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PubMed1981

D-lactate dehydrogenase of Desulfovibrio vulgaris.

D-Lactate dehydrogenase, the starting enzyme for carbon and energy metabolism in dissimilatory sulfate-reducing bacteria, has been purified 36-fold from the soluble fraction of the sonicate of Desulfovibrio vulgaris, Miyazaki. The enzyme is specific for D-lactate (Km = 0.8 mM) and DL-2-hydroxybutyrate (probably its D-isomer) as the electron donor substrate. It reduces, in the presence of lactate, various artificial electron acceptors such as 1-methoxyphenazinium methyl sulfate, ferricyanide, tetrazolium dyes, methylene blue, and 2,6-dichlorophenol-indophenol. When 2 mol of ferricyanide was reduced, 1 mol of pyruvate was produced during the reaction. Among natural electron carriers, only cytochrome c-553 isolated from the same organism can be reduced by the enzyme. The ferric complex of pyridine-2,6-dicarboxylate can act as an electron acceptor if cytochrome c-553 is present in the reaction system. NAD+, NADP+, FAD, FMN, cytochrome c3, high-molecular-weight cytochrome, eucaryotic cytochromes c (yeast and horse) and O2 could not be reduced. The enzyme does not have any diaphorase activity. The D-lactate dehydrogenase of D. vulgaris must therefore be named D-lactate:ferricytochrome c-553 oxidoreductase [EC subclass 1.1.2]. A similar enzyme exists in the formate dehydrogenase-less mutant of D. vulgaris, Miyazaki, and in D. vulgaris, Hildenborough.

Desulfovibrio↗
PubMed1981

Two cytochromes c of Methylomonas J.

Two kinds of c-type cytochromes, cytochrome c-551 (I), and cytochrome c-551 (II), were highly purified and crystallized from cell-free extract of methanol-grown Methylomonas J (formerly Pseudomonas sp. J) and their physiochemical and biochemical properties were studied. Cytochrome c-551 (I) had an absorption peak at 409 nm in the oxidized form and peaks at 417, 523, 551 nm, and a shoulder at 532 nm in the reduced form. The millimolar extinction coefficient of the alpha-peak of the reduced form was 25.3. The isoelectric point was at pH 5.3 and its standard redox potential was 0.29 V at pH 7.0. The molecular weight was estimated to be 16,000. Cytochrome c-551 (II) had absorption maxima at 409 nm in the oxidized form, and at 416, 521, and 551 nm in the reduced form. The millimolar extinction coefficient of the alpha-peak of the reduced form was 22.4. The isoelectric point was at pH 4.3 and its standard redox form was 22.4. The isoelectric point was at pH 4.3 and its standard redox potential was 0.24 V at pH 7.0. The molecular weight was estimated to be 12,500. The two cytochromes were reduced by methanol dehydrogenase [EC 1.1.99.8] of this bacterium, and formaldehyde was detected as an oxidation product. Ammonium chloride was not essential for reduction of the cytochromes. No significant reduction of the cytochromes was observed by methylamine dehydrogenase isolated from methylamine-grown cells or by 2,6-dichlorophenol-indophenol (DCPIP)-dependent aldehyde dehydrogenase of the methanol-grown cells. The reduced forms of the cytochromes were oxidized by blue copper protein of the methanol-grown cells.

Alcohol Oxidoreductases↗
PubMed1981

Partial purification and properties of respiratory chain-linked l-glycerol 3-phosphate dehydrogenase from a marine bacterium, Vibrio alginolyticus.

Respiratory chain-linked L-glycerol 3-phosphate (G3P) dehydrogenase [EC 1.1.99.5] of marine bacterium, Vibrio alginolyticus, was extracted from the membrane fraction by treatment with Tween 20, and fractionation on DEAE-Sephacel and QAE-Sephadex in the presence of 0.05% Liponox DCH(alkyl polyoxyethylene ether) yielded a preparation having a specific activity of 22.1 units/mg protein when assayed by phenazine methosulfate (PMS)-coupled reduction of thiazolyl blue tetrazolium (MTT). The purified enzyme had an apparent molecular weight of 300,000 as determined by chromatography on Sepharcyl S-300 in 0.05% Liponox DCH, and had noncovalently bound FAD as its coenzyme. The enzyme had a pH optimum of 8.5-9.0, and required 200 mM NaCl or KCl and an appropriate detergent (such as Tween, Brij or Liponox DCH) for maximum activation. The activating effect of NaCl was due to a decrease in Km for G3P and that of Tween 20 was due to both a decrease in Km and an increase in Vm. Triton X-100 could not activate the enzyme and was inhibitory in the presence of phospholipids. The reaction followed a ping-pong mechanism. IN addition to PMS, 2,6-dichlorophenol indophenol and duroquinone, the enzyme could reduce ubiquinone-5 (Q-5) in the presence of Liponox DCH at a rate of 46% of the PMS reductase activity. The enzyme was strongly inhibited by heavy metal ions and by p-chloromercuribenzoate. The activity for Q-5, but not for PMS, was inhibited by o-phenanthroline and bathophenanthroline, suggesting the participation of nonheme iron protein in the Q-5 reduction.

