ATP generation by the plasma membranes of human placental cells as affected by insulin and epidermal growth factor

Transient ATP synthesized by preparations enriched with plasmatic membranes of particles from the human placenta in the presence of insulin (4 micrograms/ml) and epidermal growth factor (1 microgram/ml) within 1 min after the addition of hormones at 30 degrees C, was isolated by means of chromatography on Dowex 1 X 8. ATP was synthesized in a medium containing Tris-HCl buffer, pH 7.5, ADP, Mg2+, and Pi during NADH-dependent oxidation in the presence of cytochrome C and oxygen. The amount of ATP was 10(-9) mole/mg protein/min. Quantitative assessment of ATP in lyophilized product was carried out by means of fluorimetry (excitation wavelength--360 nm; emission wavelength--460 nm) of NADH formed during coupled enzymatic reactions involving hexokinase and glucose-6-phosphate dehydrogenase. A possible biological role of peptide growth factor-stimulated formation of transient ATP in plasmatic membranes is discussed.     

Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP

NADH oxidase activity of rat liver plasma membranes was inhibited by lowconcentrations (1-100 nM) of ATP. The inhibition was amplified by additionof nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition wascomplex and related to a marked increase in the Km for NADH at high NADHconcentrations together with a concomitant decrease in the Vmax. In theabsence of added or residual ATP, cyclic AMP was without effect. Theresponse of cyclic AMP + ATP was inhibited by low concentrations of theselective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not bystaurosporin. The Vmax but not the Km was modified by treating the plasmamembranes with a mild oxidizing agent, N-chlorosuccinamide, or with thereducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmaxwas reduced by cyclic AMP + ATP. In contrast, in the presence ofN-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative tocyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmaxcould be either an increase or a decrease depending on whether the membraneswere oxidized or reduced. The results demonstrate regulation of NADH oxidaseactivity of rat liver plasma membranes through cyclic AMP-mediatedphosphorylation by membrane-located protein kinase activities where thefinal response is dependent on the oxidation-reduction status of the plasmamembranes.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants

Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 mM ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.     

Purification and characterization of NADH dehydrogenase from Bacillus subtilis

NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.     

Oxidation of reduced pyridine nucleotides by plasma membranes of soybean hypocotyl

Highly purified plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis or by a two-phase polymer separation system oxidize reduced pyridine nucleotides, NADH or NADPH, at rates of 2-5 nanomoles/mg protein/min. These rates are not influenced by mitochondrial inhibitors or by inhibitors of the alternate respiratory pathway. The NADH oxidase has a Km of 200 microM NADH. The enzyme activity is stimulated by Ca2+ and Mg2+ ions. The function of this enzyme is unknown at present, but it may represent a redox-controlled proton pump linked to acidification.     

Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.     

Mobilization of iron from endocytic vesicles. The effects of acidification and reduction

The factors necessary to dissociate iron from transferrin in endocytic vesicles and to mobilize the iron across the vesicle membrane were studied in a preparation of endocytic vesicles markedly enriched in transferrin-transferrin receptor complexes isolated from rabbit reticulocytes. Vesicles were prepared with essentially fully saturated transferrin by incubating the reticulocytes with the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prior to incubation with 59Fe, 125I-transferrin with or without fluorescein isothiocyanate labeling. Initiation of acidification by the addition of ATP was sufficient to achieve dissociation of 59Fe from transferrin with a rate constant of 0.054 +/- 0.06 s-1. Mobilization of 59Fe out of the vesicles required, besides ATP, the addition of a reductant with 1 mM ascorbate, allowing approximately 60% mobilization at 10 min with a rate constant of 0.0038 +/- 0.0006 s-1. An NADH:ferricyanide reductase activity could be demonstrated in the vesicles with an activity of 7.1 x 10(-9) mol of NADH reduced per min/mg of vesicle protein. Both dissociation and mobilization were inhibited by N-ethylmaleimide, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and monensin. Mobilization, but not dissociation, was inhibited by the permeant Fe(II) chelator alpha,alpha'-dipyridyl. The Fe(III) chelators deferoxamine, diethylenetriaminepentaacetic acid, and apotransferrin did not promote mobilization of dissociated iron in the absence of a reductant. This study establishes the basis for the cellular incorporation of iron through the endocytic pathway in which the endocytic vesicle membrane utilizes, in a sequential way, an acidification system, an iron reduction system, and an Fe(II) transporter system.     

Respiratory components and oxidase activities in Alcaligenes eutrophus.

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H2 oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H2 oxidase by 50% and solubilizes the bulk of the ATPase. 2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa3, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm). 3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2--3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H2 oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20-100% stimulation of oxygen uptake). 4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.     

