Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase

Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.     

Mammalian dihydroorotate dehydrogenase: physical and catalytic properties of the primary enzyme.

A number of physical and catalytic properties of purified dihydroorotate dehydrogenase from rat liver mitochondria are described. The only potentially reducible cofactor detected was iron. The enzyme was also found to contain zinc. The primary enzyme does not contain FAD, FMN, covalently bound flavin, ubiquinone, or labile sulfide. Certain metal chelators were shown to behave as noncompetitive inhibitors of dihydroorotate oxidation and as competitive inhibitors of the reduction of phenazine methosulfate. The purified preparation can use oxygen as sole electron acceptor, although the reaction rate is relatively slow. The activity of the purified enzyme differs from that of the membrane bound form in a number of ways: the pH maximum is apparently shifted, the effect of thenoyltrifluoroacetone and its K_i are markedly changed and the mode of electron transfer to dichlorophenolindophenol is altered. Therefore, only tentative extrapolations to the membrane system regarding activities such as Superoxide production can be made.     

Ubiquinone is reduced by lipoamide dehydrogenase and this reaction is potently stimulated by zinc

Ubiquinol is an endogenously synthesized lipid-soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPH-dependent reduction more than 10-fold. The concentration ubiquinone resulting in the half-maximal rate of reduction was approximately 5 microM in the presence and 4 microM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.     

Sodium-dependent steps in the redox reactions of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluster. An E(m) of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration. The spin concentration of the radical in the enzyme was approximately the same under a variety of redox conditions. The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species. Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium. Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM. This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme. These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN.     

Purification and properties of L-3-glycerophosphate dehydrogenase from pig brain mitochondria.

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.     

Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae

Saccharomyces cerevisiae mitochondria contain an NADH:Q6 oxidoreductase (internal NADH dehydrogenase) encoded by NDI1 gene in chromosome XIII. This enzyme catalyzes the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. From a structural point of view, the mature enzyme has a single subunit of 53 kDa with FAD as the only prosthetic group. Due to the fact that S. cerevisiae cells lack complex I, the expression of this protein is essential for cell growth under respiratory conditions. The results reported in this work show that the internal NADH dehydrogenase follows a ping-pong mechanism, with a Km for NADH of 9.4 microM and a Km for oxidized 2,6-dichorophenolindophenol (DCPIP) of 6.2 microM. NAD+, one of the products of the reaction, did not inhibit the enzyme while the other product, reduced DCPIP, inhibited the enzyme with a Ki of 11.5 microM. Two dead-end inhibitors, AMP and flavone, were used to further characterize the kinetic mechanism of the enzyme. AMP was a linear competitive inhibitor of NADH (Ki = 5.5 mM) and a linear uncompetitive inhibitor of oxidized DCPIP (Ki = 11.5 mM), in agreement with the ping-pong mechanism. On the other hand, flavone was a partial inhibitor displaying a hyperbolic uncompetitive inhibition regarding NADH, and a hyperbolic noncompetitive inhibition with respect to oxidized DCPIP. The apparent intercept inhibition constant (Kii = 5.4 microM) and the slope inhibition constant (Kis = 7.1 microM) were obtained by non linear regression analysis. The results indicate that the ternary complex F-DCPIPox-flavone catalyzes the reduction of DCPIP, although with lower efficiency. The effect of pH on Vmax was studied. The Vmax profile shows two groups with pKa values of 5.3 and 7.2 involved in the catalytic process.     

Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli

The respiratory NADH dehydrogenase of Escherichia coli has been further amplified in vivo by genetic methods. The enzyme, a single polypeptide of Mr 47 200 of known amino acid sequence [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. 116, 165-170], constitutes 10-15% of the total protein in the amplified membranes. In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH:ubiquinone-1 oxidoreductase activity of approximately 1100-1200 units mg-1 at 30 degrees C, pH 7.5. The purified enzyme contains phospholipid, which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (w/v) cholate at low ionic strength. Under these conditions the enzyme is extensively aggregated (apparent Mr greater than 10(6]. This procedure yielded enzyme with a specific activity of 980 units mg-1, similar to the value observed in the membrane. This preparation contained less than 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme. Neutron activation analysis of purified enzyme has demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu, and Fe. The enzyme polypeptide, prepared completely free of phospholipid, FAD, and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, has been reactivated. The results show that the only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system are the enzyme polypeptide, FAD, and phospholipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress

The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.     

Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum

Methylenetetrahydrofolate reductase in Clostridium formicoaceticum has been purified to a specific activity of 140 mumol min-1 mg-1 when assayed at 37 degrees C, pH 7.2, in the direction of oxidation of 5-methyltetrahydrofolate with benzyl viologen as electron acceptor. The purified enzyme is judged to be homogeneous by polyacrylamide disc-gel electrophoresis and gel filtration. The enzyme which is an octamer has a molecular weight of about 237,000 and consists of four each of two different subunits having the molecular weights 26,000 and 35,000. The octameric enzyme contains per mol 15.2 +/- 0.3 iron, 2.3 +/- 0.2 zinc, 19.5 +/- 1.3 acid-labile sulfur, and 1.7 FAD. The UV-visible absorbance spectrum has a peak at 385 nm and a shoulder at 430 nm and is that of a flavoprotein containing iron-sulfur centers. The reductase, which is sensitive to oxygen, must be handled anaerobically and is stabilized by 2 mM dithionite. It catalyzes the reduction of methylene blue, menadione, benzyl viologen, rubredoxin, and FAD with 5-methyltetrahydrofolate and the oxidation of reduced ferredoxin and FADH2 with 5,10-methylenetetrahydrofolate. No activity was observed with pyridine nucleotides. It is suggested that the physiologically important reaction catalyzed by the enzyme is the reduced ferredoxin-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.     

Characterization of a dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri

The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD(+) as a specific electron acceptor, and the apparent K(m) values for DLA and NAD(+) were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K(m) values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 degrees C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 degrees C.     

Reactions of NADH Oxidation by Tetrazolium and Ubiquinone Catalyzed by Yeast Alcohol Dehydrogenase

The processes of NADH oxidation by p-NTF violet and ubiquinone catalyzed by isolated yeast alcohol dehydrogenase in aqueous and water-alcohol buffer solutions were studied. In the presence of p-NTF in aqueous solution at a pH of 6–7, NADH oxidation was extremely slow due to inhibition of the enzyme by the remaining enzyme-bound hydrophobic product, formazan, which forms during the reduction of p-Nitrotetrazolium. However, when the medium was alkalinized to a pH of 8–9 or when alcohol (ethanol or isopropanol) was added, formazan was desorbed from the enzyme, leading to an increase in the NADH oxidation rate. It was assumed that this redox reaction can be used as the basis for colorimetric measurement of the activity of different alcohol dehydrogenases. Tetrazolium reduction by alcohol was not observed at any value within the entire pH range. NADH oxidation in the presence of the enzyme and ubiquinone was also slow, even with the addition of alcohol, but its rate increased when the medium was acidified to a pH of 5.5–6. When a Tris-phosphate buffer was replaced with HEPES, a quasi-vibrational process was observed: NADH oxidization with ubiquinone to NAD⁺ and its subsequent reverse recovery with alcohol to NADH.     

Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase.

Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the alpha-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn(2+) (K(i) approximately 0.15 microm) in both directions. Steady-state kinetic studies revealed that Zn(2+) competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn(2+) with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn(2+) accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 microm. Activation is a consequence of Zn(2+) binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn(2+) on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn(2+) can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.     

Purification and properties of xanthine dehydrogenase from Streptomyces cyanogenus.

Xanthine dehydrogenase has been purified to a homogeneous state from cell-free extracts of a strain of Streptomyces. The enzyme has a molecular weight of 125,000 and consists of two subunits with a molecular weight of 67,000. The isoelectric point is at pH 4.4. The enzyme exhibits absorption maxima at 273, 355, and 457 nm and contains FAD, iron, and labile sulfide in a molar ratio of 1 : 7 : 1 per subunit. Little molybdenum could be detected. The enzyme is most active at pH 8.7 and at 40 degrees C, and is stable between pH 7 and 12 (at 4 degrees C for 24 h) and below 55 degrees C (at pH 9 for 10 min). The activity is stimulated by K+ at a concentration of 50 mM or more and also by keeping the enzyme at pH 9 to 11. The activity is inhibited by cyanide, Tiron, and p-chloromercuribenzoate and by adenine and urate. Among the compounds tested, hypoxanthine, guanine, xanthine 2-hydroxypurine, and 6,8-dihydroxypurine are oxidized at considerable rates; hypoxanthine is the best substrate. NAD+ is the preferred electron acceptor. Km values of the enzyme for hypoxanthine, guanine, xanthine, and NAD+ are 0.055, 0.015, 0.15, and 0.11 mM, respectively. Marked differences in the properties of this enzyme compared to others are the activity towards guanine, which has a higher affinity for the enzyme than hypoxanthine and xanthine, and a higher reactivity with hypoxanthine than xanthine. The organism has been identified as Streptomyces cyanogenus.     

