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PubMed1994

Purification and properties of a novel cytochrome: flavocytochrome c from Shewanella putrefaciens.

The major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.

Amino Acids↗
PubMed1995

Cofactor role for 10-formyldihydrofolic acid.

10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.

Acyltransferases↗
PubMed1998

Characterization of a cellobiose dehydrogenase from Humicola insolens.

The major cellobiose dehydrogenase (oxidase) (CBDH) secreted by the soft-rot thermophilic fungus Humicola insolens during growth on cellulose has been isolated and purified. It was shown to be a haemoflavoprotein with a molecular weight of 92 kDa and a pI of 4.0, capable of oxidizing the anomeric carbon of cellobiose, soluble cellooligosaccharides, lactose, xylobiose and maltose. Possible electron acceptors are 2,6-dichlorophenol-indophenol (DCPIP), Methylene Blue, 3,5-di-t-butyl-1,2-benzoquinone, potassium ferricyanide, cytochrome c and molecular oxygen. The oxidation of the prosthetic groups by oxygen was monitored at 449 nm for the flavin group and at 562 nm for the haem group. The curves were very similar to those of the cellobiose dehydrogenase from Phanerochaete chrysosporium, suggesting a similar mechanism. The pH-optima for the oxidation varied remarkably depending on the electron acceptor. For the organic electron acceptors, the pH-optima ranged from pH 4 for Methylene Blue to pH 7 for DCPIP and the benzoquinone. In the case of the FeIII-containing electron acceptors, the enzyme displayed alkaline pH-optima, in contrast to the properties of cellobiose dehydrogenases from Phanerochaete chrysosporium and Myceliophthora (Sporotrichum) thermophila. The enzyme has optimal activity at 65 degrees C.

Amino Acids↗
PubMed2001

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed.

Amino Acid Sequence↗
PubMed2000

Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and their role in catalysis.

The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (alpha2beta2) of E1alpha and E1beta polypeptide chains. The domain structure of the E1alpha and E1beta chains, and the protein-protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1alpha in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1alpha, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (alpha2beta2). As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged. The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1. However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.

Amino Acid Sequence↗
PubMed1971

Genetics of somatic mammalian cells: lethal antigens as genetic markers for study of human linkage groups.

The antigen that causes killing of at least 98% of a human cell population treated with a 1% solution of a specific rabbit antiserum in the presence of complement is a sensitive genetic marker. The rapid loss of human chromosomes in human-Chinese hamster cell hybrids makes possible a convenient test of linkage relationships with this marker. Hybrid clones with and without the lethal antigen were isolated and analyzed. In 76 clones and subclones studied, 41 carried both the lethal antigen and the lactic dehydrogenase-A marker, 35 carried neither, and no clones contained only one of the two markers. In contrast to this clear demonstration of linkage, absence of linkage was found between the lethal antigen and the following markers: Lactic dehydrogenase B, NAD-dependent malic dehydrogenase, NADP-dependent malic dehydrogenase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, glutamate oxaloacetate transaminase, indophenol oxidase, glucose phosphate isomerase, proline, inositol, hypoxanthine B, and glycine A. This lethal antigen appears to be carried on a single human autosome.

Animals↗
PubMed1974

Human antigen and enzyme markers in man-Chinese hamster somatic cell hybrids: evidence for synteny between the HL-A, PGM3, ME1, and IPO-B loci.

In man-Chinese hamster somatic cell hybrids the segregation of the loci for 27 human enzyme markers and the species-specific surface antigens, including the HL-A histocompatibility antigens, was studied. The results show a synteny of the human loci for phosphoglucomutase 3, cytoplasmic malic enzyme, tetrameric indophenol oxidase, and HL-A. Furthermore, evidence is presented that the loci for the human species-specific antigens are distributed over several chromosomes.

Animals↗
PubMed1984

Photoreduction of ferredoxin with various electron donors: Support for the Z scheme of photosynthetic electron transport.

