Inhibition studies on the involvement of flavoprotein reductases in menadione- and nitrofurantoin-stimulated oxyradical production by hepatic microsomes of flounder (Platichthys flesus)

Inhibitors of mammalian cytochrome P450 and P450 reductase were used to investigate the enzymes in flounder (Platichthys flesus) hepatic microsomes involved in the stimulation of NAD(P)H-dependent iron/EDTA-mediated 2-keto-4-methiolbutyric acid (KMBA) oxidation (hydroxyl radical production) by the redox cycling compounds menadione and nitrofurantoin. Inhibitors were first tested for their effects on flounder microsomal P450 and flavoprotein reductase activities. Ellipticine gave type II difference binding spectra (app. K_s 5.36 μM; ΔA max 0.16 nmol-1 P450) and markedly inhibited NADPH-cytochrome c reductase, NADPH-cytochrome P450 reductase, and monooxygenase (benzo[a]pyrene metabolism) activities. 3-aminopyridine adenine dinucleotide phosphate (AADP; competitive inhibitor of P450 reductase) inhibited NADPH-cytochrome c but not NADH-cytochrome c or NADH-ferricyanide reductase activities. Alkaline phosphatase (inhibitor of rabbit P450 reductase) stimulated NADPH-cytochrome c reductase activity seven fold but had less effect on NADH-reductase activities. AADP inhibited nitrofurantoin- and menadione-stimulated KMBA oxidation by 45 and 17%, respectively, indicating the involvement of P450 reductase at least in the former. In contrast, ellipticine had relatively little effect, possibly because, unlike cytochrome c, the smaller xenobiotic molecules can access the hydrophilic binding site of P450 reductase. Alkaline phosphatase stimulated NAD(P)H-dependent basal and xenobiotic-stimulated KMBA oxidation, showing general consistency with the results for reductase activities. Overall, the studies indicate both similarities (ellipticine, AADP) and differences (alkaline phosphatase) between the flounder and rat hepatic microsomal enzyme systems.     

Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways

The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl₃. Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent K_m for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.     

Characterization of the NADH dehydrogenase and fumarate reductase of Fibrobacter succinogenes subsp. succinogenes S85

Conditions promoting maximal in vitro activity of the particulate NADH:fumarate reductase from Fibrobacter succinogenes were determined. This system showed a pH optimum of 6.0 in K⁺ MES buffer only when salt (NaCl or KCl) was present. Salt stimulated the activity eightfold at the optimal concentration of 150m M. This effect was due to stimulation of fumarate reductase activity as salt had little effect on NADH: decylubiquinone oxidoreductase (NADH dehydrogenase). The stimulation of fumarate reductase by salt at pH 6.0 was not due to removal of oxaloacetate from the enzyme. Kinetic parameters for several inhibitors were also measured. NADH dehydrogenase was inhibited by rotenone at a single site with a K _i of 1 M. 2-Heptyl-4-hydroxyquinonline-N-oxide (HOQNO) inhibited NADH: fumarate reductase with a K _i of 0.006 M, but NADH dehydrogenase exhibited two HOQNO inhibition constants of approximately 1 M and 24 M. Capsaicin and laurylgallate each inhibited NADH dehydrogenase by only 20% at 100 M. NADH dehydrogenase gave K _m values of 1 M for NADH and 4 M for reduced hypoxanthine adenine dinucleotide.Published with the approval of the Director of the Agricultural Experiment Station, North Dakota State University, as journal article no. 2201     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Capacity for NADPH/NADP turnover in the cytosol of barley seed endosperm: The role of NADPH-dependent hydroxypyruvate reductase

