Ferredoxin-sulfite Reductase from Spinach

Ferredoxin-sulfite reductase (Fd-SiR) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from spinach leaves has been purified to homogeneity by a new procedure. Subunit analysis by sodium dodecyl sulfate gel electrophoresis yielded a single protein band with a molecular weight of 71,000. Gel electrophoresis in non-denaturing media at different acrylamide concentrations gave a molecular weight of 270,000, suggesting that the native enzyme was composed of four identical subunits. In the presence of 0.2 m sodium chloride, however, gel filtration produced a value of 136,000, indicating the presence of dimer in this ionic environment. A plot of substrate (sulfite) concentration versus enzymatic (Fd-SiR) activity yielded a sigmoidal curve, giving a Hill coefficient (n?) of 2.1. Purified Fd-SiR, in the oxidized form, had absorption maxima at 279, 385, 588 and 714nm. Thus the enzyme has the property of a siroheme-containing protein.     

Ferredoxin-dependent Nitrite Reductase from Spinach Leaves

A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.

The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A₃₉₀: A₂₇₈ and A₆₇₃: A₃₉₀ were 0.61 and 0.37, respectively.

By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel.     

Studies on the Sulfite Reducing System of Algae

Sulfite reductase using reduced methyl viologen as an electron donor was purified about 94-fold from a red alga, Porphyra yezoensis. The enzyme was ultracentrifugically homogenous and could reduce sulfite to sulfide quantitatively with an uptake of six electrons. The enzyme had a pH optimum in the vicinity of 7.5. The Km for sulfite was determined to be 6.5×l0^?4m. The purified preparation of the algal reductase showed its absorption maximum at 385 mμ and slight shoulders at 408, 456, 485, 600 and 664 mμ in addition to an intense peak at 278 mμ. Metal analysis of the purified enzyme suggested the presence of iron and copper in the molecule. NADPH, NADH or the reduced form of spinach ferredoxin could not be a direct electron donor for the purified algal sulfite reductase.     

Purification and characterization of ferredoxin-sulfite reductases from leek (Allium tuberosum) leaves

Ferredoxin-sulfite reductases (Fd-SiRs) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from leek leaves have been purified to homogeneity. The enzymes (SiR 1, SiR 2 and SiR 3) were separated by Mono Q chromatography. The collective molecular mass of the enzymes was estimated to be 65 kDa by gel filtration. In all three cases, subunit analysis by SDS-PAGE yielded a single protein band corresponding to a molecular mass of 64 kDa, indicating that the enzymes each exist as a monomer. In the oxidized forms, SiR 1, SiR 2 and SiR 3 all exhibited nearly identical absorption maxima at 279∼280, 389∼390, 588 and 714 nm, indicating that siroheme is involved in the catalysis of sulfite reduction. On enzymatic properties, SiR 1, SiR 2 and SiR 3 could only react with the physiological electron donor, feriedoxin. The enzymes exhibited different heat stabilities. The pH active curve obtained from SiR 2 was different from the others. Moreover, SiR 1 exhibited a lower Km value for ferredoxin than SiR 2. Although the N-terminal sequence was the same, the results of some enzymatic properties, amino acid analysis, and peptide mapping suggested the presence of the Fd-SiR isozymes in leek leaves.     

Phosphatidylcholine biosynthesis in castor bean endosperm : purification and properties of cytidine 5'-triphosphate:choline-phosphate cytidylyltransferase

Cytidine 5′-triphosphate:choline-phosphate cytidylyltransferase (EC 2.7.7.15) has been purified to near homogeneity (3350-fold) from castor bean (Ricinus communis L. var Hale) endosperm. The steps of purification included a differential solubilization of this enzyme with n-octyl β-d-glucopyranoside (OGP) and column chromatography on sequential DEAE-sepharose, sepharose-6B, and second DEAE-sepharose columns. The uses of appropriate concentrations of the detergent, OGP, in each step were crucial to obtain the highly purified enzyme. The purified enzyme gave a single protein band on nondenaturing polyacrylamide gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed one major protein band of 40 kilodaltons. Gel filtration chromatography indicated that native cytidylyltransferase was approximately 155 kilodaltons, suggesting that it exists naturally as a tetramer. The purified enzyme used methylethanolamine-phosphate as a substrate but not ethanolamine-phosphate and dimethylethanolamine-phosphate. ATP and other nucleotides tested showed little effect on the purified enzyme. The purified enzyme activity was stimulated by both phospholipids extracted from castor bean endosperm and phosphatidylcholineoleate vesicles.     