The currently accepted scheme for photosynthetic electron flow from water to ferredoxin (and subsequently to NADP), known as the Z scheme, envisions a linear electron flow that requires two photosystems joined by several electron carriers. The observation that the extent of photoreduction of ferredoxin depends on whether electrons are provided by water (a donor to photosystem II) or by artificial electron donors to photosystem I led Arnon et al. [Arnon, D. I., Tsujimoto, H. Y. & Tang, G. M.-S. (1980) Proc. Natl. Acad. Sei. USA 77, 2676-2680] to question the validity of the Z scheme for photosynthetic electron transport. Our results show that this difference is not due to any inherent difference in electron transport but to the fact that when electron donors are added to chloroplasts they oxidize (in their oxidized state) reduced ferredoxin. Different electron donors oxidize reduced ferredoxin to a different extent; dehydroascorbate is a more potent oxidant than dithiothreitol. Dichlorophenol-indophenol is also a potent oxidant of reduced ferredoxin. The rate of NADP reduction and the K(m) for NADP with various electron donors reflect the oxidative capacities of the electron donors and mediators. These results also explain the fact that NADP reduction with electron donors to photosystem I is less than that with water, despite the fact that electron flow from donor to artificial dyes can proceed at high rates.

Journal Article↗
PubMed1995

The structure of chicken liver xanthine dehydrogenase. cDNA cloning and the domain structure.

The amino acid sequence of chicken liver xanthine dehydrogenase (EC 1.1.1.204) was determined by cDNA cloning and partial amino acid sequencing of the purified enzyme. The enzyme consisted of 1358 amino acids with calculated molecular mass of 149,633 Da. In order to compare the structure of the chicken and rat enzymes, limited proteolysis was performed with the purified chicken liver xanthine dehydrogenase. When the enzyme was digested with subtilisin, it was not converted from the NAD-dependent dehydrogenase type to the O2-dependent oxidase type, in contrast with the mammalian enzyme. However, the enzyme was cleaved mainly into three fragments in a manner similar to that for the rat enzyme at pH 8.2 (20, 37, and 84 kDa) and retaining a full complement of redox centers. The cleavage sites were identified by determination of amino-terminal sequences of the produced fragments. It was concluded that the 20-kDa fragment was amino-terminal, the 84-kDa fragment carboxyl-terminal, and the 37-kDa fragment an intermediate portion in the enzyme protein. On the other hand, when the enzyme was digested with the same protease at pH 10.5, the sample contained only the 20- and 84-kDa portions and lacked the 37-kDa portion. The resultant sample possessed xanthine dichlorophenol indophenol reductase activity, indicating that the molybdenum center remained intact. The absorption spectrum showed the sample was very similar to deflavo-enzyme. From these results and sequence analyses, the domain structure of the enzyme is discussed.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of a novel oxidoreductase KDRF from a human bone marrow-derived stromal cell line KM-102.

A cDNA clone coding for a novel oxidoreductase was cloned from a human bone marrow-derived stromal cell line KM-102. We screened a cDNA library constructed from the mRNA of KM-102 cells stimulated with phorbol 12-myristate 13-acetate and calcium ionophore A23187 using a 32P-labeled 15-mer synthetic oligonucleotide (5'-TAAATAAATAAATAA-3') probe. This probe was designed as a complementary sequence to the three reiterated AUUUA sequences, which are contained in the 3'-untranslated regions of cytokine and some proto-oncogene mRNAs and correlate with rapid mRNA turnover. Then, we obtained one cDNA clone, and further sequence analysis revealed that it coded for a new protein exhibiting 30 to approximately 40% homology with glutathione reductase. By fusion protein analysis, this protein showed reducing activities on 2, 6-dichlorophenol-indophenol and 5,5'-dithio-bis(2-nitrobenzoic acid) but only a weak reducing activity on oxidized glutathione. Although it lacked a stretch of hydrophobic amino acids in its N terminus, it was secreted by monkey kidney-derived COS-1 cells when we introduced the expression plasmid into them and also secreted by a human lung carcinoma cell line A549. Northern blot analysis revealed that the mRNA turnover of this protein was regulated by inflammatory stimuli in KM-102 cells. These results show that this protein may have scavenging enzyme properties and has its mRNA expression regulated in a similar fashion to cytokine genes or proto-oncogenes. Thus, we named it KDRF (KM-102-derived reductase-like factor), and KDRF may play a role in scavenging reactive oxygen intermediates, which are possibly toxic to cells, in response to inflammatory stimuli.

