scholarly journals Purification and Characterization ofO-Methyltransferase I Involved in Conversion of Demethylsterigmatocystin to Sterigmatocystin and of Dihydrodemethylsterigmatocystin to Dihydrosterigmatocystin during Aflatoxin Biosynthesis

1998 ◽  
Vol 64 (1) ◽  
pp. 166-171 ◽  
Author(s):  
Kimiko Yabe ◽  
Ken-ichiro Matsushima ◽  
Takumi Koyama ◽  
Takashi Hamasaki
Keyword(s):  
Molecular Mass ◽  
Aspergillus Parasiticus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Aflatoxin Biosynthesis ◽  
Purification And Characterization ◽  
Cytosol Fraction ◽  
Apparent Homogeneity

ABSTRACT O-Methyltransferase I, which catalyzes conversions both of demethylsterigmatocystin (DMST) to sterigmatocystin (ST) and of dihydrodemethylsterigmatocystin (DHDMST) to dihydrosterigmatocystin (DHST) during aflatoxin biosynthesis, was purified to apparent homogeneity from the cytosol fraction of the mycelia ofAspergillus parasiticus NIAH-26 through the following chromatography series: phenyl-Sepharose, DEAE-Sepharose, phenyl-Sepharose, Sephacryl S-300, and Matrex gel Green A. The apparent molecular mass was estimated at 150 kDa based on Sephacryl S-300 gel filtration chromatography, and the denaturing molecular mass was 43 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI of the enzyme was 4.4, and the optimal pH for activity was broad, from 6.5 to 9.0. In competition experiments using the purified enzyme, the formation of ST from DMST was suppressed when DHDMST was added to the reaction mixture and DHST was newly formed. These results indicate that DMST and DHDMST commonly serve as substrates for the enzyme. TheKm of the enzyme for DMST was 0.94 μM, and that for DHDMST was 2.5 μM. Interestingly, MT-I kinetics deviated substantially from standard Michaelis-Menten kinetics, demonstrating substrate inhibition at a higher substrate concentration.

scholarly journals Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4

2007 ◽  
Vol 189 (7) ◽  
pp. 2660-2666 ◽  
Author(s):  
Vandana P. Swetha ◽  
Aditya Basu ◽  
Prashant S. Phale
Keyword(s):  
Molecular Mass ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sole Source ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Phenol Derivatives

ABSTRACT Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants Km and V max for 1-naphthol and NADPH were determined to be 9.6 and 34.2 μM and 9.5 and 5.1 μmol min−1 mg−1, respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The Ki for 1-naphthol was determined to be 79.8 μM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.

Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus

1996 ◽  
Vol 42 (8) ◽  
pp. 804-810 ◽  
Author(s):  
Ken-Ichi Kusumoto ◽  
Dennis P. H. Hsieh
Keyword(s):  
Aspergillus Parasiticus ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Preparative Chromatography ◽  
Aflatoxin Biosynthesis ◽  
Anion Exchange Column ◽  
Versiconal Hemiacetal Acetate

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40–55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5–5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatogaphy. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.Key words: aflatoxin biosynthesis, esterase, versiconal hemiacetal acetate, Aspergillus parasiticus.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

Purification and characterization of ferro- and cobalto-chelatases

1988 ◽  
Vol 66 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Eduardo T. Cánepa ◽  
Elena B.C. Llambías
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Purification And Characterization ◽  
Degree Of Unsaturation ◽  
Apparent Homogeneity ◽  
Optimum Ph ◽  
Single Protein ◽  
Metal Insertion

Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40 000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240 000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h. but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 μM for the metal and 30.3 μM for mesoporphyrin. and for cobaltochelatase activity. 27 and 45.5 μM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

1998 ◽  
Vol 64 (2) ◽  
pp. 789-792 ◽  
Author(s):  
Giuliano Degrassi ◽  
Benedict C. Okeke ◽  
Carlo V. Bruschi ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acetyl Xylan Esterase ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Naphthyl Acetate