Barley (Hordeum vulgare L.) endosperm from developing seeds was found to contain relatively high activities of cytosolic NAD(P)H-dependent hydroxypyruvate reductase (HPR-2) and isocitrate dehydrogenase (ICDH). In contrast, activities of peroxisomal NADH-dependent hydroxypyruvate reductase (HPR-1) and glycolate oxidase as well as cytosolic NAD(P)H-dependent glyoxylate reductase were very low or absent in the endosperm both during maturation and seed germination, indicating the lack of a complete glycolate cycle in this tissue. In addition, activities of cytosolic glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were low or absent in the endosperm. The endosperm HPR-2 exhibited similar properties to those of an earlier described HPR-2 from green leaves, e.g. activities with both hydroxypyruvate and glyoxylate, utilization of both NADPH and NADH as cofactors, and a strong uncompetitive inhibition by oxalate (K_i in the order of micromolar). In etiolated leaves, both HPR-1 and HPR-2 were present with the same activity as in green leaves, indicating that the lack of HPR-1 in the endosperm is not a general feature of non-photosynthetic tissues. We conclude that the endosperm has considerable capacity for cytosolic NADP/NADPH cycling via HPR-2 and ICDH, the former being possibly involved in the utilization of a serine-derived carbon.     

The reduction of nitrous oxide to dinitrogen by Escherichia coli

Escherichia coli K12 reduces nitrous oxide stoichiometrically to molecular nitrogen with rates of 1.9 mol/h x mg protein. The activity is induced by anaerobiosis and nitrate. N₂+formation from N₂O is inhibited by C₂H₂ (K _i 0.03 mM in the medium) and nitrite (K _i=0.3 mM) but not by azide. A mutant defective in FNR synthesis is unable to reduce N₂O to N₂. The reaction in the wild type could routinely be followed by gas chromatography and alternatively by mass spectrometry measuring the formation of ¹⁵N₂ from ¹⁵N₂O. The enzyme catalyzing N₂O-reduction in E. coli could not be identified; it is probably neither nitrate reductase nor nitrogenase. E. coli does not grow with N₂O as sole respiratory electron acceptor. N₂O-reduction might not have a physiological role in E. coli, and the enzyme involved might catalyze something else in nature, as it has a low affinity for the substrate N₂O (apparent K _m3.0 mM). The capability for N₂O-reduction to N₂ is not restricted to E. coli but is also demonstrable in Yersinia kristensenii and Buttiauxella agrestis of the Enterobacteriaceae. E. coli is able to produce NO and N₂O from nitrite by nitrate reductase, depending on the assay conditions. In such experiments NO _inf2 ^sup- is not reduced to N₂ because of the high demand for N₂O of N₂O-reduction and the inhibitory effect of NO _inf2 ^sup- on this reaction.Dedicated to Professor L. Jaenicke, Köln, on the occassion of his 70th birthday     

Nitrate reductase from yeast: cultivation,partial purification and characterization

Summary Several yeast strains were assayed for occurence of nitrate reductase after growth in a defined medium with nitrate as the sole nitrogen source, Candida boidinii DSM 70026, showing the highest specific activity, was further investigated. The procedures for yeast fermentation and nitrate reductase purfication are described in detail. Nitrate reductase from this yeast was characterized as NAD(P)H: nitrate oxidoreductase (E.C.1.6.6.2). The enzyme activity with NADH (NADPH) was highest at pH 7.0 (7.1) and 30° C (25° C). The values of K _m determinations with NADH/NADPH were both 4 × 10^–4 mol/l; values for the substrate inhibition constant (K _i) were 6 × 10^–4 mol/l. The molecular mass of the native enzyme was estimated by gel permeation chromatography to be approximately 350 kDa. Offprint requests to: R. Gromes     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

Respiratory physiology and cytochrome content ofLegionella pneumophila

The cytochrome composition of membrane vesicles ofLegionella pneumophila has been examined by low temperature (77°K) and room temperature difference spectroscopy, and cytochromes of thec, b, a, andd types have been detected. The presence ofc-type cytochrome was verified by formation of the pyridine ferrohemochromogen. A carbon monoxide-bindingc-type cytochrome was detected in CO-reduced minus reduced difference spectra and may also function in cytochromec reductase activity. Respiratory activities were determined for membrane vesicles, and reduced nicotinamide adenine dinucleotide (NADH) was the most rapidly oxidized substrate (199 nmol per min per mg protein), followed by succinate and malate. Cytochrome oxidase activity was demonstrated using ascorbate andN,N,N,N-tetramethyl-p-phenylenediamine (TMPD) (39 nmol per min per mg of protein). High levels of cyanide (K _i =10 mM) inhibited NADH oxidation, while low levels of 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO, 18 and 37 M) inhibited NADH oxidation by nearly 90%. The respiratory chain appeared to be complex and terminated by at least three terminal oxidases. Superoxide dismutase activity, but not catalase activity, was detected in cellular extracts.     