Cell Membrane↗
PubMed1986

A novel FAD-protein that allows effective reduction of methyl viologen by NADH (NADH-methyl viologen reductase) from photosynthetic bacterium, Rhodospirillum rubrum: purification and characterization.

It was found that the cytoplasm of light-grown cells of Rhodospirillum rubrum could catalyze the reduction of methyl viologen (MV) (Em, 7 = -0.44 V) by NADH and NADPH. In the present study, the enzyme capable of catalyzing MV reduction by NADH (NADH-MV reductase) was purified 1,500-fold from an extract of cells with a yield of 4.4%. The purification procedure comprised (NH4)2SO4 fractionation, and chromatographies on Sepharose CL-6B, DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Blue-Cellulofine, and TSK-Gel G3000SW. Two NADPH-MV reductases were separated during the purification. The NADH-MV reductase obtained was nearly homogeneous, as judged on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The enzyme has a molecular weight of 220,000 and an isoelectric point of 4.8; it is composed of four subunits with a molecular weight of 57,000, and is bound with about 1 mol FAD/mol subunit. The activity is optimum at pH 8. The Km values for NADH and MV are 115 microM and 1.3 mM, respectively, with a molecular activity of 13,000 min-1. The activity was stimulated 2.4-fold in the presence of 20-100 mM ammonium ions. The enzyme also catalyzed the reduction of benzyl viologen, methylene blue and 2,6-dichlorophenol-indophenol (Em, 7 = -0.36, +0.011, and +0.217 V, respectively) at comparable rates. The ratios of the activity with NADH to that with NADPH were 80, 133, 41, and 5.5 with MV, benzyl viologen, methylene blue and 2,6-dichlorophenolindophenol, respectively. The enzyme was significantly stable in the presence of both 5mM 2-mercaptoethanol and 20% (w/v) glycerol. The activity was not appreciably influenced by the presence of 2 M urea, although the reagent caused dissociation to the subunits.

Chromatography↗
PubMed1986

NADPH-dependent superoxide-forming oxidase in phagocytic vesicles of human monocytes.

Phagocytic vesicles with superoxide-forming NADPH oxidase activity were obtained from human monocytes phagocytosing oil droplets. The superoxide-forming activity in the monocyte vesicles increased for the first 5 min during incubation with oil droplets and remained constant for 30 min. NADPH-dependent activities of 2,6-dichlorophenol-indophenol (DCIP) reduction and ubiquinone-1 (Q1) reduction were found in the vesicles and the activities were closely associated with the superoxide-forming oxidase. The values of apparent Km for NADPH of these three activities were essentially the same and the activities were inhibited with a similar pattern by p-chloromercuribenzoate and a cationic detergent, cetyltrimethylammonium bromide. The activities were extremely labile and the DCIP reductase activity was most labile. The superoxide-forming oxidase and the Q1 reductase could be extracted with a mixture of deoxycholate and Tween-20. The extracted activities were not enhanced by the addition of FAD.

2,6-Dichloroindophenol↗
PubMed1980

Plesiomonas (Aeromonas) shigelloides septicemia and meningitis in a neonate.

Serious Plesiomonas (Aeromonas) shigelloides infections have rarely been reported, and have probably been missed because this organism is very similar to the Enterobacteriaceae in associated clinical disease, and in properties investigated in the diagnostic laboratory. A case of overwhelming neonatal meningitis and sepsis is discussed, and the use of the simple indophenol oxidase test on laboratory isolates of gram-negative rods is urged to distinguish this organism and its close relatives from the Enterobacteriaceae.

Aeromonas↗
PubMed2004

Production of ammonia by Tritrichomonas foetus and Trichomonas vaginalis.

