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)     

Further characterization of two different,reversible aldehyde oxidoreductases from Clostridium formicoaceticum,one containing tungsten and the other molybdenum

The tungsten- and the molybdenum-containing aldehyde oxidoreductases from Clostridium formicoaceticum show, for aldehydes, K _m values<30 M and K _i values of millimolar concentrations. The tungsten-containing aldehyde oxidoreductase is inactivated to 50% by 3 mM KCN within 1 min, by 1 mM ferricyanide within 5 min, and by 0.05 mM chloralhydrate within 30 s. The molybdenum-containing AOR shows 50% inactivation within 1 min only with 70 mM KCN. The tungsten-containing enzyme is very sensitive to oxygen, especially in the reduced state, whereas the molybdenum-containing enzyme exhibits only moderate oxygen sensitivity without being markedly influenced by the redox state of the enzyme. The tungsten in the aldehyde oxidoreductase is bound to a pterin cofactor (Wco) of the mononucleotide form that is known for molybdopterin cofactor (Moco). The nature of the molybdenum cofactor in the molybdenum-containing aldehyde oxidoreductase is still unclear. The UV/VIS spectrum of the tungsten-containing aldehyde oxidoreductase shows a broad absorption in the range of 400 nm with a millimolar absorption coefficient of 18.1 (reduced form) and 24.8 (dehydrogenated form) at 396 nm. The epr spectrum exhibits two different W(V) signals with the following g values for signal A: 2.035, 1.959, 1.899 and signal B: 2.028, 2.017, 2.002. Dithionite-reduced enzyme shows signals of 4Fe–4S or 2Fe–2S clusters. Initial rate studies with different substrates for the carboxylate reduction led to a Bi Uni Uni Bi mechanism.Abbreviations AOR aldehyde oxidoreductase - NH ₂ CO-MV 1,1-carbamoylmethylviologen - MV methylviologen - TMV 1,1,2,2-tetramethylviologen     

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH,NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8–9 U mg^?1 protein (µmoles mg^?1 min^?1) for NADPH and 3–4 U mg^?1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^?1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^?1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^?1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.     

Sulfate reduction in a cell-free system ofChlorella

Summary During sulfate reduction in a cell-free system ofChlorella activated sulfate of APS is transferred to a thiosulfonate reductase. The sulfate thus bound to the thiosulfonate reductase (i.e. bound sulfite) is reduced to bound sulfide in a ferredoxin dependent reaction. This bound sulfide can be used with O-acetylserine as acceptor for cysteine biosynthesis; serine and O-phosphoserine are not used. An assaysystem for thiosulfonate reductase activity using methylviologen dependent reduction of S₂O₄ ^2– to S^2– is developed and a procedure for isolating thiosulfonate reductase fromChlorella cells is presented. During isolation of thiosulfonate reductase a low weight molecular factor, needed for optimal enzyme activity was lost. The bound sulfite seems to be attached to this factor. Reduction of APS or GS-SO₃H to the level of S^2– is inhibited by cysteine. 50% inhibition of GS-SO₃H reduction was found at a molar cysteine concentration of 6.8×10^–5.Abbreviations APS adenosine-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - GSH reduced glutathion - GS-SO₃H S-sulfoglutathion - fd ferredoxin - Mv methylviologen - DTT dithiothreitol     

On a reversible molybdenum-containing aldehyde oxidoreductase from Clostridium formicoaceticum