Studies on the Sulfite Reducing System of Algae

Sulfite reductase activity by algal extracts was investigated using reduced methylviologen as a hydrogen donor. Sulfite reductase appears to be widely distributed in various algae, but the enzymatic activity was not detected in the brown algae examined. The addition of phosphate buffer to the reaction mixture caused a marked decrease in activity. Sulfite reductase was partially purified from the autolysate of Porphyra tenera and some properties were studied. The optimal pH was 7.5 to 8.5 in Tris-HGl buffer system. The Km for sulfite was 6.65 × 10^?4m. The enzymatic activity was completely inhibited by potassium cyanide at 5 × 10^?4m. The enzyme catalyzed the reduction of sulfite to sulfide. Neither NADPH nor NADH acts as a hydrogen donor. However, it was revealed that ferredoxin can act as an electron carrier in sulfite reduction to sulfide in Porphyra extract.     

Characterization of a Vitamin B12 Compound in the Edible Purple Laver,Porphyra yezoensis

The edible purple laver, Porphyra yezoensis, contained 51.49±1.51 μg of vitamin B₁₂ compounds per 100 g dry weight of the laver (mean±SEM, n=4). A vitamin B₁₂ compound was purified from the lyophilized purple laver and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B₁₂, but not to those of vitamin B₁₂ analogues inactive for humans.     

Purification and Characterization of an Acid Phosphatase from Mycoplasma fermentans

An acid phosphatase associated with the cell membranes of Mycoplasma fermentans was released from the membranes with Triton X-100, then purified by ion-exchange chromatography on DEAE-Sephacel and CM-Sepharose, followed by affinity chromatography on Con A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single band with a molecular mass of 31.2 kilodaltons. The enzyme activity toward p-nitrophenyl phosphate was enhanced remarkably by Cu²⁺, Co²⁺ and Mg²⁺, but the activity was not inhibited by EDTA. The enzyme dephosphorylated O-phospho-l -tyrosine as well as p-nitrophenyl phosphate, but not O-phospho-l -threonine, O-phospho-l -serine, glucose-1-phosphate, phosphoryl choline and adenosine triphosphate. The level of the O-phospho-l -tyrosine phosphatase activity was the highest in Mycoplasma faucium and the second highest in Mycoplasma fermentans of all tested human mycoplasmas.     

Chemical modification studies of tryptophan,arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (E _m) values of the siroheme and [4Fe–4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.     

Purification of phycoerythrin from <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> Ueda (Bangiales,Rhodophyta) using expanded bed absorption

R-phycoerythrin was purified by means of phenyl-sepharose expanded bed absorption and DEAE-sepharose ion-exchange chromatography from Porphyra yezoensis, one of the largest and important aquaculture species in China. Final R-phycoerythrin preparation was characterized by purity ratio above 4 and the homogeneity in native PAGE, respectively. The results of absorption spectrum, fluorescence spectrum and SDS-PAGE were in agreement with previous reports on R-PE. The yield of R-phycoerythrin was 0.82 mg g⁻¹ wet leafy gametophyte of P. yezoensis. This method is a high-protein recovery technology while reducing processing time, and is suitable for the large batch production of R-phycoerythrin, which will enhance the value of P. yezoensis in China, especially the inferior P. yezoensis which can not be used for flake processing.     

The partial purification and characterization of a gibberellin C-20 hydroxylase from immature Pisum sativum L. seeds

A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA₅₃ to GA₄₄ was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 M and the isoelectric point was 5.6–5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe²⁺ stimulated the reaction, but externally added Fe²⁺ was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe²⁺ in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA₅₃ was converted to both GA₄₄ and GA₁₉. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.Abbreviation BSA bovine serum albumin - DEAE diethylaminoethyl - DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Purification and Properties of Sulfite Reductase from Desulfovibrio vulgaris,Miyazaki F

The sulfite reductase of Desulfovibrio vulgaris, strain Miyazaki F (MF), was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Ultrogel AcA34, and hydroxylapatite. The molecular weight was estimated to be 180,000 by gel filtration. It had a subunit structure of α₂β₂; the molecular weight of the α subunit was 50,000 and that of β, 39,000. The absorption spectrum with characteristic peaks at 629 and 409 nm and the amino acid composition resembled those of the sulfite reductase from D. vulgaris, Miyazaki K. The MF enzyme reduced sulfite to trithionate, thiosulfate, and sulfide by hydrogen when coupled with a hydrogenase-methyl viologen system, like other sulfite reductases from Desulfovibrio.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Purification and characterization of an arginine-specific carboxypeptidase from Mycoplasma salivarium.

