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.     

Existence of ether bond-cleaving enzyme in a polyethylene glycol-utilizing symbiotic mixed culture

Abstract Diglycolic acid dehydrogenase activity linked with 2,6-dichlorophenolindophenol and phenazine methosulfate was found in the particulate fraction of the cell-free extract of a mixed culture of Flavobacterium and Pseudomonas species grown on polyethylene glycol 6000. The amount of glyoxylic acid formed increased with the increase in reaction time and enzyme concentration. Horse heart cytochrome c , 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl 2H-tetrazolium bromide, and nitro blue tetrazolium, served as hydrogen acceptors in the presence of phenazine methosulfate. Enzyme activity was competitively inhibited by 1,4-benzoquinone. The enzyme was also active on tetraethylene glycol dicarboxylic acid, a metabolite of tetraethylene glycol, and on methoxy- or ethoxyacetic acid.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.     

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.     

Oxidation of 1,5-anhydro-D-glucitol to 1,5-anhydro-D-fructose catalyzed by an enzyme from bacterial membranes

Bacteria which grow on 1,5-anhydro-D-glucitol (AG) were isolated from soil. One such strain showing the highest AG-assimilating activity was further characterized and identified as a new strain of the Pseudomonas family (named Pseudomonas sp. NK-85001). A subcellular membranous fraction obtained from this strain catalyzed the oxidation of AG to 1,5-anhydro-D-fructose. This oxidation reaction consumed molecular oxygen as the terminal electron acceptor. The AG-oxidizing activity was further purified after solubilization. The AG oxidation catalyzed by this solubilized enzyme utilized molecular oxygen only in the presence of an electron mediator such as 2,6-dichlorophenolindophenol or phenazine methosulfate. Thus, the enzyme was suggested to be a dehydrogenase rather than an oxidase. The solubilized enzyme preparation also showed a strict substrate specificity. The observed specificity indicated that application of the enzyme for AG assay in clinical samples might be possible.     

Ethanol dissimilation in Desulfovibrio

During growth of ethanol plus sulfate Desulfovibrio gigas and three other Desulfovibrio strains tested contained high NAD-dependent alcohol dehydrogenase activities and dye-linked aldehyde dehydrogenase activities. In lactate-grown cells these activities were lower or absent. In D. gigas an NADH dehydrogenase activity was found which was higher during growth on ethanol than during growth on lactate. The NADH dehydrogenase activity appeared to consist of at least three different soluble enzymes. The aldehyde dehydrogenase activity in D. gigas was highest with benzylviologen as an acceptor and was strongly stimulated by potassium ions. Coenzyme A or phosphate dependency could not be shown, indicating that acetyl-CoA or acetyl phosphate are not intermediates in the conversion of acetaldehyde to acetate.In the absence of sulfate D. gigas was able to convert ethanol to acetate by means of interspecies hydrogen transfer to a methanogen. This conversion, however, did not lead to growth of the Desulfovibrio.Abbreviations DH dehydrogenase - BV²⁺/BV⁺ oxidized/reduced benzylviologen - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide - MV²⁺/MV⁺ oxidized/reduced methylviologen - PMS phenazine methosulfate     

Fluorimetric assay of tobacco leaf dehydrogenases with resazurin.

A versatile fluorimetric assay based on the reduction of resazurin to resorufin demonstrated high specific activities for a number of important pyridine nucleotide-linked dehydrogenases in tobacco leaves. The Michaelis constant for the important photosynthetic enzyme, D-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (EC 1.2.1.13), determined by the fluorimetric method, was considerably lower than constants determined by conventional extraction and assay methods reported for the enzyme from other plants. The sensitivity of the fluorimetric method enabled the use of dilute enzyme preparations with resultant low background and high substrate specificity. Inclusion of the anti-oxidant diethyldithiocarbamate in the extraction medium preserved the enzymes during extraction. Primary amines inhibited competitively, and phenazine methosulfate non-competitively each of the eight dehydrogenases tested with the fluorimetric assay. The Mn2+ dependence of NADP-linked dehydrogenases specific for isocitrate and malate was confirmed. The method is rapid, requires a simple combination of ingredients and should be useful for surveying dehydrogenase activity in leaves.     

Ziram, a sulfhydryl reagent and specific inhibitor of yeast mitochondrial dehydrogenases.

