpha-ketoglutarate as well as the next inhibitor succinate dehydrogenase 2 mmol/l malonate. It was shown that physical work combined with intermittent hypoxic training led to the increase of mitochondrial respiration effectiveness in muscle energy supply under alpha-ketoglutarate oxidation in comparison with succinate oxidation as well as to the decrease of succinate dehydrogenase activity and lipid peroxidation. The study suggested that these changes may correct mitochondrial dysfunction under intensive muscular work.     

Effect of substrate concentration,inorganic nitrogen,O2 concentration,temperature and pH on dehydrogenase activity in soil

Summary Dehydrogenase activity was measured in a sandy loam soil under a variety of incubation conditions using the reduction of 2-(p-iodophenyl-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan). There was a high positive correlation between dehydrogenase activity and substrate concentration, incubation temperature, and soil pH. Dehydrogenase activity also displayed a high negative correlation with O₂ concentrations. Ammonium sulfate at concentrations from 40 to 120 g/g soil had no significant effect on dehydrogenase activity. However, at concentrations of 160 and 200 g/g, dehydrogenase activity was significantly reduced. Potassium nitrate at concentrations ranging from 40 to 200 g/g had no significant effect on soil dehydrogenase activity, whereas sodium nitrite significantly inhibited activity at concentrations of 120 and 160 g/g soil.     

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.     

Characterization of an inducible,membrane-bound iminodiacetate dehydrogenase from Chelatobacter heintzii ATCC 29600

Iminodiacetate (IDA) is a xenobiotic intermediate common to both aerobic and anaerobic metabolism of nitrilotriacetate (NTA). It is formed by either NTA monooxygenase or NTA dehydrogenase. In this paper the detection and characterization of a membrane-bound iminodiacete dehydrogenase (IDA-DH) from Chelatobacter heintzii ATCC 29600 is reported, which oxidizes IDA to glycine and glyoxylate. Out of 15 compounds tested, IDA was the only substrate for the enzyme. Optimum activity of IDA-DH was found at pH 8.5 and 25°C, respectively, and the K_m for IDA was found to be 8mM. Activity of the membrane-bound enzyme was inhibited by KCN, antimycine and dibromomethylisopropyl-benzoquinone. When inhibited by KCN IDA-DH was able to reduce the artificial electron acceptor iodonitrotetrazolium (INT). It was possible to extract IDA-DH from the membranes with 2% cholate, to reconstitute the enzyme into soybean phospholipid vesicles and to obtain IDA-DH activity (more than 50% recovery) using ubiquinone Q₁ as the intermediate electron carrier and INT as the final electron acceptor. Growth experiments with different substrates revealed that in all NTA-degrading strains tested both NTA monooxygenase and IDA-DH were only expressed when the cells were grown on NTA or IDA. Furthermore, in Cb. heintzii ATCC 29600 growing exponentially on succinate and ammonia, addition of 0.4 g l^-1 NTA led to the induction of the two enzymes within an hour and NTA was utilized simultaneously with succinate. The presence of IDA-DH was confirmed in ten different NTA-degrading strains belonging to three different genera.Abbreviations cA component A - cB component B - DBMIB dibromomethylisopropyl-benzoquinone - HEPES hydroxyethylpiperazinethanesulfonic acid - IDA iminodiacetate, HN(CH₂COOH)₂ - IDA-DH iminodiacetate dehydrogenase - INT iodonitrotetrazolium chloride - NTA nitrilotriacetate, N(CH₂COOH)₃ - NTA-MO nitrilotriacetate monooxygenase - PMS phenazine methosulphate - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Suc-DH succinate dehydrogenase     

Metabolic Status of Bacteria and Fungi in the Rhizosphere of Ponderosa Pine Seedlings

We determined the quantity and metabolic status of bacteria and fungi in rhizosphere and nonrhizosphere soil from microcosms containing ponderosa pine seedlings. Rhizosphere soil was sampled adjacent to coarse, fine, or young roots. The biovolume and metabolic status of bacterial and fungal cells was determined microscopically and converted to total and active biomass values. Cells were considered active if they possessed the ability to reduce the artificial electron acceptor 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan. A colorimetric assay of INT formazan production was also used to assess dehydrogenase activity. INT-active microorganisms made up 44 to 55% of the microbial biomass in the soils studied. The proportion of fungal biomass that exhibited INT-reducing activity (40 to 50%) was higher than previous estimates of the active proportion of soil fungi determined by using fluorescein diacetate. Comparison between soils from different root zones revealed that the highest total and INT-active fungal biomass was adjacent to fine mycorrhizal roots, whereas the highest total and active bacterial biomass was adjacent to the young growing root tips. These observations suggest that fungi are enhanced adjacent to the fine roots compared with the nonrhizosphere soil, whereas bacteria are more responsive than fungi to labile carbon inputs in the young root zone. Colorimetric dehydrogenase assays detected gross differences between bulk and rhizosphere soil activity but were unable to detect more subtle differences due to root types. Determination of total and INT-active biomass has increased our understanding of the role of spatial compartmentalization of bacteria and fungi in rhizosphere carbon flow.     

Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli

The effects of substrates of primary aerobic dehydrogenases, and inorganic phosphate on aerobic INT and CTC reduction in Escherichia coli were examined. In general, INT produced less formazan than CTC, but INT (+) cell counts remained near values of CTC (+) cells. INT and CTC (+) cell numbers were higher than plate counts on R2A medium using succinate, formate, lactate, casamino acids, glucose, glycerol (INT only) and no substrate. Formate resulted in the greatest amount of INT and CTC formazan. Reduction of both INT and CTC was inhibited above 10 mmol 1^-1 phosphate, and this appeared to be related to decreased rates of O₂ consumption. Formation of fluorescent CTC (+), but not INT (+) cells was also inhibited in a concentration dependent manner by phosphate above 10 mmol 1^-1. From light microscopic observations it appeared CTC formed increasing amounts of poorly or non-fluorescent formazan with increasing phosphate. Therefore, use of phosphate buffer in excess of 10 mmol 1^-1 may not be appropriate in CTC and INT reduction assays.     

Localization of the Enzymes Involved in the Photoevolution of H(2) from Acetate in Chlamydomonas reinhardtii

The localization of a series of enzymes involved in the anaerobic photodissimilation of acetate in Chlamydomonas reinhardtii F-60 adapted to a hydrogen metabolism was determined through the enzymic analyses of the chloroplastic, cytoplasmic, and mitochondrial fractions obtained with a cellular fractionation procedure that incorporated cell wall removal by treatment with autolysine, digestion of the plasmalemma with the detergent digitonin, and fractionation by differential centrifugation on a Percoll step gradient. The sequence of events leading to the photoevolution of H₂ from acetate includes the conversion of acetate into succinate via the extraplastidic glyoxylate cycle, the oxidation of succinate to fumarate by chloroplastic succinate dehydrogenase, and the oxidation of malate to oxaloacetate in the chloroplast by NAD dependent malate dehydrogenase. The level of potential activity for the enzymes assayed were sufficient to accommodate the observed rate of the photoanaerobic dissimilation of acetate and the photoevolution of H₂.     

Quantitative succinate-dehydrogenase histochemistry. I. A Methodological study on mammalian and fish muscle.

In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are chosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.     

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide.

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.     

Quantitative succinate-dehydrogenase histochemistry

Summary In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are choosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.