Altered Diaphorase Activity in Optochin-resistant Pneumococci

Methylene blue (MB) reductase activities of washed cell suspensions and of cell-free extracts prepared from optochin-resistant mutant pneumococci were two times greater than and four to eight times more resistant to optochin inhibition than those of similar preparations from the optochin-sensitive parent strains. With whole cells, optochin hydrochloride was approximately four times more potent than quinine hydrochloride in inhibiting MB reductase activity. However, with cell-free extracts, both drugs had similar inhibitory activities. Glucose uptake was not affected by optochin hydrochloride, and both optochin-sensitive and optochin-resistant pneumococci had similar glucose uptake patterns. Diaphorase activities of cell-free extracts prepared from optochin-resistant pneumococci were two times greater than and four to eight times more resistant to optochin inhibition than those of cell-free extracts prepared from the optochin-sensitive parent strains. Flavin concentrations of cell-free extracts prepared from optochin-sensitive and optochin-resistant strains were similar.     

Evaluation of gene-technological and conventional methods in the identification of Streptococcus pneumoniae

To find reliable methods able to identify the "difficult" Streptococcus pneumoniae isolates, an in-house polymerase chain reaction (PCR) for pneumolysin gene (Ply-PCR) and a commercial RNA hybridisation test (AccuProbe) were evaluated. Selected isolates of suspected pneumococci, sent for confirmation of identification and for serotyping, were classified into four groups based on their optochin sensitivity and capsule reaction. All isolates in Group 1, which consisted of 24 typical, optochin-sensitive, encapsulated pneumococcal strains, were positive in the Ply-PCR and AccuProbe tests. In Group 2, which consisted of 25 optochin-sensitive, but unencapsulated pneumococcal strains, all the isolates were positive in the Ply-PCR test, and 23 were positive in the AccuProbe test. In Group 3, which consisted of 15 atypical, optochin-resistant but encapsulated pneumococci, 12 of the isolates were positive in the Ply-PCR and 12 in the AccuProbe test, and 11 of these 12 strains were positive in both tests. In Group 4, which consisted of 36 equivocal optochin-resistant, unencapsulated isolates, 15 strains were positive in the Ply-PCR test and 8 strains in the AccuProbe test. As a conclusion, the Ply-PCR and AccuProbe tests identified similarly typical optochin-sensitive pneumococci, but gave partly controversial results about atypical pneumococci. Thus, they did not reliably help in the identification of suspected pneumococcal isolates lacking the conventional characteristics of pneumococcus.     

Purification and properties of the phosphoglycerate mutase isozymes from the mouse

The two homodimeric isozymes of phosphoglycerate mutase have been purified from murine kidney and muscle. No differences were observed in the Michaelis-Menten constant for the substrate 2-phospho-D-glycerate, in molecular weight, temperature and pH optima, when the purified isozymes were compared. The isozymes differ in their inhibition constants for phosphoenolpyruvate, in their Michaelis constants for 3-phospho-D-glycerate and 2,3-bisphospho-D-glycerate, their thermal and pH lability and in their sensitivity towards mercury ions.     

A new flavin enzyme catalyzing the reduction of dihydrodipicolinate in sporulating Bacillus subtilis I. Purification and properties.

A dihydrodipicolinate reductase containing flavin was purified from sporulating Bacillus subtilis PCI 219. The purified enzyme appeared homogeneous by dise gel electrophoresis. Its molecular weight was estimated as 74,000 by gel filtration on Sephadex G-200, and as 18,500 by electrophoresis on sodium dodecylsulfate polyacrylamid gel. These results suggest that the enzyme is composed of four subunits. The prosthetic group was identified as FMN, and one mole of the enzyme contained two moles of FMN. Both NADPH and NADH acted as coenzyme, though NADH was less effective. The enzyme also exhibited diaphorase activity. The pH optimum was 6.1. The enzyme was inhibited by dipicolinate but not by lysine or alpha, epsilon-diaminopimelate.     