The fungicide zinc dimethyldithiocarbamate (ziram) is a sulfhydryl reagent which inhibits specifically the growth of the yeast Saccharomyces cerevisiae on nonfermentable substrates. In isolated mitochondria, the uncoupled as well as the state 3 oxidations of succinate, α-ketoglutarate, ethanol, and malate plus pyruvate are sensitive to ziram concentrations of 10 to 30 μm. The oxidations of isocitrate, of external NADH, of α-glycerophosphate, and of ascorbate plus tetramethylphenylenediamine exhibit a lower sensitivity to ziram. Succinate, α-ketoglutarate, and pyruvate dehydrogenases activities are 50% inhibited by concentration of ziram lower than 10 μm. At the same concentrations, neither the mitochondrial transports of succinate, ADP, or phosphate nor oxidative phosphorylation and adenosine triphosphatase activities are modified. The kinetic study of the inhibition by ziram of succinate dehydrogenase activity shows that ziram is noncompetitive with succinate and produces sigmoidal inhibitions of state 3 and of uncoupled oxidation of succinate by intact mitochondria. Inhibition of succinate:phenazine methosulfate oxidoreductase activity yields exponential kinetics. However sigmoidal-type inhibition is observed when succinate dehydrogenase activity is stimulated by ATP.     

A new technique for the precise location of lactate and malate dehydrogenases in goat, boar and water buffalo spermatozoa using gel incubation film.

Gel incubation film, which contained gelatin to prevent the diffusion of enzyme during chemical reaction and phenazine methosulfate to operate as a hydrogen acceptor between NADH and tetrazolium, was used and light microscopy revealed that lactate dehydrogenase was located in the head and tail of the spermatozoa as well as in the midpiece, whereas malate dehydrogenase was confined to the midpiece in spermatozoa of the animals examined. In goat spermatozoa, lactate dehydrogenase was associated mainly with the inner acrosomal membrane in the head, the mitochondrial matrix in the midpiece and with flagellar fibrils in the tail, whereas malate dehydrogenase was present only in the mitochondrial matrix.     

Preparation and partial fractionation of nonhistone chromosomal proteins from human diploid fibroblasts

2,6-Dichlorophenolindophenol reacts with the sulfhydryl group at the active site of apo-glyceraldehyde-3-phosphate dehydrogenase isolated from chicken muscle to form a leuco dye-enzyme adduct which is yellow. The leuco dye-enzyme adduct is oxidized by 2,6-dichlorophenolindophenol to form an oxidized dye-enzyme adduct which is blue. NADH converts the oxidized enzyme-dye adduct to the leuco enzymedye adduct. The enzyme-dye adducts catalyze the oxidation of NADH by 2,6-dichlorophenolindophenol in a reaction which exhibits “ping-pong” kinetics. The pH rate behavior of the reaction catalyzed by the enzyme-dye adduct differs considerably from the non-enzymatic oxidation of NADH by 2,6-dichlorophenolindophenol. A scheme for the reaction catalyzed by the enzyme-dye adduct which is consistent with the experimental observations is presented.     

Functional mosaicism of membrane proteins in vesicles of Escherichia coli.

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.     

Oxidation of C1-compounds in Pseudomonas C.

Extracts of Pseudomonas C grown on methanol as a sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts. The addition of D-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when D-ribulose 5-phosphate was present in the assay mixtures. The amount of radioactivity found in CO2, was 6;8-times higher when extracts of methanol-grown Pseudomonas C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate. These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Interaction between NAD-dependent isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and NADH:ubiquinone oxidoreductase

Interaction between the alpha-ketoglutarate dehydrogenase complex and NAD+-dependent isocitrate dehydrogenase was detected with a variety of techniques including polyethylene glycol precipitation, ultracentrifugation, and centrifugal gel filtration on a Sepharose 6B column. The interaction was specific in that citrate synthase, cytosolic malate dehydrogenase, and NADP-dependent isocitrate dehydrogenase did not interact with alpha-ketoglutarate dehydrogenase complex. The interaction was not inhibited by either 0.1 M KCl or 0.4 M (NH4)2SO4, but was completely prevented by 5% glycerol. A new method for the preparation of NADH: ubiquinone oxidoreductase resulted in an enzyme having a protein subunit composition similar to that of classical complex I preparation. Evidence is given for the existence of ternary complexes containing NADH:ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-NAD-dependent isocitrate dehydrogenase and NADH: ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-succinate thiokinase. These data suggest that a part of the citric acid cycle may be located in the vicinity of NADH: ubiquinone oxidoreductase. These complexes may facilitate the transport of metabolites among these enzymes without their equilibrating with the whole compartment.     

Electrochemical bioreactor with regeneration of NAD+ by rotating graphite disk electrode with PMS adsorbed.