One- and two-electron reduction of ubiquinone homologs by NADH- dehydrogenase preparations from the mitochondrial respiratory chain

The mechanism of ubiquinone homologs reduction by different preparations of mitochondrial NADH dehydrogenase: complex I within submitochondrial particles, isolated NADH-ubiquinone oxidoreductase and soluble low molecular weight NADH dehydrogenase, has been investigated. It has been shown that NADH oxidation via the rotenone-insensitive reaction is associated with one-electron reduction of low molecular weight ubiquinone homologs (Q0, Q1, Q2) to semiquinone with subsequent fast oxidation of the latter by atmospheric oxygen to form a superoxide radical. The two-electron ubiquinone reduction to quinol in the rotenone-sensitive reaction is unaccompanied by the semiquinone release from the enzyme active center into the surrounding solution.     

Purification and properties of L-alpha-glycerophosphate oxidase from Streptococcus faecium ATCC 12755.

A procedure was developed to purify the Streptococcus faecium ATCC 12755 L-alpha-glycerophosphate oxidase. The molecular weight of the purified enzyme was 131,000 and the subunit molecular weight was 72,000. Two moles of FAD were bound/mol of enzyme. Apo-L-alpha-glycerophosphate oxidase displayed physical properties similar to the holoenzyme as judged by electrophoresis in 10% buffer gels at pH 8.5 and by centrifugation in a 5 to 20% linear sucrose gradient. The apoenzyme was completely reactivated by incubation with FAD. L-alpha-Glycerophosphate oxidase was specific for L-alpha-glycerophosphate when compared with several other pohsphorylated glycerol and sugar derivatives. Oxygen was the preferred electron acceptor. At 10 mM DL-alpha-glycerophosphate (below the Km of 26 mM for L-alpha-glycerophosphate), activity was increased from 2.6- to 10-fold by increasing the buffer concentration from 0.01 to 0.1 m. This buffer effect was observed with potassium phosphate and other anionic buffers. In 0.001 m potassium phosphate buffer, pH 7.0, activity was increased by several divalent metal ions, including 10 mM CaCl2 (7.7-fold activation) and 10 mM MgCl, (6.8-fold activation). Fructose 6-phosphate and fructose1-phosphate were inhibitors of the L-alpha-glycerophosphate oxidase.     

Purification and properties of 4-hydroxycyclohexanecarboxylate dehydrogenase from Corynebacterium cyclohexanicum

4-Hydroxycyclohexanecarboxylate dehydrogenase, which requires NAD as a cofactor, was detected in crude soluble extracts of Corynebacterium cyclohexanicum grown on cyclohexanecarboxylic acid as the sole carbon source. The dehydrogenase was purified from extracts to an electrophoretically homogenous state by ammonium sulfate precipitation and chromatography on DEAE-650s, agarose-NAD and hydroxyapatite. The enzyme consisted of two identical subunits and had a native relative molecular mass of 53,600. There were two residues each of cysteine and tryptophan in the enzyme molecule. Oxo acid rather than hydroxy acid was routinely used as substrate for assay of the enzyme. The enzyme is highly specific for 4-oxocyclohexanecarboxylic acid: the carboxyl group is essential and the position of carbonyl group is important; neither the 2-oxo nor the 3-oxo homologue was used as substrate. A methyl substitution on the ring of 4-oxocyclohexanecarboxylate resulted in an almost complete loss of its activity. The reduction product was identified as trans-4-hydroxycyclohexanecarboxylic acid by gas-liquid chromatography and mass spectrometry. It was used as a substrate for the reverse reaction in the presence of NAD but not its cis-isomer. The enzyme was specific for the B-side (pro-S) hydrogen of NADH in the hydrogen transfer from NADH to 4-oxocyclohexanecarboxylate. The Km values for 4-oxocyclohexanecarboxylate and NADH in the reduction reaction at pH 6.8 were 0.50 mM and 0.28 mM, respectively, whereas those for trans-4-hydroxycyclohexanecarboxylate and NAD in the oxidation reaction at pH 8.8 were 0.51 mM and 0.23 mM, respectively. The equilibrium constant of the reaction was 1.79 x 10(-10) M. The enzyme was strongly inhibited by N-bromosuccinimide.     

