The intramitochondrial localization of succinate-yellow tetrazolium reductase with the electron microscope

Summary A new tetrazolium salt, yellow tetrazolium, has been used to localise succinatetetrazolium reductase with the electron microscope. As expected, the formazan did not give high contrast in the optical microscope, but localization with the EM was good. The size of the formazan granules was 60–100Å; lead staining was essential to secure good contrast.     

NADPH: nitroblue tetrazolium reductase found in plasma membrane of human neutrophil

After phorbol 12-myristate 13-acetate (PMA) stimulation the increase of NADPH:nitroblue tetrazolium reductase activity in the plasma membrane almost corresponded with the stimulated activity of respiratory burst oxidase. Solubilization of plasma membranes from PMA-activated neutrophils with n-octyl glucoside resulted in high recoveries of the two enzymatic activities. When solubilized plasma membrane was subjected to non-denaturing polyacrylamide gel electrophoresis in the presence of 35 mM n-octyl glucoside, we could see three major bands stained with NADPH-dependent nitroblue reductase activity giving molecular masses of approx. 95, 45 and 40 kDa, respectively. Activity was specific for NADPH but not for NADH. These bands also stained weakly in the plasma membranes obtained from resting cells. The activities for NADPH oxidase and nitroblue tetrazolium reductase were found to elute as a very similar protein peak on an anion-exchange HPLC, at about 0.32 M KCl. This elution peak also contains 45 and 40 kDa proteins showing NADPH:nitroblue tetrazolium reductase activity.     

Characterization of a yeast phase specific protein from a fungus, Histoplasma capsulatum

We have characterized an unusual yeast phase specific protein from Histoplasma capsulatum. The protein, which we have called protein 6, is produced by the yeast cells which have been derepressed for sulfite reductase, and it can account for more than 40% of the total extract protein. Synthesis of both sulfite reductase and protein 6 is subject to cysteine repression. However, sulfite reductase activity is maximal in logarithmically growing cells whereas protein 6 is synthesized de novo and accumulated by stationary phase cells. The following are the major physicochemical properties of protein 6: (1) the native protein has a molecular weight of about 15 000; (2) electrophoresis on a sodium dodecyl sulfate polyacrylamide gel yielded a single band with a molecular weight 7600; (3) protein 6 is capable of reducing the dye, nitroblue tetrazolium, and cytochrome c, a property that has been found to be shared by a number of trypsin inhibitors, and (4) the molecule is negatively charge and is relatively resistant to proteolysis. The amino acid composition of protein 6 has been determined.     

Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1.

The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells. The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates. The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method. The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups. Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor.     

Polarographic investigation on tetrazolium reductase activity

Summary An attempt was made to elaborate a simple method of tetrazolium reductase activity assay in biological material. The method is based on recording of changes in the amperage of threshold current, occuring in course of the reduction of tetrazolium salts. — The method could be employed by histochemists working on the reduction of tetrazolium salts.     

Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H₂O₂ oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC₅₀ 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC₅₀ 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).     

Multiple forms of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from spinach.

Crude extracts of ferredoxin-NADP reductase prepared from spinach by three different methods consistently contained two molecular weight forms of the enzyme: P-1, 117,500, and P-2, 50,000. The lower molecular weight form was purified and shown to consist of two different ionic forms. These three forms of the flavoprotein are immunologically identical. A third molecular weight form of the reductase, excluded by Sephadex G-100, generated P-1 and P-2 on rechromatography. Other experiments demonstrated that this enzyme has NADPH-tetrazolium reductase activity and it accounts for essentially all of the tetrazolium reductase activity of isolated chloroplasts.     

Modifications of the active center of T4 thioredoxin by site-directed mutagenesis

The active site sequence of T4 thioredoxin, Cys-Val-Tyr-Cys, has been modified in two positions to Cys-Gly-Pro-Cys to mimic that of Escherichia coli thioredoxin. The two point mutants Cys-Gly-Tyr-Cys and Cys-Val-Pro-Cys have also been constructed. The mutant proteins have similar reaction rates with T4 ribonucleotide reductase as has the wild-type T4 thioredoxin. Mutant T4 thioredoxins with Pro instead of Tyr at position 16 in the active site sequence have three to four times lower apparent KM with E. coli ribonucleotide reductase than wild-type T4 thioredoxin. The KM values for these mutant proteins which do not have Tyr in position 16 are thus closer to E. coli thioredoxin than to the wild-type T4 thioredoxin. The bulky tyrosine side chain probably prevents proper interactions to E. coli ribonucleotide reductase. Also the redox potentials of these two mutant thioredoxins are lower than that of the wild-type T4 thioredoxin and are thereby more similar to the redox potential of E. coli thioredoxin. Mutations in position 15 behave more or less like the wild-type protein. The kinetic parameters with E. coli thioredoxin reductase are similar for wild-type and mutant T4 thioredoxins except that the apparent kcat is lower for the mutant protein with Pro instead of Tyr in position 16. The active site sequence of T4 thioredoxin has also been changed to Cys-Pro-Tyr-Cys to mimic that of glutaredoxins. This change does not markedly alter the reaction rate of the mutant protein with T4 ribonucleotide reductase or E. coli thioredoxin reductase, but the redox potential is lower for this mutant protein than for wild-type T4 thioredoxin.     