Production of ammonia is difficult to find among the various studies of amino acid metabolism in protozoa. Several studies suggest that catabolism of arginine to ammonium is important for the growth of trichomonads. Trichomonads are amitochondriate zooflagellates that thrive under microaerophilic and anaerobic conditions. The authors were able to detect accumulation of ammonium ions and ammonia in cultures of Tritrichomonas foetus and Trichomonas vaginalis, including those resistant to metronidazole. Ammonium ions and ammonia were detected using the indophenol colorimetric method. Cells incubated overnight under an ambient oxygen gas phase had 0.9 mM soluble ammonium (NH(4)(+) and NH(3)) or a 20 % greater concentration of ammonium relative to sterile growth medium that had been incubated similarly. Production of ammonia itself was confirmed by analysis of a wick that was moistened with sulfuric acid (20 mM) and placed above the liquid in sealed cultures of a strain of Trichomonas vaginalis. The wicks from these cultures captured the equivalent of 0.048 mM volatile ammonia (NH(3)) from the liquid as compared to 0.021 mM volatile ammonia from sterile medium after overnight incubation. Intact trichomonads, 0.7 x 10(6) cells ml(-1) equivalent to 0.7 mg protein ml(-1), incubated in Doran's buffer with or without (1 mM) L-arginine produced significant amounts of soluble ammonium (0.07 mM and 0.04 mM, respectively) during 60 min. The results indicate that ammonium ions and the more irritating ammonia are significant metabolites of trichomonads. In addition, based upon end-product amounts, it appears that the rate of arginine metabolism is of the same order of magnitude as that for carbohydrate metabolism by trichomonads.

Ammonia↗
PubMed1966

Plastoquinone B.

A compound found in spinach and other higher plants previously referred to as R 263 has now been found to be a breakdown product of plastoquinone B. This quinone, PQ B, is found with 8 other quinones in spinach chloroplasts. These 9 quinones are PQ A, PQ B, PQ C, PQ D (7, 8, 15) Vitamin K(1) (10, 12), an unknown naphthoquinone (13) and alpha-, beta- and gamma-tocopherylquinones (7, 12). An improved method for purification of plastoquinone B is described. Previous confusion of this compound with other quinoid material on silica gel is described and corrected R(F) values are given. The activity of PQ B is similar to the activity of PQ C in restoration studies of the photo-reduction of ferricyanide and indophenol.

Journal Article↗
PubMed1966

Differential effects of desaspidin on photosynthetic phosphorylation.

Sensitivity to low concentrations of desaspidin (5 x 10(-7)m) sharply distinguishes the photophosphorylations associated with the photooxidation of water from all other types of photophosphorylation by isolated chloroplasts. Contrary to recent reports in the literature, the effects of desapidin were not altered by changes in the redox conditions as influenced by the concentration of ascorbate and by the presence or absence of oxygen. Desaspidin consistently inhibited all types of cyclic photophosphorylation and the photophosphorylation coupled with the reduction of NADP by ascorbate-dichlorophenol indophenol. The same concentration of desaspidin gave little or no inhibition of photophosphorylation that are coupled with the photooxidation of water.

Butyrophenones↗
PubMed1967

An Electron Spin Resonance Study of Manganese in Wild-Type and Mutant Strains of Chlamydomonas reinhardi.

Changes in the intensity of the electron spin resonance signal of divalent manganese were found to occur in suspensions of wild-type Chlamydomonas reinhardi. The observed manganese signal decreased in the light and increased in the dark. Through the use of a continuous-flow system it was possible to determine that the manganous ions responsible for the observed signal were localized solely in the medium. Changes in the signal intensity associated with wild-type cells were independent of the ability of fragments prepared from these cells to perform the Hill reaction with 2,6-dichlorophenol-indophenol (DPIP) as the oxidant.The manganese signal changes were still evident, though smaller, in cell suspensions of wild-type cells treated with 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, and in mutant strains unable to carry out the Hill reaction, ac-115 and ac-141.From these data it is concluded that the changes in intensity of the manganese resonance are not related to the function of manganese in photosynthesis but may reflect the capacity of cells for ion uptake in the light.

Journal Article↗
PubMed1968

Photoreduction and photophosphorylation with tris-washed chloroplasts.

The artificial electron donor compounds p-phenylenediamine (PD), N, N, N', N'-tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 m tris (hydroxymethyl) aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 mum PD plus 330 mum ascorbate was largely inhibited by 1 mum 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) while that supported by 33 mum TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 mum donor plus 330 mum ascorbate) photophosphorylation, which occurred with P/e(2) ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited.

Chloroplasts↗
PubMed1970

Some effects of hydrolytic enzymes on coupled and uncoupled electron flow in chloroplasts.