Studies on the biosynthesis of cytochrome P-450 in rat liver--a probe with phenobarbital.

The reaction of NAD(P)H:flavin oxidoreductase (flavin reductase) from Photobacterium fischeri is proposed to follow a ping-pong bisubstrate-biproduct mechanism. This is based on a steady-state kinetic analysis of initial velocities and patterns of inhibition by NAD⁺ and AMP. The double reciprocal plots of initial velocities versus concentrations of FMN or NADH show, in both cases, a series of parallel lines. The Michaelis constants for NADH (FMN saturating) and FMN (NADH saturating) are 2.2 and 1.2 × 10^?4m, respectively. The product NAD⁺ has been found to be an inhibitor competitive with FMN but non-competitive with NADH. Using AMP as an inhibitor, noncompetitive inhibition patterns were observed with respect to both NADH and FMN as the varied substrate. In addition, the reductase was not inactivated by treatment with N-ethylmaleimide either alone or in the presence of FMN, but the enzyme was inactivated by N-ethylmaleimide in the presence of NADH. These findings suggest that flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis.     

Reduced pyridine nucleotide oxidases of Eugena gracilis var. bacillaris.

Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (Km) determined were as follows: NADH diaphorase, 350 muM; NADPH oxidase 150 muM ; NADH lipoyl dehydrogenase, 0.35 muM. Enzyme activities after storage at -5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

Purification and properties of a NAD(P)H:flavin oxidoreductase from the luminous bacterium, Beneckea harveyi.

A NAD(P)H:flavin oxidoreductase, which produces FMNH2, one of the substrates for the luciferase reaction in bioluminescent bacteria, has been purified with the aid of affinity chromatography on epsilon-aminohexanoyl-FMN-Sepharose. The purified enzyme, isolated from Beneckea harveyi, had a specific activity of 89 mumol of NADH oxidized/min/mg of protein at 23 degrees in the presence of saturating FMN and NADH and appeared homogeneous by several criteria on polyacrylamide gel electrophoresis. A molecular weight of 24,000 was estimated both by gel filtration and and sodium dodecyl sulfate gel electrophoresis indicating that the enzyme is composed of a single polypeptide chain. Kinetic studies showed that the higher specificity of the enzyme for NADH than NADPH and for riboflavin and FMN than FAD was primarily due to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.     

Reduction of 2-methoxy-1,4-naphtoquinone by mitochondrially-localized Nqo1 yielding NAD+ supports substrate-level phosphorylation during respiratory inhibition

Provision of NAD⁺ for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD⁺, provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1^?/? and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD⁺ to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.     

Occurrence of an NADH diaphorase activity associated with xanthine dehydrogenase in Chlamydomonas reinhardtii

Wild-type Chlamydomonas reinhardtii cells exhibited a peculiar NADH-nitrobluetetrazolium reductase (NADH diaphorase) activity when grown under conditions in which xanthine dehydrogenase (XDH) is present. This XDH-coinduced diaphorase was electrophoretically distinguishable from constitutive diaphorases, showed the same mobility as XDH and could be assayed in vitro with dichlorophenol indophenol. Mutant strains 102, 104 and 307 of Chlamydomonas which lack XDH did not exhibit XDH-coinduced diaphorase. Heat treatment of crude extracts or partial purification of XDH inactivated or removed all constitutive diaphorases and left significant levels of XDH-coinduced diaphorase which remained always associated with XDH. These results demonstrate that XDH from C. reinhardtii, like other organisms, is also capable of catalyzing NADH oxidation.     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

NADH and NADPH-viologen reductases from Acinetobacter calcoaceticus.

Three pyridine nucleotide-dependent diaphorases have been isolated from Acinetobacter calcoaceticus cells and partially characterized. Two of them, with molecular weights of 165,000 and 57,000, utilize NADPH as electron donor whereas the third one (MW = 57,000) is specific for NADH. Oxidized viologen dyes, flavin nucleotides, dichlorophenol indophenol and ferricyanide can act with efficiency as acceptors in the reaction mediated by these diaphorases. The diaphorase activities have been characterized kinetically, and the effect of different inhibitors and cofactors has been also studied. The diaphorases seem to be subjected to metabolic control by oxidation and reduction.     

