Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

Isolation and properties of glycerol-3-phosphate oxidoreductase from human placenta

Glycerol-3-phosphate oxidoreductase (sn-glycerol 3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from human placenta has been purified by chromatography on 2,4,6-trinitrobenzenehexamethylenediamine-Sepharose, DEAE-Sephadex A-50 and 5'-AMP-Sepharose 4B approximately 15800-fold with an overall yield of about 19%. The final purified material displayed a specific activity of about 88 mumol NADH min-1 mg protein-1 and a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The native molecular mass, determined by Ultrogel AcA 44 filtration, was 62000 +/- 2000 whereas the subunit molecular mass, established on polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate, was 38000 +/- 500. The isoelectric point of the enzyme protein, determined by column isoelectric focusing, was found to be 5.29 +/- 0.09. The pH optimum of the placental enzyme was in the range 7.4-8.1 for dihydroxyacetone phosphate reduction and 8.7-9.2 for sn-glycerol 3-phosphate oxidation. The apparent Michaelis constants (Km) for dihydroxyacetone phosphate, NADH, sn-glycerol 3-phosphate and NAD+ were 26 microM, 5 microM, 143 microM and 36 microM respectively. The activity ratio of cytoplasmic glycerol-3-phosphate oxidoreductase to mitochondrial glycerol-3-phosphate dehydrogenase in human placental tissue was 1:2. The consumption of oxygen by human placental mitochondria incubated with the purified glycerol-3-phosphate oxidoreductase, NADH and dihydroxyacetone phosphate was similar to that observed in the presence of sn-glycerol 3-phosphate. The possible physiological role of glycerol-3-phosphate oxidoreductase in placental metabolism is discussed.     

Protein-protein interactions and antigenic relationships between the components of 4-methoxybenzoate monooxygenase and of benzene 1,2-dioxygenase from Pseudomonas putida

The investigations presented in this paper were performed on two enzyme systems from Pseudomonas putida: (a) 4-methoxybenzoate monooxygenase, consisting of a NADH: putidamonooxin oxidoreductase and putidamonooxin, the oxygen-activating component, and (b) benzene 1,2-dioxygenase, a three-component enzyme system with an NADH: ferredoxin oxidoreductase, functioning together with a plant-type ferredoxin as electron-transport chain, and an oxygen-activating component similar to putidamonooxin in its active sites. The influence of temperature, ionic strength, and pH on the activities of 4-methoxybenzoate monooxygenase and of NADH: putidamonooxin oxidoreductase were investigated. The studies revealed that the activity of 4-methoxybenzoate monooxygenase is determined by the behaviour of the reductase. Spectroscopic measurements showed that the interaction between the two components of 4-methoxybenzoate monooxygenase influences the optical-absorption behaviour of one or both components. As a criterion for the affinity between the two components of 4-methoxybenzoate monooxygenase, the Km value of the reductase for putidamonooxin was determined and found to be 31 +/- 11 microM. Antibodies against both components of 4-methoxybenzoate monooxygenase were obtained from rabbits. The antibodies against putidamonooxin inhibited the O-demethylation reaction (up to 80%) and also the reduction of putidamonooxin by the reductase (up to 40%). The antibodies against putidamonooxin did not interact with the oxygen-activating component of benzene 1,2-dioxygenase. The electron-transport chains of 4-methoxybenzoate monooxygenase and benzene 1,2-dioxygenase could not be replaced by one another without a complete loss of enzyme activity.     

Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b.

A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase.     

Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 +/- 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.     

Purification and properties of an NADP-specific 6-phosphogluconate dehydrogenase from Streptococcus faecalis.

A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.     