Kinetic constants were compared among p-quinone, 2,6-dichlorophenolindophenol, phenazine methosulfate (PMS), methylene blue, and FAD in the oxidation of NADH. Among those, PMS was selected for its highest rate constant as a mediator for the electrochemical oxidation of NAD. The PMS could be stably immobilized on a graphite electrode surface by adsorption. The PMS adsorbed and that in the solution showed distinctly separated peaks in the cyclic voltammogram. The immobilized PMS functioned as an immobilized mediator to reduce the overpotential in the electrochemical oxidation of NAD so that the electrode could be used as an NAD regenerator. For the construction of an electrochemical bioreactor, a specially designed rotating disk graphite electrode was used. In spite of its extraordinarily large surface area, the behavior of the rotating disc electrode was described well by the Levich law. The NAD oxidation system of the rotating graphite disk electrode with PMS adsorbed was combined with glucose-6-phosphate dehydrogenase reaction, which reduced NAD with the consumption of glucose-6-phosphate. The electrochemical bioreactor system worked well with recycling of NAD at a high current efficiency.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.     

Effects of pH, NADH, succinate and malate on the oxidation of glycine in spinach leaf mitochondria

The effect of external pH on several reactions catalyzed by glycine decarboxylase in spinach leaf mitochondria was investigated. Glycine-dependent oxygen consumption showed a pH optimum at 7.6, whereas the release of CO₂ and NH3 from glycine in the presence of oxaloacetate both showed pH maxima at 8.1. Glycine-dependent reduction of 2,6-dichlorophenolindophenol. on the other hand showed a pH optimum at 8.4. It is concluded that these three reactions have different rate-limiting steps. The rate of the glycine-bicarbonate exchange reaction catalyzed by glycine decarboxylase showed no optimum in the pH range investigated, pH 7–9, but increased with decreasing pH. This suggests that CO₂ may be the true substrate in this reaction.
The oxidation of glycine inhibited the oxidation of both malate, succinate and external NADH since the addition of malate, succinate or NADH to mitochondria oxidizing glycine in state 3 resulted in a rate of oxygen consumption which was lower than the sum of the rates when the substrates were oxidized individually. The addition of malate, succinate or NADH did not, however, decrease the rate of CO₂ or NH, release from glycine. It is suggested that the preferred oxidation of glycine by-spinach leaf mitochondria may constitute an important regulatory mechanism for the function of leaf mitochondria during photosynthesis.     

Sources of reducing power for nitrate reduction in spinach leaves

The possible source of NADH, the energy donor for nitrate reductase (EC 1.6.6.1), has been studied using an in vivo assay involving freezing the material (leaves of Spinacea oleracea L.) in liquid nitrogen in order to render the tissue permeable to added substrates. Glycolysis and the pentose phosphate pathway were capable of generating NADH through glyceraldehyde-3-phosphate dehydrogenase. Malate and isocitrate were also capable of generating NADH white other organic acids tested were not, including glycolate which was ineffective even under anaerobic conditions.     

D-fructose dehydrogenase of Gluconobacter industrius: purification, characterization, and application to enzymatic microdetermination of D-fructose.

D-Fructose dehydrogenase was solubilized and purified from the membrane fraction of glycerol-grown Gluconobacter industrius IFO 3260 by a procedure involving solubilization of the enzyme with Triton X-100 and subsequent fractionation on diethylaminoethyl-cellulose and hydroxylapatite columns. The purified enzyme was tightly bound to a c-type cytochrome and another peptide existing as a dehydrogenase-cytochrome complex. The purified enzyme was deemed pure by analytical ultracentrifugation as well as by gel filtration on a Sephadex G-200 column. The molecular weight of the enzyme complex was determined to be about 140,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of three components having molecular weights of 67,000 (dehydrogenase), 50,800 (cytochrome c), and 19,700 (unknown function). Only D-fructose was readily oxidized by the enzyme in the presence of dyes such as ferricyanide, 2,6-dichlorophenolindophenol, or phenazine methosulfate. Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and oxygen did not function as electron acceptors. The optimum pH of D-fructose oxidation was 4.0. The enzyme was stable at pH 4.5 to 6.0 Stability of the purified enzyme was much enhanced by the presence of detergent in the enzyme solution. Removal of detergent from the enzyme solution facilitated the aggregation of the enzyme and caused its inactivation. An apparent Michaelis constant for D-fructose was observed to be 10(-2) M with the purified enzyme. D-Fructose dehydrogenase was shown to be a satisfactory reagent for microdetermination of D-fructose.