Molecular cloning of the gene for dihydrofolate reductase from Lactobacillus casei

The Lactobacillus casei gene for dihydrofolate reductase has been cloned in Escherichia coli using the multicopy vector pBR322. A restriction map of the cloned DNA has been prepared. The cloned DNA directs the synthesis of L. casei dihydrofolate reductase in E. coli and confers trimethoprim and methotrexate resistance.     

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.     

Thioredoxin/Glutaredoxin System of Chlorella: CHLORELLA ADENOSINE 5'-PHOSPHOSULFATE SULFOTRANSFERASE CANNOT USE THIOREDOXIN OR GLUTAREDOXIN AS COFACTORS

Using the thioredoxin/glutaredoxin-dependent adenosine 3'-phosphate 5'-phosphosulfate reductase coupled assay system, the Chlorella thioredoxin/glutaredoxin system has been partially purified and characterized. A NADPH-thioredoxin reductase and two thioredoxin/glutaredoxin activities, designated as Chlorella thioredoxin/glutaredoxin protein I and II (CPI and CPII), were found in crude extracts of Chlorella. Similar to their counterparts from Escherichia coli, both CPI and CPII are heat-stable low molecular proteins of approximately 14,000. While CPI (but not CPII) is a substrate for its homologous NADPH-thioredoxin reductase as well as for E. coli NADPH-thioredoxin reductase, CPII is better than CPI as a substrate for reduction by the glutathione system. Based on these properties, CPI and CPII may be classified as Chlorella thioredoxin and Chlorella glutaredoxin, respectively. The Chlorella NADPH-thioredoxin reductase (M(r) = 72,000, with two 36,000-dalton subunits) resembles E. coli-thioredoxin reductase in size. Besides Chlorella thioredoxin, the Chlorella thioredoxin reductase will also use E. coli thioredoxin, but not glutaredoxin, as a substrate. Although a thioredoxin-like protein has been implicated in higher plant light-dependent sulfate reaction, neither Chlorella thioredoxin nor glutaredoxin can stimulate the thiol-dependent adenosine 5'-phosphosulfate-sulfotransferase reaction. Furthermore, Chlorella thioredoxin and glutaredoxin, in conjunction with an appropriate reductase system, cannot replace the thiol requirement of Chlorella adenosine 5'-phosphosulfate-sulfotransferase. The exact physiological roles and subcellular localization of the Chlorella thioredoxin and glutaredoxin systems remain to be determined.     

The gene-enzyme relationships of proline biosynthesis in Escherichia coli

A simple chromatographic procedure has been devised to separate gamma-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase, allowing the measurement of the former in crude Escherichia coli extracts. Analysis of a number of strains of E. coli has demonstrated that gene proA codes for gamma-glutamyl phosphate reductase and proB for gamma-glutamyl kinase. Introduction of a ColE1 hybrid plasmid containing the proA,B region into a strain with a chromosomal deletion of proA,B led to 3- and 17-fold increases in the specific activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, respectively.     

Reduction of tetrazolium salts catalyzed by soluble diaphorase in rat liver and embryos of Vicia faba

Summary The presence of two diaphorases has been shown in rat liver and embryos of Vicia fdba. One of these, the NAD(P)H tetrazolium reductase, was firmly bound in the section and was not lost into the incubation medium under conditions of histochemical assay The second diaphorase (soluble diaphorase) was lost from the section into the incubation medium during the first five minutes of incubation. This soluble diaphorase from both rat liver and embryos of V. faba is capable of transferring electrons from NAD(P)H to MTT, INT, NBT and TNBT, but not to tellurite, TTC, BT and NT. The behaviour of the soluble diaphorase in histochemical reactions involving tetrazolium salts as electron acceptors is discussed.     