Digestion of spinach chloroplasts with pancreatic lipase or trypsin effectively uncoupled electron transport. Continued digestion led to inhibition of saturated rates of Hill reaction activity and a decrease in quantum yield. Irradiation with ultraviolet light decreased the quantum yield and inhibited Hill activity, but did not uncouple. Ascorbate-dichlorophenol-indophenol-mediated reduction of nicotinamide adenine dinucleotide phosphate was not appreciably inhibited by treatment with either of the enzymes or by ultraviolet irradiation.Carbonylcyanide m-chlorophenylhydrazone became a potent inhibitor of electron transport after trypsin treatment of chloroplasts. It also inhibited, rather than uncoupled, electron transport in glutaraldehyde-fixed chloroplasts. No other uncouplers tested showed these effects. Glutaraldehyde fixation of chloroplasts also greatly reduced the inhibitory effects of lipase and trypsin digestion but not the inhibition by ultraviolet irradiation.The inhibitory effects of trypsin and pancreatic lipase, and probably ultraviolet irradiation as well, appear to be due to a general breakdown of the membrane structure rather than inactivation of specific sites in the electron transport chain.

Journal Article↗
PubMed1972

The problem of reduced nicotinamide adenine dinucleotide oxidation in glyoxysomes.

NADH is generated in glyoxysomes both in the glyoxylate cycle and in beta-oxidation. No system has yet been described which would oxidize NADH in these organelles. A series of oxidants which might function by coupling NADH oxidation to O(2) through endogenous carriers in the glyoxysomes was examined. Oxidation was brought about by ferricyanide or dichlorophenol-indophenol, but it was shown that this "diaphorase" activity is probably a contaminant. Hydroxypyruvate reductase (NAD-linked) is present in the glyoxysomes, and at very high substrate concentrations (>10 mm) this enzyme can also transfer electrons from NADH to glyoxylate. However, it is most unlikely that this concentration of glyoxylate is ever approached in glyoxysomes, where the malate synthetase would compete on much superior terms. The maximum rates of NADH oxidation observed in the presence of ferricyanide or glyoxylate are only a fraction of those required to reoxidize NADH at the rate occurring in vivo.

Journal Article↗
PubMed1972

Inhibition of Photosystem II in Isolated Chloroplasts by Lead.

Inhibition of photosynthetic electron transport in isolated chloroplasts by lead salts has been demonstrated. Photosystem I activity, as measured by electron transfer from dichlorophenol indophenol to methylviologen, was not reduced by such treatment. However, photosystem II was inhibited by lead salts when electron flow was measured from water to methylviologen and Hill reaction or by chlorophyll fluorescence. Fluorescence induction curves indicated the primary site of inhibition was on the oxidizing side of photosystem II. That this site was between the primary electron donor of photosystem II and the site of water oxidation could be demonstrated by hydroxylamine restoration of normal fluorescence following lead inhibition.

Journal Article↗
PubMed1974

Studies on the Energy-coupling Sites of Photophosphorylation: V. Phosphorylation Efficiencies (P/e(2)) Associated with Aerobic Photooxidation of Artificial Electron Donors.

The rate of Hill reaction can be measured accurately as O(2) uptake (the Mehler reaction) if a rapidly autoxidizable electron acceptor (e.g., methylviologen) is used. However, when an artificial electron donor-ascorbate couple (or ascorbate alone) replaces the natural donor, water, the rate of O(2) consumption is no longer a reliable measure of the electron flux, because superoxide radical reactions contribute to O(2) uptake. Such radical reactions, however, can be suppressed by adding enough superoxide dismutase to the reaction mixture. Indeed in all of the photosystem I- and photosystem II-donor reactions tested (except with benzidine which was tested without ascorbate added), the O(2) uptake was inhibited by 30 to 50% by the addition of superoxide dismutase. The rate of phosphorylation was totally unaffected by the enzyme. The reasessment of the phosphorylation efficiencies thus made by the use of superoxide dismutase led us to the following conclusions. The phosphorylation efficiency associated with the transfer of electrons from a donor to methlylviologen (than to O(2)) through both photosystems II and I is practically independent of the donor used-catechol, benzidine, p-aminophenol, dicyanohydroquinone, or water. The P/e(2) ratio is 1.0 +/- 0.1. Only ascorbate gives a slightly lower value (P/e(2) = 0.9). (NH(2)OH-treated, non-water-splitting chloroplasts were used for reactions with these artificial donors.) The phosphorylation efficiency associated with DCMU-insensitive, photosystem I-mediated transfer of electrons from a donor to methylviologen (then to O(2)) is again largely independent of the donor used, such as diaminodurene, diaminotoluene, and reduced 2,6-dichlorphenol-indophenol. The P/e(2) ratio is 0.6 +/- 0.08.