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K_m = 7.9 mM; k_cat/K_m = 0.73 mM⁻¹ s⁻¹). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k_cat/K_m = 450 mM⁻¹ s⁻¹) than with NADPH (k_cat/K_m = 5.5 mM⁻¹ s⁻¹), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu²⁺ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.     

A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity

Summary A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed. For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor. The apparentK _m of the reaction is 0.83 mM. Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP⁺ (K _i=1.50mm) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity. An NADP⁺ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition). The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal.Continuous microdensitometric monitoring of the reaction product as it is formed has shown that the specific reaction is linear with incubation up to 10 min, thus allowing end-point measurements to be used for cytophotometric analysis.     

Identification and functional reconstitution of phosphate: Sugar phosphate antiport ofStaphylococcus aureus

Summary Resting cells ofStaphylococcus aureus displayed a phosphate (P_i) exchange that was induced by growth with glucose 6-phosphate (G6P) orsn-glycerol 3-phosphate (G3P). P_i-loaded membrane vesicles from these cells accumulated³²P_i, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of³²P_i (andl-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (dl-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both P_i anddl-lactate to establish an internal pool of P_i. Thistrans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal P_i.Reconstitution of membrane protein allowed a quantitative analysis of P_i-linked exchange. P_i-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous³²P_iP_i exchange (K _i's of 2.2 and 1.4mm;V _max's of 180 and 83 nmol P_i/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (K _i=27 m) over G3P (K _i=1.3mm) and P_i (K _i=2.2mm), suggesting that the same antiporter was induced in both cases. We conclude that³²P_iP_i exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate. In this respect, P_i-linked antiport inS. aureus resembles other examples in a newly described family of bacterial transporters that use anion exchange as the molecular basis of solute transport.     

Synthesis and enzymology of modifiedN-benzyloxycarbonyl-L-cysteinylglycyl-3,3-dimethylaminopropylamide disulphides as alternative substrates for trypanothione reductase fromTrypanosoma crud: Part 3

Summary Kinetic data for alternative substrates of recombinant trypanothione reductase fromTrypanosoma cruzi were measured for a series ofN-substituted-L-cysteinylglycyl-3-dimethylaminopropylamides, in which the cysteineN-substituent was either a variant of the benzyloxycarbonyl group or was L-phenylalanine or L-tryptophan. Replacing the benzylic ether oxygen atom by CH₂. or NH had relatively minor effects on k_cat, but raised the value of K_m, 4.5- and 10-fold, respectively. Similarly, relative to the carbobenzoxy group, anN-L-phenylalanyl orN-L-tryptophanyl replacement on the cysteine hardly altered k_cat, but increased K_m, values by 16.6 and 7.4 fold, respectively. These observations were consistent with the K_m, values referring primarily to binding for this series of nonspecific substrates.Abbreviations DCC N,N-dicyclohexylcarbodiimide - dmapa dimethylaminopropylamine - DMF dimethylformamide - GR glutathione reductase - GSSG glutathione disulphide - GSH reduced glutathione - T[S]₂ trypanothione disulphide - Hbt hydroxybenzotriazole - TFA trifluoroacetic acid - TLC thin layer chromatography - T[SH]₂ reduced trypanothione as dithiol - TR trypanothione reductase - Z.cys.gly.dmapa N-benzyloxycarbonyl-Lcysteinylglycyl-3-dimethylpropylamide     

Distribution and properties of NADPH-linked aldehyde reductases from rat brain synaptosomes