Changes in enzyme histochemical profiles of identified spinal motoneurons of the European eel,Anguilla anguilla, following cordotomy

Summary The enzyme histochemical profiles of glucose-6-phosphate dehydrogenase (a marker of synthetic performance), succinate dehydrogenase (an indicator of oxidative metabolism), and NADH tetrazolium reductase (a marker of overall neuronal activity) were determined for identified white muscle motoneurons in six control and six cordotomized eels. Images were digitized and mean integrated absorbances obtained using appropriate hardware and software. For motoneurons caudal to the transection site there was a significant decrease in the mean absorbance value for NADH tetrazolium reductases which declines from 0.28 a.u. (arbitrary units) in control animals to 0.23 a.u. in cordotomized animals. However, no significant changes were detected in the activities of glucose-6-phosphate and succinate dehydrogenases. The cross-sectional area of the motoneuronal cell body was not affected by cordotomy. The decrease by around 20% in overall neuronal activity, as expressed by NADH tetrazolium reductase activity, might be expected from the decline in body motility that follows cordotomy. Changes in SDH and G6PDH activities would also be expected to follow this surgery, but none were seen, perhaps because they are compensated for by changes in neuronal metabolism that result from deafferentation.     

NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.     

Purification and characterization of a soybean flour-inducible ferredoxin reductase of Streptomyces griseus.

We have purified an NADH-dependent ferredoxin reductase from crude extracts of Streptomyces griseus cells grown in soybean flour-enriched medium. The purified protein has a molecular weight of 60,000 as determined by sodium dodecyl sulfate gel electrophoresis. The enzyme requires Mg2+ ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotide-containing flavoproteins. A relatively large number of hydrophobic amino acid residues are found by amino acid analysis, and beginning with residue 7, a consensus flavin adenine dinucleotide binding sequence, GXGXXGXXXA, is revealed in this protein. In the presence of NADH, the ferredoxin reductase reduces various electron acceptors such as cytochrome c, potassium ferricyanide, dichlorophenolindophenol, and nitroblue tetrazolium. However, only cytochrome c reduction by the ferredoxin reductase is enhanced by the addition of ferredoxin. In the presence of NADH, S. griseus ferredoxin and cytochrome P-450soy, the ferredoxin reductase mediates O dealkylation of 7-ethoxycoumarin.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8? resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.     

On-bead combinatorial techniques for the identification of selective aldose reductase inhibitors

Aldose reductase (AKR1B1; ALR2; E.C. 1.1.1.21) is an NADPH-dependent carbonyl reductase which has long been associated with complications resulting from the elevated blood glucose often found in diabetics. The development of effective inhibitors has been plagued by lack of specificity which has led to side effects in clinical trials. To address this problem, a library of bead-immobilized compounds was screened against fluorescently labeled aldose reductase in the presence of fluorescently labeled aldehyde reductase, a non-target enzyme, to identify compounds which were aldose reductase specific. Picked beads were decoded via novel bifunctional bead mass spec-based techniques and kinetic analysis of the ten inhibitors which were identified using this protocol yielded IC50 values in the micromolar range. Most importantly, all of these compounds showed a preference for aldose reductase with selectivities as high as approximately 7500-fold. The most potent of these exhibited uncompetitive inhibition versus the carbonyl-containing substrate D/L-glyceraldehyde with a Ki of 1.16 microM.     

The monocytic differentiation of HL60 induced by rat kidney NADPH-linked high-Km aldehyde reductase protein

The human promyelocytic leukemia cell line HL60 differentiates to monocyte/macrophage cells when incubated with NADPH-linked high-Km aldehyde reductase (EC 1.1.1.2) purified from the cytosol of rat kidney. Differentiation was assessed by cell growth, morphology, adhesiveness, nitro blue tetrazolium reduction, and nonspecific esterase activity. The extent of differentiation induced by the reductase and measured at 4 days by nitro blue tetrazolium reduction is dose-dependent with an ED50 (dose required for half-maximal effect) of 71 nM. In the presence of 10 nM retinoic acid the ED50 for reductase is reduced to 18 nM and an isobologram analysis of this effect indicates that the combination is synergistic. Inactivation of the enzymatic activity is not associated with a decrease in differentiation-induced activity. These results suggest that the structure of the enzyme protein and not its enzymatic activity is involved with induction of differentiation. This view is supported by the demonstration that aldehyde reductase binds specifically to HL60 cells with a KD of 70 nM and that there are 13,000 binding sites/cell. Thus, the extent of differentiation induced by various concentrations of aldehyde reductase are directly related to the expected level of receptor occupancy.