Properties of formate dehydrogenase from Desulfovibrio gigas

1986 ◽  
Vol 32 (5) ◽  
pp. 430-435 ◽  
Author(s):  
Mary Ann Riederer-Henderson ◽  
Harry D. Peck Jr.
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Ph Optimum ◽  
Benzyl Viologen ◽  
Desulfovibrio Gigas ◽  
Diaphorase Activity ◽  
Lag Period ◽  
Molecular Radius

The formate dehydrogenase from extracts of Desulfovibrio gigas was partially purified to a specific activity of 5600 nmol CO2 ∙ min−1 ∙ mg protein−1. Uniquely for a formate dehydrogenase from anaerobes, the enzyme was stable when stored aerobically. Nevertheless, thiols were required in the assay mixture for enzymatic activity. If the enzyme first catalyzed the transfer of electrons from thiols to benzyl viologen (a diaphorase activity), then formate was oxidized rapidly without a lag period. The enzyme had a molecular weight of approximately 240 000, a pH optimum of 7.5–8.0, and a temperature optimum of 56 °C. Activity with cytochrome c3 (molecular radius (Mr) = 13 000) was about twice that with ferredoxin or flavodoxin as the electron acceptor. These results suggest that the formate dehydrogenase from D. gigas can be activated by transferring electrons from thiols to an electron acceptor and uses cytochrome c3 as the natural electron carrier for the oxidation of formate.

scholarly journals Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans

1977 ◽  
Vol 161 (2) ◽  
pp. 357-370 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Transport Chain ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.

In vitro requirements for formate dehydrogenase activity from Desulfovibrio

1986 ◽  
Vol 32 (5) ◽  
pp. 425-429 ◽  
Author(s):  
Mary Ann Riederer-Henderson ◽  
Harry D. Peck Jr.
Keyword(s):  
Methylene Blue ◽  
Cytochrome C ◽  
Dehydrogenase Activity ◽  
Electron Acceptor ◽  
Formate Dehydrogenase ◽  
Direct Reduction ◽  
Protein S ◽  
Benzyl Viologen

In Desulfovibrio the protein(s) involved in formate dehydrogenase activity have not been identified or characterized. In situ assays in polyacrylamide gels demonstrated that formate dehydrogenase from either D. gigas or D. vulgaris catalyzed the direct reduction of either methylene blue or benzyl viologen in the presence of formate. Thus, the same protein was active with either electron acceptor. Although the enzyme could be stored in air without irreversible inactivation by O2, activity with either dye was stimulated by the addition of thiols to the assay mixture. In the absence of formate the thiols served as a substrate for the in situ reduction of methylene blue or benzyl viologen by the enzyme. Ammonium sulfate fractionation revealed the presence of a fraction which selectively stimulated activity with either benzyl viologen or cytochrome c3 as the electron acceptor. The stimulating fraction was nondialyzable, heat labile, and unstable upon storage. The fraction from either species could stimulate the formate dehydrogenase activity of the other species. The protein may be of physiological signficance as it increased when the cells were grown on formate, and it stimulated the formate hydrogenlyase system with cytochrome c3 as the electron carrier.

Enzymes involved in the metabolism of thiosulfate by Thiobacillus thioparus. II. Properties of adenosine-5′-phosphosulfate reductase

1970 ◽  
Vol 48 (3) ◽  
pp. 344-354 ◽  
Author(s):  
Ronald M. Lyric ◽  
Isamu Suzuki
Keyword(s):  
Molecular Weight ◽  
Cytochrome C ◽  
Electron Acceptor ◽  
Desulfovibrio Desulfuricans ◽  
Thiobacillus Denitrificans ◽  
Ph Optimum ◽  
Thiobacillus Thioparus ◽  
Aps Reductase

Adenosine-5′-phosphosulfate (APS) reductase was purified from Thiobacillus thioparus extracts 25- to 46-fold and the properties were studied. The molecular weight was 170 000 and the enzyme had 1 mole of FAD, 8–10 moles of iron, and 4–5 moles of labile sulfide. Cytochrome c as well as ferricyanide served as the electron acceptor. The pH optimum shifted from 7.4 to 9.5 when cytochrome c was used instead of ferricyanide. The Km values for sulfite and AMP were reduced from 2.5 mM and 100 μM to 17 μM and 2.5 μM, respectively, with cytochrome c as electron acceptor. Properties of the T. thioparus enzyme were compared to those of APS reductase isolated from Thiobacillus denitrificans and Desulfovibrio desulfuricans.