Studies on the subcellular distribution of NADPH-linked aldehyde reductase from rat brain showed that 10% of the total reductase activity is located in the mitochondrial-synaptosomal fraction. There are differences in the percentages of reductase activity found in the synaptosomes compared to cytosol in various regions of the brain. The NADPH-linked aldehyde reductase from the synaptosomal fraction exhibited a nonlinear Lineweaver-Burk plot. This nonlinearity is due to the presence of two distinct aldehyde reductases, which can be distinguished by Michealis constants forp-nitrobenzaldehyde of 4.1×10^–5 M and 2.6×10^–6 M. The two NADPH-linked aldehyde reductases isolated from synaptosomes were further characterized according to pH optima, andK _i values for inhibition by barbiturates. In addition regional distributions for the two enzymes were determined. TheK _i values for pentobarbital for the highK _m enzyme and the lowK _m enzyme were estimated to be 2×10^–5 M and 6×10^–5 M, respectively. It was concluded from the above studies that the lowK _m reductase is probably responsible for 3,4-dihydroxyphenylglycoaldehyde (derived from norepinephrine) reduction in brain and a role of the highK _m enzyme for protection of neurons from high concentrations of chemically reactive aldehydes was proposed.This work was supported in part by Grants from the National Institute of Mental Health, MH 18948 from the University of Colorado Council on Research and Creative Work and by an MBS Program Grant #081-39.This work was performed in partial fulfillment of the requirements for the Ph. D. thesis.     

Studies on the regulation of assimilatory nitrate reductase in Ankistrodesmus braunii

In the green alga Ankistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH₂-or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg²⁺, presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH.Abbreviations ADP Adenosine-5-diphosphate - AMP Adenosine-5-monophosphate - ATP Adenosine-5-triphosphate - FAD Flavin adenine dinucleotide - FMNH₂ Flavin adenine mononucleotide, reduced form - GDP Guanosine-5-diphosphate - MVH Methyl viologen, reduced form - NADH Nicotinamide adenine dinucleotide, reduced form - NADPH Nicotinamide adenine dinucleotide phosphate, reduced form     

Studies of the ferricyanide reductase activities of the mitochondrial reduced nicotinamide adenine dinucleotide-ubiquinone reductase (complex I) utilizing arylazido-β-alanyl NAD+ and arylazido-β-alanyl NADP+

The NADH and NADPH ferricyanide reductase activities present in mitochondrial NADH-CoQ reductase preparations have been studied utilizing two photoaffinity pyridine nucleotide analogues: arylazido--alanyl NAD⁺ (A3-O-{3-[N-(4-azido-2-nitrophenyl)amino]propionyl}NAD⁺) and arylazido--alanyl NADP⁺ (N3-O-{3-[N-(4-azido-3-nitrophenyl)amino]-propionyl}NADP⁺). For the NADH-K₃Fe(CN)₆ reductase activity, arylazido--alanyl NAD⁺ was found to be, in the dark, a competitive inhibitor with respect to both NADH and K₃Fe(CN)₆ withK _i,app values of 9.7 and 15.5 µM, respectively. In comparison the NADP⁺ analogue exhibited weak noncompetitive inhibitor activity for this reaction against both substrates. Upon photoirradiation arylazido--alanyl NAD⁺ inhibited NADH-K₃Fe(CN)₆ reductase up to 70% in the presence of a 25-fold molar excess of analogue over the enzyme concentration. This photodependent inhibition could be prevented by the presence, during irradiation, of the natural substrate NADH. In contrast complex kinetic results were obtained with studies of the effects of the pyridine nucleotide analogues of NADPH-K₃Fe(CN)₆ reductase activity in the dark. Photoirradiation of either analogue in the presence of the enzyme complex resulted in an activation of NADPH-dependent activity. The possibility that the NADPH-K₃Fe(CN)₆ reductase activity of complex I represents a summation of the combined ferricyanide reductase activity of the NADPH-NAD⁺ transhydrogenase and NADH oxidoreductase is suggested.     

Competition between β-ketothiolase and citrate synthase during poly(β-hydroxybutyrate) synthesis inMethylobacterium rhodesianum

The enzymes -ketothiolase and citrate synthase from the facultatively methylotrophicMethylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The -ketothiolase had a relatively highK _m for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (K _i 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (K _m 0.07 mM) and was significantly inhibited by NADH (K _i 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined inM. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)^-1 during growth to the detection limit when poly(-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6–0.7, and 1.0 nmol (mg dry mass)^-1, respectively, were determined during growth and PHB synthesis. During growth, the -ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the -ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (K _mNADPH 18 M). PHB synthesis is probably mainly controlled by the CoA concentration inM. rhodesianum MB 126.Abbreviation PHB Poly(-hydroxybutyrate)     