Clostridium formicoaceticum grown in the presence of 1 mM molybdate and about 1.5×10^-5 mM tungsten (present in the 5 g yeast extract/l of the growth medium) forms two reversible aldehyde oxidoreductases in an activity ratio of about 45:55. The fraction of 45% does not bind to the octyl-Sepharose column, whereas the 55% aldehyde oxidoreductase binds to this column. From cells grown on a synthetic medium without the addition of tungstate only about 2% of the aldehyde oxidoreductase of the crude extract binds to octyl-Sepharose. The enzyme not binding to octyl-Sepharose has been purified as judged by electrophoresis. It is pure after about 50 fold enrichment. According to SDS gel electrophoresis the enzyme consists of identical 100 kD subunits. Based on gel chromatography it seems to be a trimer. Per subunit 0.6 molybdenum, 7 iron, 6.6 acid labile sulphur, about 0.1 pterin-6-carboxylic and <0.05 tungsten have been found. The first 13 amino acids from the amino end show no similarity with the W-containing aldehyde oxidoreductase from the same bacterium. With reduced tetramethylviologen (E₀=–550 mV) the new molybdenum containing enzyme can reduce various aliphatic and aromatic acids to aldehydes. The pH optimum is at 6.0. For the dehydrogenation of butyraldehyde a rather broad pH region from pH 6 to 10 shows almost no variation of rate. From 15 different aldehydes acetaldehyde exhibits the highest rate. The K_m value for butanal is 0.002 and for propionate 7.0 mM. Compared with the tungsten enzyme the molybdenum enzyme is only moderately oxygen-sensitive.Abbreviations AOR aldehyde oxidoreductase - BV benzylviologen - MV methylviologen - NH₂CO-MV 1,1-carbamoylmethylviologen - TMV 1,1,2,2-tetramethylviologen     

Ethanol dissimilation in Desulfovibrio

During growth of ethanol plus sulfate Desulfovibrio gigas and three other Desulfovibrio strains tested contained high NAD-dependent alcohol dehydrogenase activities and dye-linked aldehyde dehydrogenase activities. In lactate-grown cells these activities were lower or absent. In D. gigas an NADH dehydrogenase activity was found which was higher during growth on ethanol than during growth on lactate. The NADH dehydrogenase activity appeared to consist of at least three different soluble enzymes. The aldehyde dehydrogenase activity in D. gigas was highest with benzylviologen as an acceptor and was strongly stimulated by potassium ions. Coenzyme A or phosphate dependency could not be shown, indicating that acetyl-CoA or acetyl phosphate are not intermediates in the conversion of acetaldehyde to acetate.In the absence of sulfate D. gigas was able to convert ethanol to acetate by means of interspecies hydrogen transfer to a methanogen. This conversion, however, did not lead to growth of the Desulfovibrio.Abbreviations DH dehydrogenase - BV²⁺/BV⁺ oxidized/reduced benzylviologen - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide - MV²⁺/MV⁺ oxidized/reduced methylviologen - PMS phenazine methosulfate     

Characterization of a malate dehydrogenase in the cyanobacterium Coccochloris peniocystis

A malate dehydrogenase (MDH) was characterized from the cyanobacterium Coccochloris peniocystis. The enzyme was purified approximately 180-fold and had a molecular weight of about 90000. The enzyme had a pH optimum of pH 6.7 to 7.5; a K_m (malate) of 5.6 mM and K_ms for NAD and NADP of 24 M and 178 M, respectively, although similar V_max were obtained with either pyridine nucleotide. Enzyme activity was inhibited by ATP, citrate, oxalacetate, acetyl CoA and CoA. Enzyme assays with uniformly ¹⁴C-labelled malate caused no ¹⁴CO₂ release, indicating this MDH is not a malic enzyme. Electrophoresis and S-200 gel filtration of the partially purified enzyme indicated a single MDH was present in this preparation. A second, less abundant, MDH was present in crude extracts. The presence of MDH in this organism is consistent with the operation of a glyoxylate cycle which, in the absence of a TCA cycle, would provide organic acids required in secondary carbon metabolism. ATP inhibition of MDH may allow for light regulation of MDH activity since, in the light, oxaloacetic acid is generated by phosphoenolpyruvate carboxylase activity.Abbreviations MDH malate dehydrogenase - PEPcase phosphoenolpyruvate carboxylase - MOPS 3-[N-Morpholino] propane sulfonic acid - TRIS Tris(hydroxymethyl)-aminomethane - EDTA Disodium Ethylenadiamine Tetraacetate - MES 2[N-Morpholino]-ethane Sulfonic Acid - EPPS N-2-Hydroxyethylpiperazine Propane - MW Molecular weight - OAA Oxaloacetic acid     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Partial purification and characterization of a coniferyl alcohol dehydrogenase fromRhodococcus erythropolis