The carboxypeptidase which had been shown to be present exclusively in nonfermentative mycoplasmas was found to be associated with cell membranes of Mycoplasma salivarium. The enzyme was released from the membranes with Triton X-100 and purified by ion-exchange chromatography on DEAE-Sephacel, affinity chromatography on arginine-Sepharose 4B, and chromatofocusing. The purified enzyme had a molecular mass of 218 kilodaltons, as estimated by gel filtration through Sepharose CL-6B, and yielded one band of activity in analytical disc-polyacrylamide gel electrophoresis performed in the presence of 0.5% (wt/vol) Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme treated in the presence or absence of 2-mercaptoethanol revealed one band with a molecular mass of 87 kilodaltons. The enzyme catalyzed selectively the cleavage of the C-terminal arginine residue of peptides such as N-benzoylglycyl-L-arginine, tuftsin, and bradykinin and was inhibited considerably by o-phenanthroline and EDTA but only slightly by NiCl2. The inhibition of the enzyme by EDTA was fully reversed by the addition of ZnCl2, whereas the addition of CoCl2 activated the enzyme.     

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin was 55°C and the optimal pH for sucrose was 4.75. The apparent K _m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides. The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69. Received 17 November 1999/ Accepted in revised form 30 May 2000     

Purification of pyruvate kinase from germinating castor bean endosperm

Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.     

Purification and properties of cytochrome P-450(11beta) from adrenocortical mitochondria.

A cytochrome P-450, which is functional in the steroid methylene 11β-hydroxylation (P-450_11β), has been purified to a protein weight of 85 kg per heme from bovine adrenocortical mitochondria. The purification is accomplished in the presence of deoxycorticosterone as a substrate stabilizer. The procedure involved solubilization of sonicated mitochondrial pellets, ammonium sulfate fractionation, alumina Cγ gel treatment and aniline-substituted Sepharose 4B chromatography.The purified preparation when freed from deoxycorticosterone, has a low spin type absorption spectrum which can rapidly be converted into a typical high spin substrate-bound form by the addition of an 11β-hydroxylatable steroid, either deoxycorticosterone or testosterone. The preparation exhibits high 11β-hydroxylase activity and is free from the cholesterol side-chain cleavage cytochrome P-450 (P-450_scc).The purified P-450_11β, when submitted to SDS-polyacrylamide gel electrophoresis, exhibits a single protein band (molecular weight of 46 kilodaltons) which is clearly distinguished from P-450_scc. As determined by the sedimentation equilibrium method, the molecular weight of the guanidine-treated P-450_11β is estimated to be 43 kilodaltons.     

Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues

An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The K_m values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP⁺ in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP⁺ oxidoreductase. The effect of NaCl and MgCl₂ concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP⁺ oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues.     

Purification and characterization of X-prolyl dipeptidyl peptidase fromLactobacillus casei subsp.casei LLG

X-Prolyl dipeptidyl peptidase, which hydrolysed X-Pro-Y almost specifically, has been purified to homogeneity from crude cell-free extracts ofLactobacillus casei subsp.casei LLG using fast protein liquid chromatography equipped with preparative and analytical anion exchange columns. The enzyme was purified to 274-fold by ammonium sulphate fractionation, and by two successive ion-exchange chromatographies with a recovery of 34%. The purified enzyme appeared as a single band on both native-polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate (SDS)-PAGE and had a molecular mass of 79 kDa. The pH and the temperature optima by the purified enzyme were 7.0 and 50°C, respectively. X-PDP was a serine-dependent enzyme, as both diisopropylfluorophosphate and phenylmethylsulphonylfluoride caused complete inhibition of the enzyme activity. The Michaelis constant (K _m ) and maximum reaction velocity (V _max ) values were 0.2 mm and 43 mm per milligram, respectively.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.