Effect of phospholipids,Triton X-100 and biological membranes on redox systems involving tetrazolium salt reduction. Its implications for the assay of enzymatic activities

The influence of phospholipids and Triton X-100 on the time course of chemical and enzyme-mediated reductions of a commonly used tetrazolium salt, MTT, was studied. MTT reduction was followed by the absorbance changes at 570 nm. With ascorbate as reducing agent, a 3-fold increase in the initial rates of the absorbance changes and a 24 % increase in the final absorbance values were observed in the presence of Triton X-100 micelles or phospholipid vesicles. The enzyme-mediated reduction of MTT with NADH generated by the NAD-dependent lactate dehydrogenase was also enhanced in the presence of Triton X-100, phospholipids or erythrocyte membranes. No enhancement was observed following the enzymatic generation of NADH at 340 nm in the absence of MTT. The above findings were interpreted as arising from: a) solubilization or reduced MTT in the detergent micelles or phospholipid vesicles which favors the redox reaction occurring in the aqueous fase, and b) changes in the spectral properties of reduced MTT in aqueous and lipid-like media.     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

Pyridine nucleotide oxidation by a plasma membrane fraction from red beet (Beta vulgaris L.) storage tissue

The potential role of pyridine nucleotide oxidation in the energization and/or regulation of membrane transport was examined using sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue. In this system, pyridine nucleotide oxidation, which was enhanced in the presence of ferricyanide, occurred. In the presence or absence of ferricyanide, the oxidation of NADH was several-fold greater than the oxidation of NADPH, indicating that it was the preferred substrate for oxidation in this system. Ferricyanide reduction coupled to NADH oxidation did not require the transmembrane movement of reducing equivalents since ferricyanide incorporated inside the vesicles could not be reduced by NADH added externally to the vesicles, unless the vesicles were made leaky by the addition of 0.05% (v/v) Triton X-100. Using fluorescent probes for the measurement of transmembrane pH gradients and membrane potentials, it was determined that NADH oxidation did not result in the production of a proton electrochemical gradient or have any effect upon the proton electrochemical gradient produced by the plasma membrane H+-ATPase. The oxidation of NADH in the presence of ferricyanide did result in the acidification of the reaction medium. This acidification was unaffected by the addition of Gramicidin D and stimulated by the addition of 0.05% (v/v) Triton X-100, suggesting a scalar (nonvectorial) production of protons in the oxidation/reduction reaction. The results of this study suggest that the oxidation of pyridine nucleotides by plasma membrane vesicles is not related to energization of transport at the plasma membrane or modulation of the activity of the plasma membrane H+-ATPase.     

Redox reactions of tonoplast and plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis

Redox reactions were studied in more than 90% pure tonoplast and plasma membranes isolated by free-flow electrophoresis from soybean (Glycine max) hypocotyls. Both types of membrane contained a b-type cytochrome (_max = 561 nm) and a noncovalently bound flavin, two possible components of a transmembrane electron-transport chain. Isolated tonoplast and plasma membranes reduced ferricyanide, indophenol and various iron complexes with NADH or NADPH as electron donors. The redox activity was inhibited in tonoplast membranes by about 60% by 10 μM p-chloromercuribenzene sulfonate, 8% by 500 μM lanthanum nitrate and 10% by 100 μM nitrophenyl acetate. In contrast, the redox activity of isolated plasma membranes was inhibited by about 60% by 500 μM lanthanum nitrate or 100 μM nitrophenyl acetate, but only 25% by 10 μM p-chloromercuribenzene sulfonate. The results show that both tonoplast and plasma membranes of soybean contain active electron-transport systems, but that the two systems respond differently to inhibitors.     

ATP-dependent acidification of membrane vesicles isolated from purified rat liver lysosomes. Acidification activity requires phosphate

Membrane vesicles were isolated from purified liver lysosomes of rats treated with Triton WR-1339. In order to preserve ATP-dependent acidification activity, proteolysis of membranes was minimized by adding protease inhibitors and by centrifuging to form dilute bands of vesicles rather than highly concentrated pellets. The membrane vesicle fraction represented about 20% of the total lysosomal protein, 80% of the ATPase activity, and 3% of the solute proteins as marked by N-acetylglucosaminidase. About one-half of the membranes were oriented right side out. The space unavailable to [14C]sucrose corresponded to 3 microliters/mg of membrane protein which indicates that the membranes form vesicles about one-tenth the size of lysosomes. Uptake of either [14C]methylamine or [14C]chloroquine by lysosomal membrane vesicles was ATP-dependent, indicating acidification of the intravesicle space. The acidification activity was inhibited when either 1.5 microM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, 100 microM dicyclohexylcarbodiimide, or millimolar concentrations of such permeant weak bases as ammonium sulfate and dansyl cadaverine were added. Acidification of lysosomal vesicles by ATP occurred electroneutrally. This acidification activity was not dependent on added salts but was inhibited by the anion transport inhibitors pyridoxal phosphate and diisothiocyanostilbene disulfonic acid, thus suggesting co-transport of protons and anions. Results which indicate that phosphate is the transported anion included (a) ATP-dependent uptake of [32P]phosphate by lysosomal membrane vesicles and (b) stimulation of ATP-dependent acidification of these vesicles by added phosphate. These observations provide further evidence that maintenance of the acid intralysosomal pH necessary for activation of lysosomal hydrolases is due to an ATP-driven proton pump located in the lysosomal membrane.     