Amino Acid Sequence↗
PubMed1997

Eales' disease: accumulation of reactive oxygen intermediates and lipid peroxides and decrease of antioxidants causing inflammation, neovascularization and retinal damage.

PURPOSE: To measure the blood levels of oxygen and lipid-free radicals as lipid peroxidation products and of vitamins E, C and A, in order to explain intraocular inflammation, retinal neovascularization and detachment in Eales' disease. METHODS: Levels of the lipid peroxidation products produced by oxygen and lipid free radicals (estimated as thiobarbituric acid reactive substances-TBARS), vitamin E with ferric chloride, vitamin C with 2,6-dichlorophenol-indophenol and vitamin A with antimony trichloride were all evaluated in three groups of subjects between 20 and 40 years of age. Twenty three were patients with Eales' disease in the active perivasculitis stage (Group I), 19 were patients with Eales' disease in the healed-perivasculitis stage (Group II), and 46 were healthy volunteers (Group III), used as normal controls. Patients in each group were subdivided into two age groups, 20-30 and 31-40 years, each group containing both sexes. RESULTS: The results showed a 4.6- and 5.6-fold increase in the levels of TBARS in erythrocytes of patients with Eales' disease in the active perivasculitis stage (Group I) and a 2-fold increase in patients with Eales' disease in the healed perivasculitis stage (Group II), when compared to levels in the controls (Group III). On the other hand, there was a decrease of 75 and 76.2% in the levels of vitamin E in serum, 34 and 40.9% of vitamin C in plasma and 72.8 and 67% of vitamin A in serum in patients with Eales' disease in the active perivasculitis stage (Group I), as compared to the controls (Group III). Also decreases of 56 and 43.5% of vitamin E in serum, 26.8 and 12.5% of vitamin C in plasma and 50.5 and 49.4% of vitamin A in serum were found in patients with Eales' disease in the healed perivasculitis stage (Group II) as compared to the healthy controls (Group III). The two values of variations between the patients and the normal control group given above for TBARS, vitamins E,C and A are for the two age groups, 20-30 and 31-40 respectively. The differences were statistically significant (P < 0.01 to 0.001). CONCLUSIONS: Lowered levels of antioxidant vitamins E and C and consequent accumulation of oxygen and lipid free radicals, or vice versa, could explain the inflammation, neovascularization and retinal pathology in patients with Eales' disease. Also, vitamin A deficiency could aggravate retinal illness.

Adult↗
PubMed2003

Glycinebetaine protects the D1/D2/Cytb559 complex of photosystem II against photo-induced and heat-induced inactivation.

The presence of 1.0 mol/L glycinebetaine during isolation of D1/D2/Cytb559 reaction centre (RC) complexes from photosystem II (PSII) membrane fragments preserved the photochemical activity, monitored as the light-induced reduction of pheophytin and electron transport from diphenylcarbazide to 2.6-dichlorophenol-indophenol.-Glycinebetaine also protected the D1/D2/Cytb559 complexes against strong light-induced damage to the photochemical reactions and the irreversible bleaching of beta-carotene and chlorophyll. The presence of glycinebetaine also enhanced thermotolerance of the D1/D2/Cytb559 complexes isolated in the presence of 1.0 mol/L betaine with an increase in the temperature for 50% inactivation from 29 degrees C to 35 degrees C. The results indicate an increased supramolecular structural stability in the presence of glycinebetaine.

2,6-Dichloroindophenol↗
PubMed1993

Effect of reductant level in skeletal muscle and liver on the rate of loss of thiamin due to gamma-radiation.

A study was made of thiamin content of the skeletal muscles and livers of pork, chicken and beef after gamma-irradiation. gamma-Radiation from a 137Cs source was used to irradiate the samples with doses of 0, 1.5, 3, 6 and 10 kGy at 2 degrees C. Samples were also titrated with dichlorophenol-indophenol to determine the reducing capacity of the tissues. The rate of loss of thiamin upon irradiation was found to be about three time as as fast in skeletal muscle as it was in liver, and to be a function of the reducing capacity of the tissues, the loss decreasing with increasing reductant titer. For the same amount of thiamin loss, liver could be irradiated to three times the dose as could muscle.