The purification and characterization of acetoacetyl-coenzyme A reductase from Azotobacter beijerinckii

A soluble acetoacetyl-CoA reductase (EC 1.1.1.36) was purified 54-fold from Azotobacter beijerinckii N.C.I.B. 9067 and the reaction product identified as d(-)-beta-hydroxybutyryl-CoA. The Michaelis constants for acetoacetyl-CoA, NADPH and NADH were determined and the reaction rate was found to be some fivefold greater with NADPH than with NADH. At neutral pH the equilibrium greatly favours the formation of the reduced product. Substrate specificity was in the order: acetoacetyl-CoA>acetoacetylpantetheine>acetoacetyl-(acyl-carrier protein). The enzyme possesses a functional thiol group, suffers inactivation by oxygen and is inhibited by thiol-blocking reagents. Inhibition by p-chloromercuribenzoate is reversed by excess of dithiothreitol, which also protects the enzyme from inactivation by oxygen.     

The effect of luciferase and NADH:FMN oxidoreductase concentrations on the light kinetics of bacterial bioluminescence

The effects of NADH:FMN oxidoreductase and luciferase concentrations on the light kinetics of the bacterial bioluminescent reaction were investigated. Light emission with low decay rates was obtained by regulating the conversion of NADH to NAD+ by controlling oxidoreductase activity. Constant light emission can be obtained when the oxidoreductase activity is below 2.5 U/1 in the assay system. The luciferase concentration affects the light intensity but it has no effect on the decay rate of light emission. The substrate decanal and the end-products NAD+ and capric acid had no effect on the light kinetics. The Michaelis constants of bacterial luciferase for FMNH2 and decanal were 3 X 10(-6) M and 8 X 10(-7) M, respectively, and those of oxidoreductase for FMN and NADH were 6.1 X 10(-6) M and 1.6 X 10(-5) M, respectively.     

Alteration in Bacterial Morphology by Optochin and Quinine Hydrochlorides

Incubation of washed bacterial and ribosomal suspensions with optochin or quinine hydrochloride caused an increase in the turbidity of the suspensions and the appearance of electron-dense cytoplasmic aggregates in the treated cells. These effects were more pronounced with optochin hydrochloride than with quinine hydrochloride, and they did not correlate with the relative sensitivities of different bacteria to growth inhibition by optochin or quinine.     

Aldehyde oxidase isoenzymes (E.C. 1.2.3.1) in potato tubers (Solanum tuberosum)

Two isoenzymes of aldehyde oxidase (E.C. 1.2.3.1 [EC] ) can be separatedfrom potato tubers (Solanum tuberosum) by polyacrylamide gelelectrophoresis. The pH optima of these two isoenzymes werepH 7.5. Both enzymes can oxidize different aldehydes, e.g. crotonaldehyde,Propionaldehyde, acetaldehyde, formaldehyde, glyoxal and benzyl-aldehyde.The isoenzymes could not use xanthine as a substrate. Formaldehydewas oxidized only in the presence of phosphate ions. A substratedependent inhibition of the enzyme activity is possible throughchloral hydrate. PMS, FMN, riboflavine, cytochrome c and O₂ serve as electronacceptors. (Received December 12, 1973; )     

Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils

Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.     