Fluorometric determination of adenine nucleotides and adenosine by ion-paired, reverse-phase, high-performance liquid chromatography

A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The 1,N6-ethenoderivatives of ATP (epsilon-ATP), ADP (epsilon-ADP), AMP (epsilon-AMP), and adenosine (epsilon-Ado), formed by reaction with chloroacetaldehyde at 37 degrees C, were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for epsilon-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. alpha, beta-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88, 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1.0, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys.     

The microbiol metabolism of C1 compounds. Oxidative phosphorylation in membrane preparations of Pseudomonas AM1

A method is described for preparation of membrane vesicles (diameter 80nm) capable of respiration-linked ATP synthesis. Vesicles prepared from succinate-grown bacteria oxidized NADH, succinate and ascorbate plus NNN'N'-tetramethylphenylenediamine; vesicles prepared from methanol-grown bacteria also oxidized methanol and formaldehyde, but they were otherwise identical. The uncoupling agent carbonyl cyanide chlorophenylhydrazone and the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide both inhibited ATP synthesis, whereas they had no effect on the rate of respiration. Rotenone inhibited ATP synthesis and respiration with NADH as substrate; antimycin A inhibited with succinate as substrate, and cyanide inhibited with all substrates. P/O ratios were usually 0.7-1.3 with NADH, 0.6-1.0 with succinate and 0.2-0.6 with reduced NNN'N'-tetramethylphenylenediamine or methanol as respiratory substrate. When 2,6-dichlorophenol-indophenol was used as an alternative electron acceptor to O(2) (NADH as donor) the P/2e ratio was 1.65. Although these P/O ratios are minimum values, because they do not take into account unknown amounts of uncoupled O(2) consumption, they are consistent with previous proposals [O'Keeffe & Anthony (1978) Biochem, J.170, 561-567] based on measurements of proton translocation in whole cells. The results also confirm that methanol dehydrogenase and cytochromes c and a/a(3) are arranged so that the first step in methanol oxidation is coupled to synthesis of ATP.     

NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain

Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity. In comparison, membranes from E. coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity. Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol. Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH. Inside-out membrane vesicles from E. coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate. Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity. The data provide a strong indication that the E. coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.     

Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to approximately 70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (approximately 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 microm, kcat = 0.03 s(-1), and Vmax = 54.5 nm min(-1). Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 microm, kcat = 0.06 s(-1), and Vmax = 105 nm min(-1). Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was > or =50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the beta-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.     

The stereospecificity,purification, and characterization of an NADH-ferricyanide reductase from spinach leaf plasma membrane

Summary The stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane, peroxisomal membrane, plasma membrane and tonoplast are all specific for the -hydrogen of NADH whereas the reductases in the ER, the Golgi and the outer mitochondrial membrane are -specific. This shows unequivocally that the NADH-ferricyanide activity in the plasma membrane is not caused by ER contamination. In all the membranes one or several polypeptides with an apparent size of 45–50 kDa cross-react with antibodies raised against a microsomal NADH-ferricyanide reductase. An NADH-ferricyanide reductase was purified from spinach leaf plasma membranes. The enzyme was released from the membrane by CHAPS solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size exclusion chromatography on FPLC. A major band of 45 kDa was detected by SDS-PAGE and it cross-reacted with the anti-NADH-ferricyanide reductase antibodies. The native size of the enzyme is 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Isoelectric focusing revealed three isoenzymes between pH 5.3 and 5.6. The enzyme shows typical FAD fluorescence spectra with excitation peaks at 371 and 468 nm and an emission peak at 525 nm. It is specific for the -hydrogen of NADH and prefers NADH over NADPH as electron donor. It is highly specific for ferricyanide as electron acceptor and it is therefore unlikely to be the enzyme responsible for iron reduction on the outer surface of the plasma membrane.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate - DQ duroquinone - FPLC fast protein liquid chromatography; Ferricyanide hexacyanoferrate(III) - NEM N-ethylmaleimide - PCMB p-chloromercurobenzoate - SHAM salicylhydroxamic acid - SMP submitochondrial particles     

Redox reactions of tonoplast and plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis

Redox reactions were studied in more than 90% pure tonoplast and plasma membranes isolated by free-flow electrophoresis from soybean (Glycine max) hypocotyls. Both types of membrane contained a b-type cytochrome (alpha max = 561 nm) and a noncovalently bound flavin, two possible components of a transmembrane electron-transport chain. Isolated tonoplast and plasma membranes reduced ferricyanide, indophenol and various iron complexes with NADH or NADPH as electron donors. The redox activity was inhibited in tonoplast membranes by about 60% by 10 microM p-chloromercuribenzene sulfonate, 8% by 500 microM lanthanum nitrate and 10% by 100 microM nitrophenyl acetate. In contrast, the redox activity of isolated plasma membranes was inhibited by about 60% by 500 microM lanthanum nitrate or 100 microM nitrophenyl acetate, but only 25% by 10 microM p-chloromercuribenzene sulfonate. The results show that both tonoplast and plasma membranes of soybean contain active electron-transport systems, but that the two systems respond differently to inhibitors.