Soybean nodule xanthine dehydrogenase: a kinetic study

Xanthine dehydrogenase was purified from soybean nodules and the kinetic properties were studied at pH 7.5. Km values of 5.0 +/- 0.6 and 12.5 +/- 2.5 microM were obtained for xanthine and NAD+, respectively. The pattern of substrate dependence suggested a Ping-Pong mechanism. Reaction with hypoxanthine gave Km's of 52 +/- 3 and 20 +/- 2.5 microM for hypoxanthine and NAD+, respectively. The Vmax for this reaction was twice that for the xanthine-dependent reaction. The pH dependence of Vmax gave a pKa of 7.6 +/- 0.1 for either xanthine or hypoxanthine oxidation. In addition the Km for xanthine had a pKa of 7.5 consistent with the protonated form of xanthine being the true substrate. Km for hypoxanthine varied only 2.5-fold between pH 6 and 10.7. Product inhibition studies were carried out with urate and NADH. Both products gave mixed inhibition with respect to both substrates. Xanthine dehydrogenase was able to use APAD+ as an electron acceptor for xanthine oxidation, with a Km at pH 7.5 of 21.2 +/- 2.5 microM and Vmax the same as that obtained with NAD+. Reduction of APAD+ by NADH was also catalyzed by xanthine dehydrogenase with a Km of 102 +/- 15 microM; Vmax was approximately 2.5 times that for the xanthine-dependent reaction, and was independent of pH between 6 and 9. Reaction with group-specific reagents indicated the possibility of an essential histidyl group. A thiol-modifying reagent did not cause inactivation of the enzyme. A role for the histidyl side chain in catalysis is proposed.     

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 inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp)

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.     

Ecdysone 3-epimerase from the midgut of Manduca sexta (L.).

Ecdysone 3-epimerase was partially purified by ammonium sulfate fractionation from the 100,000 g supernate of Manduca sexta midguts. The enzyme converts ecdysone and 20-hydroxyecdysone to their respective 3-epimers, requires NADH or NADPH and O2 for this reaction, and has the following kinetic parameters: for ecdysone, Km = 17.0 +/- 1.4 microM, Vmax = 110.6 +/- 14.6 pmol min-1 mg-1; for 20-hydroxyecdysone, Km = 47.3 +/- 7.5 microM, Vmax = 131.0 +/- 3.5 pmol min-1 mg-1: for NADPH, Km = 85.4 +/- 10.6 microM; for NADH, Km = 51.3 +/- 1.3 microM. The reaction is irreversible and can be inhibited by various ecdysteroids.     

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.     

D-1-amino-2-propanol:NAD+ oxidoreductase. Purification and general properties of the large molecular form of the enzyme from Escherichia coli K12

Growth of Escherichia coli K12 under relatively anaerobic conditions in a medium containing casein hydrolysate, 0.8% glycerol, and 0.8% hydroxyacetone has been found to induce the level of D-1-amino-2-propanol oxidoreductase activity 50- to 100-fold over that in cells grown in casein hydrolysate alone or with 0.8% glycerol added. A large molecular weight form of this oxidoreductase (designated Form L) has been purified to apparent homogeneity in good yield by three simple steps designed to obviate its conversion to a smaller species. The molecular weight of native Form L and its basic subunit are 417,000 +/- 20,700 and 50,500 +/- 2,770, respectively; hence Form L would appear to consist of eight identical subunits. The pH activity profile for Form L shows one optimum in the range of 8.3 to 8.6 and another at pH 10.0 to 10.2. This form of the oxidoreductase has no apparent requirement for added metal ions (rather, numerous divalent transition metal ions are strongly inhibitory) or thiol compounds; it catalyzes the oxidation of several vic-glycols but is completely stereospecific for the D-isomer of 1-amino-2-propanol, utilizes only NAD+ as cosubstrate in the oxidation reaction (Km for NAD+ with DL-1-amino-2-propanol = 1.23 mM), but both NADH and NADPH serve as cosubstrate in the reduction of hydroxyacetone. Oxidoreductase activity of Form L is highly sensitive to inhibition by Hg2+, p-mercuribenzoate, or dithiodipyridine; inhibition by the latter two compounds is completely reversed by adding a thiol in excess.     

Xanthine:NAD+ oxidoreductase from embryo liver of hen Gallus gallus

1. Xanthine:NAD+ oxidoreductase from chick embryo liver is unconvertible to the O2-dependent form, as is the enzyme from the adult hen. The Km for NAD+ (approximately 3 microM) of the embryonic enzyme is equal to, and the Km for xanthine (approximately 5 microM) is 2.5-fold lower, when compared with respective Km values of the "adult" hen enzyme. The inhibition of embryonic enzyme by NADH begins at 10 microM NADH and attains 13% at 35 microM NADH (respective data for the "adult" enzyme: 50 microM and 20% at 80 microM NADH). 2. The course of hypoxanthine----xanthine----uric acid hydroxylation catalyzed by the embryonic and "adult" enzymes is similar, however the rate of the first reaction is 2-fold lower for the embryonic enzyme. Under conditions of the limited nutritional system in the developing chick embryo, the low rate of hypoxanthine hydroxylation may promote reutilization of hypoxanthine for nucleotide synthesis.     

Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae

A mitochondrial NADH:Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370 mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km = 31 microM and was found to be inhibited by flavone (I50 = 95 microM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s-1). With the natural ubiquinone-6 this value was 500 s-1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0. The amount of enzyme in the cell is subject to glucose repression; it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.     

Partial purification, substrate specificity and regulation of alpha-L-glycerolphosphate dehydrogenase from Saccharomyces carlsbergensis.

alpha-L-Glycerolphosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) from Saccharomyces carlsbergensis was purified 400-fold. The enzyme preparation is free of interfering activities, such as glyceraldehyde phosphate dehydrogenase, alcohol dehydrogenase, triose phosphate isomerase and glycerolphosphatase. At pH 7.0 it is specific for NADH (Km = 0.027 mM with 0.8 mM dihydroxyacetone phosphate) and dihydroxyacetone phosphate (Km = 0.2 mM with 0.2 mM NADH). Between pH 5.0 and 6.0 the enzyme functions with NADPH, but only at 7% of the rate with NADH. Various anions (I- greater than SO42- greater than Br- greater than Cl-) act as inhibitors competing with the substrate dihydroxyacetone phosphate. Inorganic phosphate (Ki = 0.1 mM), pyrophosphate and arsenate are strong inhibitors. The nucleotides ATP and ADP are also inhibitory, but their action seems to be of the same type as the general anion competition (Ki = 0.73 mM for ATP). The results are consistent with the notion that the enzyme may regulate the redox potential of the NAD+/NADH couple during fermentation.     

Characterization of Peptostreptococcus asaccharolyticus glutamate dehydrogenase purified by dye-ligand chromatography

Glutamate dehydrogenase (L-glutamate:NAD+ oxidoreductase (deaminating); EC 1.4.1.2) has been purified from Peptostreptococcus asaccharolyticus in a single step using dye-ligand chromatography. The enzyme (GDH) was present in high yields and was stabilized in crude extracts. A subunit molecular weight of 49000 +/- 500 was determined by SDS polyacrylamide gel electrophoresis and six bands were obtained after cross-linking the subunits with dimethyl suberimidate. This bacterial GDH was predominantly NAD+-linked, but was able to utilize both NADP+ and NADPH at 4% of the rates with NAD+ and NADH, respectively. An investigation of the amino acid specificity revealed some similarities with GDH from mammalian sources and some clear differences. The values of apparent Km for the substrates ammonia, 2-oxoglutarate, NADH, NAD+ and glutamate were 18.4, 0.82, 0.066, 0.031 and 6 mM, respectively. The P. asaccharolyticus GDH was not regulated by purine nucleotides, but was subject to strong inhibition with increasing ionic strength.     

Purification and properties of the glutathione reductase of Chromatium vinosum.

The Chromatium vinosum glutathione reductase [NAD(P)H: glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000. Incubation of enzyme with NADH in the absence of GSSG resulted in a significant loss in activity. The enzyme was stimulated by phosphate and sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 X 10-3 for GSSG and 6 X 10-5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 X 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.     

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.     

Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.     

Purification of mitochondrial NADH dehydrogenase from Drosophila hydei and comparison with the 'heat-shock' polypeptides.

Mitochondrial NADH dehydrogenase (NADH:(acceptor) oxidoreductase, EC .6.99.3) from either Drosophila hydei larvae or embryos has been purified 150- and 120-fold, respectively. The purified enzyme appeared homogeneous and showed a molecular weight of 57 000. The molecular weight of the nondenatured enzyme was 79 000. On isoelectro-focussing of the preparation, two fractions were observed, a major one with an isoelectric point of 6.2 and a minor fraction with an isoelectric point of 4.9. Straight-line kinetics in Lineweaver-Burk plots were observed for the purified enzyme with a Km of 0.040 mM. The Km was not changed during the purification procedure, suggesting that the enzyme was not denatured or inactivated. The pH optimum of the purified enzyme was 5.6. The molecular weight of the purified mitochondrial NADH dehydrogenase does not correspond to that of one of the 'heat-shock' polypeptides.