The reduced nicotinamide adenine dinucleotide "oxidase" of Acholeplasma laidlawii membranes.

An NADH dehydrogenase possessing a specific activity 3-5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0% ethanol at 43 degrees C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1:2 FAD to FMN and 30-40% lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O2, ubiquinone Q10 or cytochrome c. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0% ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar Km's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller V's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg2+, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O2 uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.     

Characterization of the fructose 1,6-bisphosphate-activated, L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus.

The L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus wt was purified to a final specific activity of 598 mumol pyruvate reduced per min per mg of protein. The specific activity of the pure enzyme with L(+)-lactate was 0.79 units per mg of protein. The M(r) of the native enzyme was 134,000 containing a single subunit type of M(r) 33,500 indicating an apparent tetrameric structure. The L(+)-lactate dehydrogenase was activated by fructose 1,6-bisphosphate in a cooperative manner affecting Vmax and Km values. The activity of the enzyme was also effected by pH, pyruvate and NADH. The Km for NADH at pH 6.0 was 0.05 mM and the Vmax for pyruvate reduction at pH 6.0 was 1082 units per mg in the presence of 1 mM fructose 1,6-bisphosphate. The enzyme was inhibited by NADPH, displaying an uncompetitive pattern. This pattern indicated that NADPH was a negative modifier of the enzyme. The role of L(+)-lactate dehydrogenase in controlling the end products of fermentation is discussed.     

FAD and FMN flavoproteins participate in the sodium-transport respiratory chain NADH:quinone reductase of a marine bacterium,Vibrio alginolyticus

The sodium-transport respiratory chain NADH:quinone reductase of a marine bacterium, Vibrio alginolyti-cus, is composed of three protein subunits, α,β and γ. The β-subunit contains FAD as a prosthetic group and corresponds to NADH dehydrogenase, which catalyses the reduction of ubiquinone to ubisemiquinone. In addition to β, subunits α. and γ are essential for the quinone reductase, which catalyses the reduction of ubiquinone to ubiquinol. The α-subunit contains FMN and the reaction catalysed by subunit α is related to the coupling site of the sodium pump in the quinone reductase.     

Regulation, purification, and properties of xanthine dehydrogenase in Neurospora crassa.

Xanthine dehydrogenase (EC 1.2.1.37) is the first enzyme in the degradative pathway by which fungi convert purines to ammonia. In vivo, the activity is induced 6-fold by growth in uric acid. Hypoxanthine, xanthine, adenine, or guanine also induce enzyme activity but to a lesser degree. Immunoelectrophoresis using monospecific antibodies prepared against Neurospora crassa xanthine dehydrogenase shows that the induced increase in enzyme activity results from increased numbers of xanthine dehydrogenase molecules, presumably arising from de novo enzyme synthesis. Xanthine dehydrogenase has been purified to homogeneity by conventional methods followed by immunoabsorption to monospecific antibodies coupled to Sepharose 6B. Electrophoresis of purified xanthine dehydrogenase reveals a single protein band which also exhibits enzyme activity. The average specific activity of purified enzyme is 140 nmol of isoxanthopterine produced/min/mg. Xanthine dehydrogenase activity is substrate-inhibited by xanthine (0.14 mM), hypoxanthine (0.3 mM), and pterine (10 micron), is only slightly affected by metal binding agents such as KCN (6 mM), but is strongly inhibited by sulfhydryl reagents such as p-hydroxymercuribenzoate (2 micron). The molecular weight of xanthine dehydrogenase is 357,000 as calculated from a sedimentation coefficient of 11.8 S and a Stokes radius of 6.37 nm. Sodium dodecyl sulfate-gel electrophoresis of the enzyme reveals a single protein band having a molecular weight of 155,000. So the xanthine dehydrogenase protein appears to be a dimer. In contrast to xanthine dehydrogenases from animal sources which typically possess as prosthetic groups 2 FAD molecules, 2 molybdenum atoms, 8 atoms of iron, and 8 acid-labile sulfides, the Neurospora enzyme contains 2 FAD molecules, 1 molybdenum atom, 12 atoms of iron, and 14 eq of labile sulfide/molecule. The absorption spectrum of the enzyme shows maxima between 400 and 500 nm typical of a non-heme iron-containing flavoprotein.