Different Characteristics of the Two Glutamate Synthases in the Green Leaves of Lycopersicon esculentum

The two glutamate synthases, NAD(P)H- and ferredoxin-dependent, from the green leaves of tomato plants (Lycopersicon esculentum L. cv Hellfrucht frühstamm) differed in their chemical properties and catalytic behavior. Gel filtration of NAD(P)H enzyme gave an apparent molecular size of 158 kilodalton, whereas the ferredoxin enzyme molecular size was 141 kilodalton. Arrhenius plots of the activities of the two enzymes showed that the NAD(P)H enzyme had two activation energies; 109.6 and 70.5 kilojoule per mole; the transition temperature was 22°C. The ferredoxin enzyme however, had only one activation energy; 56.1 kilojoule per mole. The respective catalytic activity pH optima for the NAD(P)H- dependent and the ferredoxin dependent enzymes were around 7.3 and 7.8. In experiments to evaluate the effects of modulators aspartate enhanced the NAD(P)H-linked activity, with a K_a value of 0.25 millimolar, but strongly inhibited that of the ferredoxin-dependent glutamate synthase with a K_i of 0.1 millimolar. 3-Phosphoserine was another inhibitor of the ferredoxin dependent enzyme with a K_i value of 4.9 millimolar. 3-Phosphoglyceric acid was a potent inhibitor of the ferredoxin-dependent form, but hardly affected the NAD(P)H-dependent enzyme. The results are discussed and interpreted to propose different specific functions that these activities may have within the leaf tissue cell.     

Activation of spinach chloroplast glyceraldehyde 3-phosphate dehydrogenase: effect of glycerate 1,3-bisphosphate

Spinach (Spinacia oleracea L.) chloroplast NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) was purified. The association state of the protein was monitored by fast protein liquid chromatography-Superose 12 gel filtration. Protein chromatographed in the presence of NADP⁺ and dithiothreitol consisted of highly NADPH-active protomers of 160 kDa; otherwise, it always consisted of a 600-kDa oligomer (regulatory form) favoured by the addition of NAD⁺ in buffers and with low NADPH-dependent activity (ratio of activities with NADPH versus NADH of 0.2–0.4). Glycerate 1,3-bisphosphate (BPGA) was prepared enzymatically using rabbit-muscle NAD-GAPDH, and purified. Among known modulators of spinach NAD(P)-GAPDH, BPGA is the most effective on a molar basis in stimulating NADPH-activity of dark chloroplast extracts and purified NAD(P)-GAPDH (activation constant, K _a= 12 M). It also causes the enzyme to dissociate into 160-kDa protomers. The K _m of BPGA both with NADPH or NADH as coenzyme is 4–7 M. NAD⁺ and NADH are inhibitory to the activation process induced by BPGA. This compound, together with NADP(H) and ATP belongs to a group of substrate-modifiers of the NADPH-activity and conformational state of spinach NAD(P)-GAPDH, all characterized by K _a values three- to tenfold higher than the K _m. Since NADP(H) is largely converted to NAD(H) in darkened chloroplasts Heineke et al. 1991, Plant Physiol. 95, 1131–1137, it is proposed that NAD⁺ promotes NAD(P)-GAPDH association into a regulatory conformer with low NADPH-activity during dark deactivation. The process is reversed in the light by BPGA and other substrate-modifiers whose concentration increases during photosynthesis, in addition to reduced thioredoxin.Abbreviations BPGA glycerate 1,3-bisphosphate - Chl chlorophyll - DTT dithiothreitol - FPLC fast protein liquid chromatography - NAD(P)-GAPDH glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent - 3-PGA glyerate 3-phosphate - PGK phosphoglycerate kinase - Prt protein - Tricine N-tris (hydroxymethyl) methyl-glycine This work was supported by grants from the Ministero dell'Università e della Ricerca Scientifica e Technologica in years 1990–1991. We are grateful to Dr. G. Branlant (Laboratoire d'Enzymologie et de Génie Génétique, Vandoeuvre les Nancy, France) for introducing us to the BPGA purification procedure.