Animals↗
PubMed1969

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 alpha-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.

Bacterial Proteins↗
PubMed2005

Ascorbic acid supplementation and regular consumption of fresh orange juice increase the ascorbic acid content of human milk: studies in European and African lactating women.

BACKGROUND: Little is known about the influence of an increased intake of ascorbic acid (AA) on human milk AA output. OBJECTIVE: We aimed to compare human milk AA content in European and African women and to evaluate the influence of increased AA intake on human milk AA output. DESIGN: Apparently healthy lactating women were recruited. AA was analyzed by titration with 2,6-dichlorophenol-indophenol. RESULTS: Mean human milk AA was approximately 50% lower (P < 0.001) in the African women (31 mg/kg; n = 171) than in the European women (63 mg/kg; n = 142). AA supplementation (1000 mg/d for 10 d) increased mean human milk AA from 19 to 60 mg/kg (P < 0.001) and from 60 to 70 mg/kg (P = 0.03) in 18 African and 10 European women, respectively. In 11 African women, mean human milk AA increased from 17 to 36 mg/kg (P < 0.001) after intake of 100 mg AA/d for 10 d. In African women, intake of 1 serving of orange juice per week had no significant effect, whereas 3 or 5 servings/wk ( approximately 100 mg AA/serving) for 6 wk increased mean human milk AA from 16 to 32 mg/kg (n = 13) and from 21 to 46 mg/kg (n = 13), respectively (P < 0.001). CONCLUSIONS: Human milk AA can be doubled or tripled by increased intake of AA in women with low human milk AA content at baseline. The response to a relatively high dose of AA was modest in European women in contrast with the 3-fold increase in mean human milk AA content in African women. These data indicate that human milk AA content is regulated.

Adolescent↗
PubMed1997

Antibacterial efficacy of tobramycin against anaerobic Escherichia coli cultures in the presence of electron acceptors.

The antimicrobial activity of tobramycin against anaerobic cultures of Escherichia coli was tested in the presence of various electron carriers. The presence of 2,6-dichlorophenol 4-indophenol (DCIP) significantly enhanced the killing efficacy of tobramycin. Only 0.003% of the initial cell population (i.e. 10(6) cfu/mL) remained viable after exposure for 10 h to the mixture of antibiotic (20 x MIC, i.e. 40 mg/L) and electron acceptor (10(-3) M), as compared with 9% of surviving organisms in the presence of tobramycin alone. Less synergy was obtained with p-benzoquinone and 1,2-naphthoquinone. Fumarate did not affect the efficiency of the antibiotic. The mixture of tobramycin and DCIP was ineffective against agar-entrapped bacteria which, like biofilm organisms, are subject to oxygen limitation.

2,6-Dichloroindophenol↗
PubMed1975

Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.

Animals↗
PubMed1978

Properties and kinetics of salt activation of a membrane-bound NADH dehydrogenase from a marine bacterium Photobacterium phosphoreum.

A membrane-bound NADH dehydrogenase, solubilized and partially purified from a marine bacterium Photobacterium phosphoreum, contains FAD as the prosthetic group, and is specific for NADH. Ferricyanide, various other redox dyes and cytochrome c can act as electron acceptors. The enzymatic activity when assayed with electron acceptors other than cytochrome c, is activated by monovalent cations (Na+ and K+) and deactivated by high concentrations of monovalent anions (SCN-, NO3-, and Cl-) but not by phosphate ions. The enzymatic reaction follows a ping-pong mechanism and kinetic analysis of the enzyme showed that the activation by monovalent cations is due to increase of affinity of the enzyme for substrates; Vm was not affected. The increase of affinity was 62- and 46-fold for NADH and 57- and 31-fold for 2,6-dichlorophenol indophenol in the presence of Na+ and K+, respectively. On the other hand, NADH-cytochrome c reductase activity of the enzyme was strongly inhibited by these cations.

Anions↗