Journal Article↗
PubMed1976

Evidence for a Block between Plastoquinone and Cytochrome f in a Photosynthetic Mutant of Lemna with Abnormal Flowering Behavior.

Mutant strain 1073 of Lemna perpusilla is concluded to be blocked between plastoquinone and cytochrome f in the photosynthetic electron transport system. The location of the block is based on the following observations of activities in chloroplasts isolated from the mutant and wild-type plants. (a) Relative to wild type, electron flow rates from water to ferricyanide, 2,6-dichlorophenol indophenol or NADP were very low in the mutant, but rates of photosystem I-dependent electron flow and cyclic phosphorylation were high. (b) Chlorophyll a fluorescence induction curves for mutant and wild type were similar. (c) Silicomolybdate and lipophilic acceptors in the mutant were photoreduced at rates comparable to wild type. (d) Cytochrome f of the mutant chloroplasts was not reduced by red light, but was oxidized by red or far red light. (e) Reduction of the primary electron acceptor of photosystem II (Q) by ATP-driven reverse electron flow was not observed in the mutant.

Journal Article↗
PubMed1978

Low temperature spectral properties of subchloroplast fractions purified from spinach.

Spinach (Spinacia oleracea L.) chloroplasts solubilized by digitonin were separated into five fractions by sucrose density gradient centrifugation. Three of the fractions, F(I), F(II), and F(III), corresponding to photosystem I, photosystem II, and the chlorophyll a/b complex, were purified further by two steps of diethylaminoethyl-cellulose chromatography followed by electrofocusing on an Ampholine column. The polypeptide patterns of the fractions were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the spectral properties of the fractions at -196 C determined by absorption spectra, fourth derivative curves of the absorption spectra, fluorescence emission spectra, and fluorescence excitation spectra. The activity of purified F(II) (photosystem II) was also assayed by the photoreduction of dichlorophenol-indophenol at room temperature using 1,5-diphenylcarbohydrazine as the electron donor and by the photoreduction of C-550 at -196 C. The different fractions showed unique polypeptide patterns and unique sets of low temperature-absorbing forms of chlorophyll. The fluorescence emission spectra of F(I), F(II), and F(III) at -196 C were also unique with maxima at 734, 685 and 681 nm, respectively. F(I) showed negligible emission at wavelengths shorter than 700 nm and the long wavelength tails of F(II) and F(III) in the 730 nm region were relatively small (approximately 10% of emission of their wavelength maxima). Addition of 0.1% Triton to F(I) and F(II) caused the longer wavelength absorbing forms of chlorophyll to shift to 670 nm and the fluorescence emission maxima (of both fractions) to shift to 679 nm at -196 C with an increase in the yield of fluorescence especially in the case of F(I).

Journal Article↗
PubMed1979

Proline Oxidase and Water Stress-induced Proline Accumulation in Spinach Leaves.

Spinach (Spinacia oleracea L.) leaf discs accumulated free proline when exposed to polyethylene glycol solutions of water potential less than -10 bars. At -20 bars, the accumulation was 11 micromoles per gram original fresh weight in a 24-hour period.When the leaf organelles were separated on a sucrose gradient, a proline oxidase was detected in the mitochondrial fraction. Isolated mitochondria were used for the study of the properties of the enzyme which was assayed by both oxygen uptake measurement and reduction of 2,6-dichlorophenol-indophenol in the presence of phenazine methosulfate. There was a stoichiometry of one-half mole of oxygen uptake per mole of Delta(1)-pyrroline-5-carboxylate production in the enzymic reaction. The enzyme had an optimal activity at pH 8.0 to 8.5 and an apparent K(m) value of 0.028 molar for proline. MgCl(2) and flavin adenine dinucleotide were required for maximal activity. Addition of sucrose, mannitol, or polyethylene glycol to reduce the water potential of the reaction mixture to as low as -20 bars resulted in little inhibition. The enzyme preparation was unable to reduce NAD to NADH, and NAD did not inhibit the enzyme activity. The enzyme preparation reduced cytochrome c in the presence of KCN. Triton X-100 at low concentration strongly inhibited the enzyme activity. The enzyme was apparently linked to the mitochondrial electron transport system. The in vitro activity of the enzyme under optimal assay conditions was high enough to prevent proline accumulation under water stress condition; presumably this activity was restrained in vivo.

Journal Article↗