Reduced Pyridine Nucleotide Oxidases of Euglena gracilis var. bacillaris*

SYNOPSIS. Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (K_m) determined were as follows: NADH diaphorase, 350 μM; NADPH diaphorase, 200 μM; NADH cytochrome c reductase, 13 μM; NADPH cytochrome c reductase, 9 μM; NADH oxidase, 100 μM; NADPH oxidase 150 μM; NADH lipoyl dehydrogenase, 0.35 μM. Enzyme activities after storage at –5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

Cloning and expression of a Clostridium kluyveri gene responsible for diaphorase activity

A small enzyme showing diaphorase activity was purified from culture supernatant of Clostridium kluyveri and its N-terminal amino acid sequence was determined. This sequence identified a gene (diaA) encoding a protein (DiaA) of 229 amino acids with a predicted molecular weight of 24,981 in the genomic DNA sequence database of C. kluyveri constructed by the Research Institute of Innovative Technology for the Earth. The predicted protein was composed of a flavin reductase-like domain and a rubredoxin-like domain from its N-terminus. The diaA gene was cloned into an expression vector and expressed in an Escherichia coli recombinant. Recombinant enzyme rDiaA showed NADH/NADPH diaphorase activity with 2,6-dichlorophenolindophenol and nitro blue tetrazolium. The enzyme was most active at pH 8.0 at 40 degrees C. The UV-visible absorption spectrum and thin layer chromatography (TLC) analyses indicated that one rDiaA molecule contained a tightly bound FMN molecule as a prosthetic group. An iron molecule was also detected in an enzyme molecule.     

Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm

Two isozymes of creatine kinase have been purified from sperm of the sea urchin, Strongylocentrotus purpuratus. One isozyme was purified from the sperm flagellum, and the other from the head. Both require nonionic detergent for extraction from sperm. The flagellar isozyme is a monomeric species with an Mr of 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126,000 from sucrose density gradient and gel filtration analyses. Creatine kinase from sperm heads was localized to the mitochondrion by an antibody raised against mouse muscle creatine kinase. This purified mitochondrial isozyme is multimeric, with an Mr of 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but 240,000 for the native enzyme. Peptide mapping indicates that the two isozymes are not related. The following kinetic characteristics were observed for the purified flagellar and mitochondrial isozymes, respectively. In the direction of ATP formation, at pH 6.6 and 25 degrees C, specific activities were 235 and 180 units/mg; pH optima were 6.7 and 6.9 and Michaelis constants were 0.13 and 0.055 mM for ADP and 5.8 and 2.7 mM for phosphocreatine. In the direction of phosphocreatine formation, at pH 7.5 and 25 degrees C, specific activities were 29 and 47 units/mg; pH optima were 7.5 and 7.7 and Michaelis constants were 0.89 and 0.31 mM for ATP and 39 and 62 mM for creatine. These unique isozymes constitute the termini of the phosphocreatine shuttle of sea urchin sperm that is responsible for energy transport from the mitochondrion to the distal flagellum (Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334; Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys. J., 52, 75-86).     

Characterization of glutathione reductase from porcine erythrocytes.

Glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) was purified to homogeneity from porcine erythrocytes by use of affinity chromatography on 2',5'-ADP-Sepharose 4-B. Analytical ultracentrifugation experiments were analysed to give the following physical parameters for the enzyme: s20,w = 5.7 S, D20,w = 50 microgram2/s, and Mw = 103 000 (protein concentration, 0.5 mg/ml). The frictional ratio was 1.37 and the Stokes radius was 4.3 nm. The enzyme molecule is a dimer composed of subunits of equal size each containing a FAD molecule. The amino acid compositions and circular dichroism spectra of the porcine and human enzymes indicated extensive structural similarities. The isoelectric point was at pH 6.85 (at 4 degrees C). The absorption spectrum of the oxidized enzyme had maxima at 377 and 462 nm. In vivo the enzyme appears to be partially reduced. At a physiological concentration of reduced glutathione the apparent Michaelis constants for glutathione disulfide and NADPH were higher than in the absence of reduced glutathione. At 0.15 M ionic strength the catalytic activity obtained with NADPH as reductant was optimal at pH 7 and more than 200 times higher than that obtained with NADH. S-sulfoglutathione and some mixed disulfides of glutathione were poor substrates with the exception of the mixed disulfide of coenzyme A and reduced glutathione. The purified enzyme displayed low transhydrogenase activity with oxidized pyridine nucleotide analogs and diaphorase activity with 2,6-dichlorophenolindophenol as acceptor substrates; both NADPH and NADH served as donors.