AN ISOPHENOXAZINE SYNTHASE FROM PYGNOPORUS COCCINEUS (FR.) BOND. AND SING.

1964 ◽  
Vol 42 (11) ◽  
pp. 1515-1526 ◽  
Author(s):  
P. M. Nair ◽  
L. C. Vining
Keyword(s):  
Electron Acceptor ◽  
Specific Activity ◽  
Fold Increase ◽  
Maximum Activity ◽  
Flavin Mononucleotide ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Hydroxyanthranilic Acid ◽  
Pycnoporus Coccineus ◽  
Citrate Phosphate

Mycelium from cultures of the red polypore Pycnoporus coccineus (Fr.) Bond. and Sing, contains an enzyme which catalyzes the oxidative condensation of 2 molecules of 2-aminophenol to yield 2-amino-3H-isophenoxazin-3-one. Fractionation of the crude extract has given a preparation with an 893-fold increase in specific activity. The purified enzyme has a pH optimum in citrate-phosphate buffer of 5.0, and a temperature optimum of 55°. The Km value is 4.35 × 10−4 M. FMN and Mn++ ions were required for maximum activity. FAD also served as an electron acceptor. Of the metal ions tested only Mn++ activated the reaction. Hg++ and Fe++ inhibited strongly. The course of the reaction when cofactors were added separately suggested that flavin mononucleotide is the initial electron acceptor and that Mn++ ions are required for reoxidation of the flavin. The enzyme has narrow specificity, and does not catalyze the oxidation of 3-hydroxyanthranilic acid, 3-hydroxykynurenine, or pyrocatechol.

scholarly journals CO oxidoreductase from Streptomyces strain G26 is a molybdenum hydroxylase

1988 ◽  
Vol 250 (2) ◽  
pp. 605-612 ◽  
Author(s):  
J M Bell ◽  
J Colby ◽  
E Williams
Keyword(s):  
Cytochrome C ◽  
Specific Activity ◽  
Superoxide Radicals ◽  
Native Enzyme ◽  
Benzyl Viologen ◽  
Streptomyces Strain ◽  
Cytochrome C Reduction ◽  
Acid Labile

CO oxidoreductase was purified to 95% homogeneity from crude mycelial extracts of Streptomyces G26. The purified preparation has a specific activity of 25.7 units/mg, a 13-fold improvement on crude soluble mycelial extracts. The native enzyme (Mr 282,000) is composed of non-identical subunits of Mr 110,000 and 33,000. It is a molybdenum hydroxylase containing 1.6 mol of FAD, 7.3 mol of Fe, 8.3 mol of acid-labile sulphide and 1.3 mol of Mo per mol of enzyme. Purified CO oxidoreductase catalyses the reduction of benzyl viologen, confirming the previously reported ability of this enzyme to interact with low-potential acceptors. Cytochrome c reduction cannot be accounted for entirely by non-enzymic reduction by superoxide radicals. NAD+ and NADP+ are not reduced, nor is clostridial ferredoxin.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

Poly(β-L-malate) hydrolase from Plasmodia of Physarum polycephalum

1995 ◽  
Vol 41 (13) ◽  
pp. 192-199 ◽  
Author(s):  
Christian Korherr ◽  
Michael Roth ◽  
Eggehard Holler
Keyword(s):  
Hydrophobic Interaction ◽  
Malic Acid ◽  
Physarum Polycephalum ◽  
Culture Medium ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Reduced Form ◽  
Serine Esterase ◽  
Hydroxybutyric Acid ◽  
Ph Optimum

A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmax and the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.

scholarly journals Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

1991 ◽  
Vol 279 (3) ◽  
pp. 727-732 ◽  
Author(s):  
G B Sala-Newby ◽  
A K Campbell
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Live Cells ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

scholarly journals Purification and characterization of cytosolic pyruvate kinase from banana fruit

2000 ◽  
Vol 352 (3) ◽  
pp. 875-882 ◽  
Author(s):  
William L. TURNER ◽  
William C. PLAXTON
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Banana Fruit ◽  
Ph Optimum ◽  
Cytosolic Ph ◽  
Pep Carboxylase ◽  
Sds Page ◽  
Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

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