A nicotinamide adenine dinucleotide (NAD)-dependent coniferyl alcohol dehydrogenase was enriched 1,200-fold from crude extracts ofRhodococcus erythropolis. The purification procedure involved ion exchange chromotography, gel filtration on Biogel A 1,5 and Sephadex G-200, and hydroxyapatite treatment. The enzyme had a molecular weight of approximately 200,000 and displayed maximal activity at pH 9.0. The apparentK _m values for NAD and coniferyl alcohol were, respectively, 0.22 and 0.645 mM. Nicotinamide adenine dinucleotide phosphate (NADP) could only partially replace NAD. The enzyme was active with vanillyl alcohol and aromatic alcohols bearing the ,-unsaturated side chain of coniferyl alcohols. These aromatic alcohols included the dilignols dehydrodiconiferyl alcohol and guaiacylglycerol--coniferyl ether.     

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     

A F420-dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus

Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temperature optimum of 83°C, uses the 5-deazaflavin coenzyme F₄₂₀ rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nuclcotides for biosynthetic purposes. We describe here that the Archaeon contains a coenzyme F₄₂₀-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purfied 3600-fold by affinity chromatography to apparent homogeneity in a 60% yield. The native enzyme with an apparent molecular mass of 55 kDa was composed of only one type of subunit of apparent molecular mass of 28 kDa. Spectroscopic analysis of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible reduction of NADP (apparent K _M 40 M) with reduced F₄₂₀ (apparent K _M 20M) with a specific activity of 660 U/mg (apparent V _max) at pH 8.0 (pH optimum) and 80°C (temperature optimum). It was specific for both coenzyme F₄₂₀ and NADP. Sterochemical investigations showed that the F₄₂₀-dependent NADP reductase was Si face specific with respect to C5 of F₄₂₀ and Si face specific with respect to C4 of NADP.Abbreviations F₄₂₀ coenzyme F₄₂₀ - F₄₂₀H₂ 1,5-dihydrocoenzyme F₄₂₀ - H₄MPT tetrahydromethanopterin - CH=H₄MPT N⁵, N¹⁰-methylenetetrahydromethanopterin - MFR methanofuran - HPLC high performance liquid chromatography - methylene-H₄MPT dehydrogenase N⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase - 1 U = 1 mol/min     

Succinic semialdehyde dehydrogenase of wheat grain

Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain (Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6–9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K _m for SSA and for NAD are 7.4x10^-6 M and 2x10^-4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.Abbreviations GABA -aminobutyric acid - GABA-T -aminobutyric acid transaminase - ME mercaptoethanol - SSA succinic semialdehyde - SSA-DH succinic semialdehyde dehydrogenase     

Membrane-bound NADPH dehydrogenase- and ferredoxin: NADP oxidoreductase activity involved in electron transport during acetate oxidation to CO2 in Desulfobacter postgatei