ABSTRACT Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km ) andV max for α-naphthyl acetate were 1.54 mM and 360 μmol min−1 mg of protein−1, respectively.

scholarly journals Studies on the subunits of human myeloperoxidase

1984 ◽  
Vol 222 (3) ◽  
pp. 701-709 ◽  
Author(s):  
R L Olsen ◽  
C Little
Keyword(s):  
Heat Treatment ◽  
Amino Acid ◽  
Molecular Mass ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Additional Species ◽  
Main Protein

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

scholarly journals O-Acetylserine Sulfhydrylase fromMethanosarcina thermophila

2000 ◽  
Vol 182 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Birthe Borup ◽  
James G. Ferry
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cysteine Biosynthesis ◽  
High Sequence Similarity ◽  
Methanosarcina Thermophila ◽  
Moderately Thermophilic

ABSTRACT Cysteine is the major source of fixed sulfur for the synthesis of sulfur-containing compounds in organisms of the Bacteriaand Eucarya domains. Though pathways for cysteine biosynthesis have been established for both of these domains, it is unknown how the Archaea fix sulfur or synthesize cysteine. None of the four archaeal genomes sequenced to date contain open reading frames with identities to eitherO-acetyl-l-serine sulfhydrylase (OASS) or homocysteine synthase, the only sulfur-fixing enzymes known in nature. We report the purification and characterization of OASS from acetate-grown Methanosarcina thermophila, a moderately thermophilic methanoarchaeon. The purified OASS contained pyridoxal 5′-phosphate and catalyzed the formation of l-cysteine and acetate from O-acetyl-l-serine and sulfide. The N-terminal amino acid sequence has high sequence similarity with other known OASS enzymes from the Eucarya andBacteria domains. The purified OASS had a specific activity of 129 μmol of cysteine/min/mg, with a Km of 500 ± 80 μM for sulfide, and exhibited positive cooperativity and substrate inhibition withO-acetyl-l-serine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at 36 kDa, and native gel filtration chromatography indicated a molecular mass of 93 kDa, suggesting that the purified OASS is either a homodimer or a homotrimer. The optimum temperature for activity was between 40 and 60°C, consistent with the optimum growth temperature for M. thermophila. The results of this study provide the first evidence for a sulfur-fixing enzyme in the Archaea domain. The results also provide the first biochemical evidence for an enzyme with the potential for involvement in cysteine biosynthesis in theArchaea.

scholarly journals Cloning and Characterization of 1-Deoxy-d-Xylulose 5-Phosphate Synthase fromStreptomyces sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis

2000 ◽  
Vol 182 (4) ◽  
pp. 891-897 ◽  
Author(s):  
Tomohisa Kuzuyama ◽  
Motoki Takagi ◽  
Shunji Takahashi ◽  
Haruo Seto
Keyword(s):  
Molecular Mass ◽  
Mevalonate Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Initial Step ◽  
Enzymatic Properties ◽  
Isopentenyl Diphosphate ◽  
Ph Optimum ◽  
Nonmevalonate Pathway

ABSTRACT In addition to the ubiquitous mevalonate pathway,Streptomyces sp. strain CL190 utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. The initial step of this nonmevalonate pathway is the formation of 1-deoxy-d-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. The corresponding gene, dxs, was cloned from CL190 by using PCR with two oligonucleotide primers synthesized on the basis of two highly conserved regions among dxs homologs from six genera. Thedxs gene of CL190 encodes 631 amino acid residues with a predicted molecular mass of 68 kDa. The recombinant enzyme overexpressed in Escherichia coli was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of 9.0, with a V max of 370 U per mg of protein and Km s of 65 μM for pyruvate and 120 μM for d-glyceraldehyde 3-phosphate. The purified enzyme catalyzed the formation of 1-deoxyxylulose by condensation of pyruvate and glyceraldehyde as well, with aKm value of 35 mM ford-glyceraldehyde. To compare the enzymatic properties of CL190 and E. coli DXP synthases, the latter enzyme was also overexpressed and purified. Although these two enzymes had different origins, they showed the same enzymatic properties.

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