Desulfobacter postgatei grows on acetate and sulfate as energy source. The oxidation of acetate to 2 CO₂ proceeds via the citric acid cycle involving membrane-bound succinate dehydrogenase and membrane-bound malate dehydrogenase. We report here that the organism contains membrane-bound NADPH dehydrogenase and ferredoxin: NADP oxidoreductase for the reoxidation of NADPH and reduced ferredoxin generated during isocitrate- and 2-oxoglutarate oxidation, respectively. The presence of proton translocating ATPase activity is also described.NADPH dehydrogenase and succinate dehydrogenase were found to be electrically connected within the membrane and electron transfer between these two enzymes was shown to be coupled with proton translocation. The membrane fraction catalyzed the oxidation of NADPH with fumarate and the reduction of NADP with succinate. NADPH oxidation with fumarate was stimulated by protonophores and inhibited by the proton translocating ATPase inhibitor dicyclohexylcarbodiimide (DCCD) and by heptylhydroxyquinoline-N-oxide (HQNO); inhibition by DCCD was relieved by protonophores. NADP reduction with succinate was dependent on ATP and inhibited by protonophores, DCCD, and HQNO. The membrane fraction also mediated the oxidation of NADPH with the water soluble menaquinone analogue dimethylnaphthoquinone (DMN) and the reduction of fumarate with DMNH₂. Only the former reaction was stimulated by protonophores and only the latter reaction was inhibited by HQNO. This suggests that the NADPH dehydrogenase reaction is the site of energy conservation and the succinate dehydrogenase is the site of HQNO inhibition.Non-standard abbreviations APS Adenosine 5-phosphosulfate - DCCD N,N-dicyclohexylcarbodiimide - DCPIP 2,6-dichloroindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - HQNO 2(n-heptyl)-4-hydroxyquinoline-N-oxide - TCS 3,5,3,4-tetrachlorosalicylanilide - Tricine N-tris-(hydroxymethyl)methylglycine - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - SF-6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile     

Heat-stress stimulation of oxygen uptake by Photosystem I involves the reduction of superoxide radicals by specific electron donors

A Photosystem I submembrane fraction isolated from spinach was used to study the mechanism of heat-stress stimulation of oxygen uptake by the photosystem. Various artificial electron donors were shown to generate electron transport reactions with various degrees of thermally induced stimulation. A strong stimulation was observed with durohydroquinone as electron donor with a maximal effect at 50 °C. The degree of stimulation obtained was independent from the redox potential of the electron donors and from their oxidation site because the enzyme superoxide dismutase fully inhibited the stimulation. Instead, it is proposed that thermal stress causes the release of membrane bound superoxide dismutase from the thylakoids thus allowing the reduced form of electron donors with specific properties to reduce O₂ ^– radicals to H₂O₂ besides the usual disproportionation of O₂ ^– into O₂ and H₂O₂.Abbreviations: PS photosystem - DCIP 2,6-dichlorophenolindophenol - MV methylviologen - TMPD N,N,N,N-tetramethylphenylenediamine - SOD superoxide dismutase - Chl chlorophyll - DQ duroquinone - DAD N,N,N,N-tetramethyl-1,4-benzenediamine - PMS 5-methylphenazium methyl sulfate - PC plastocyanin     

Metabolic regulation in Pseudomonas oxalaticus OX1

Metabolic control associated with diauxic growth of Pseudomonas oxalaticus in batch cultures on mixtures of formate and oxalate was investigated by measuring intracellular enzyme and coenzyme concentrations and Q _{O 2}values during transition experiments from oxalate to formate and vice versa. In transition from oxalate to formate oxalyl-CoA reductase concentration declined after the exhaustion of oxalate and ribulose-1,5-diphosphate carboxylase and ¹⁴CO₂ fixation appeared upon addition of formate. In the reciprocal transition, ribulose-1,5-diphosphate carboxylase and ¹⁴CO₂ fixation rate declined sharply after formate exhaustion, and oxalyl-CoA reductase appeared only after addition of oxalate. The intracellular NAD and NADP concentrations measured in the same experiments are reported. At substrate exhaustion the proportion of NAD in the reduced form fell from 15–20% to 2%. On addition of formate to an oxalate-starved culture there was an immediate increase in the proportion of NADH to 50%; such an increase was not observed in the reverse experiment.Abbreviations RuDP ribulose-1,5-diphosphate - HEPES 2-(N-2 hydroxyethylpiperazin-N-yl) ethane sulphonic acid     

Purification and characterization of a potent hemolytic toxin with phospholipase A2 activity from the venom of Indian Russell's viper

A potent toxin with phospholipase A₂ (PLA₂) and hemolytic activity in vitro was purified from the Russell's viper venom of eastern India (RVV-EI). The purified protein (RVV-PFIIc) of 15.3 kDa molecular weight, and a lethal toxicity dose (LD₅₀ i.p.) of 0.1 mg/kg body weight, was the most toxic PLA₂ so far reported from the Indian subcontinent. The material also possessed anticoagulant activity as it enhanced the prothrombin induced plasma clotting time in vitro. The PLA₂ toxin (RVV-PFIIc) was shown to be different from other PLA₂s of RVV in respect to one or more of these parameters e.g. molecular weight, isoelectric pH, in vivo toxicity, specific activity of the enzyme and certain other biological activities. The first 19 amino terminal sequence (NLFQFAEMIVKMTGKEAVH) of RVV-PFIIc showed variable degree of homology (42.1–94.7%) with those of other RVV-PLA₂s described in the literature. Antisera raised against RVV-EI or RVV-PFIIc, though completely neutralized the in vivo lethal toxicity of RVV-EI or RVV-PFIIc, failed to inhibit their PLA₂ activity in vitro thereby suggesting that in vivo toxicity and in vitro activity of the enzyme may not be directly related. Apart from RVV-PFIIc, at least two other PLA₂ isozymes were found to be present in RVV-EI that were distinct from RVV-PFIIc in respect to their molecular, biological as well as serological properties. The significance of these and related data in antivenom therapy is discussed.     

Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H₂) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H₂) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H₂, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.Abbreviations CODH carbon monoxide dehydrogenase - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl-p-trifluormethoxyphenylhydrazon - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - pmf proton motive force     

Molecular aspects of cytochrome c oxidase: Structure and dynamics

Summary In the last few years much attention has been dedicated to the elucidation of some of the molecular aspects of cytochrome c oxidase. It has been shown conclusively that the enzyme from several sources (yeast, Neurospora, heart, liver) contains seven different subunits, which are asymmetrically inserted in the membrane. All of these are in contact with the lipid bilayer (except subunits V and VI) and to a greater or lesser extent with the water phase as well (except for subunit I). Subunit II of the enzyme appears to be involved in the formation of the binding site of cytochrome c. The location of the redox groups of the enzyme is still a matter of controversy. Their distance from the cytochrome c heme group is approximately 35 Å such that electron tunneling appears to be the only possible mechanism for transporting electrons across such a distance.A proton pump appears to be associated with electron transport and approximately one proton is extruded per electron equivalent reducing oxygen via the enzyme. N,N, dicyclohexylcarbodiimide a well-established inhibitor of H⁺-translocating ATPases inhibits the proton pump and labels specifically subunit III of the enzyme.Abbreviations CCCP carbonyl cyanide mchlorophenylhydrazone - DADH₂ diamino-durene (reduced form) - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid - NEM N-ethyl-maleimide - pH transmembrane chemical-potential gradient of H⁺ - transmembrane electricalpotential gradient - transmembrane electrochemical-potential gradient of H⁺ - Q/QH₂ oxidised/reduced form of ubiquinone - TMPD/TMPDH₂ non-protonated/protonated form of tetramethyl-p-phenylenediamine - SDS sodium dodecyl sulphate     

ADP-ribosylation of actin from the green algaChara corallina byClostridium botulinum C2 toxin andClostridium perfringens iota toxin

Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [³²P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of M_r 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP adenosine-diphosphate - EGTA ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid - NAD nicotinamide dinucleotide - pCA -log [Ca²⁺] - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Eigenschaften der NAD-spezifischen Hydrogenase aus Hydrogenomonas H 16

Zusammenfassung Aus zellfreiem Extrakt von Hydrogenomonas H 16 wurde die lösliche Hydrogenase 45 fach bis zu einer spezifischen Aktivität von 36500 E/g Protein angereichert. Das Enzym katalysiert die Reduktion von NAD mit molekularem Wasserstoff. Ein Cofaktorbedürfnis konnte nicht festgestellt werden. Der Einfluß von NADH, ATP, Bicarbonat und Magnesium auf die hydrogenasekatalysierte NAD-Reduktion war unerheblich. Das angereicherte Enzym ist flavin- und pyridinnucleotidfrei und reagiert mit NAD, nicht mit O₂, NADP, FMN, FAD oder Methylenblau. Die drei letztgenannten Wasserstoff-Acceptoren werden lediglich in Gegenwart katalytischer Mengen NAD reduziert. Die lag-Phase der Reduktion von NAD läßt sich durch Vorinkubation des Enzyms mit NADH oder Wasserstoff, nicht jedoch mit NAD eliminieren. Die Hemmung der Hydrogenasereaktion durch Sauerstoff ist gering. Reduzierende Agenzien wie Mercaptoäthanol oder Sulfid setzten die Reaktionsrate herab.Die Michaeliskonstante der löslichen Hydrogenase für molekularen Wasserstoff beträgt K _m ^H2 =1,9·10^–4 M. Die NAD-Konzentration, bei der halbmaximale Aktivität erreicht wird, beträgt [NAD]_{0,5 (V)}=1,3·10^–4 M. Das pH-Optimum wird in 0,05 M Kaliumphosphat-Puffer bei pH 8,5 und in 0,05 M Tris-HCl-Puffer bei pH 7,9 erreicht. Unter den angegebenen Bedingungen lag das Temperatur-Optimum bei 36°C. Die Aktivierungsenergie der löslichen Hydrogenase wurde als 10,4 kcal/mol ermittelt.

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Properties of the NAD-Specific Hydrogenase from Hydrogenomonas H 16

Summary A soluble hydrogenase from cell-free extracts of Hydrogenomonas H 16 has been purified 45-fold up to a specific activity of 36,500 units per g protein. The enzyme catalyzes the reduction of NAD with molecular hydrogen. It does not require cofactors. The NAD-reduction catalyzed by this enzyme is influenced to only a small extent by the presence of NADH, ATP, bicarbonate or magnesium ions. The enzyme is free from flavins and pyridine nucleotides, reacts only with NAD and not with oxygen, NADP, FMN, FAD or methylene blue. FMN, FAD and methylene blue are reduced only in the presence of catalytic amounts of NAD. The lag-phase of the reduction of NAD can be eliminated by preincubating the enzyme in the presence of NADH or molecular hydrogen; NAD is ineffective. The inhibition of the hydrogenase reaction by oxygen is negligible. Reducing agents such as mercaptoethanol or sulfide decreased the reaction rate.The Michaelis constant of the soluble hydrogenase for molecular hydrogen is K _m ^H2 =1.9·10^–4 M. Half maximal activity is attained at a NAD-concentration of [NAD]_{0.5 (V)}=1.3·10^–4 M. The pH-optimum is 8.5 in 0.05 M potassium phosphate buffer and 7.9 in 0.05 M Tris-HCl-buffer; reaction rates were maximal at 36°C under the conditions employed. The activation energy was calculated to be 10.4 kcal/mol.

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Abkürzungen E Enzymeinheit (mole Substrat/min) - E _x Extinktionsänderung bei der Wellenlänge x nm - Ext._x Extinktion bei der Wellenlänge x nm - KP Kaliumphosphat (KH₂PO₄-K₂HPO₄-Gemisch) - MB Methylenblau - NAD Nicotinamid-adenin-dinucleotid - NADH reduziertes - NAD; TEAE Triäthylaminoäthyl - Tris Tris(hydroxymethyl)aminomethan