Purification and characterization of a 4-hydroxybenzoate decarboxylase from an anaerobic coculture

2000 ◽  
Vol 46 (9) ◽  
pp. 856-859 ◽  
Author(s):  
Tong Li ◽  
Pierre Juteau ◽  
Réjean Beaudet ◽  
François Lépine ◽  
Richard Villemur ◽  
...  
Keyword(s):  
Gel Chromatography ◽  
Enterobacter Agglomerans ◽  
Anaerobic Conditions ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Cell Extracts ◽  
Sds Page ◽  
Oxygen Sensitive ◽  
Terminal Amino

The oxygen-sensitive 4-hydroxybenzoate decarboxylase (4OHB-DC) activity from a phenol-carboxylating coculture, consisting of Clostridium-like strain 6 and an unidentified strain 7, was studied. Assays done with cell extracts showed that the optimal pH was 5.0-6.5 and the Kmwas 5.4 mM. The activity decreased by 50% in the presence of 5 mM EDTA, and it was restored and even enhanced by the addition of Mg++, Mn++, Zn++, or Ca++. After purification, the molecular mass of the enzyme was estimated as 420 kDa by gel chromatography, and as 119 kDa by SDS-PAGE, suggesting a homotetrameric structure. Its pI was 5.6. The N-terminal amino acid sequence showed 95% and 76% homology with the pyruvate-flavodoxin oxidoreductase (nifJ gene product) from Enterobacter agglomerans and Klebsiella pneumoniae, respectively. The purified enzyme also slowly catalyzed the reverse reaction, that is the phenol carboxylation. These characteristics suggest that this enzyme is different from other known decarboxylases. This includes the 4OHB-DC from Clostridium hydroxybenzoicum, which is the only one that had been purified before.Key words: purification, 4-hydroxybenzoate decarboxylase, coculture, phenol carboxylation, anaerobic conditions.

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

scholarly journals Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats

1986 ◽  
Vol 235 (3) ◽  
pp. 859-868 ◽  
Author(s):  
S L Seidel ◽  
T K Shires
Keyword(s):  
Polyclonal Antibodies ◽  
The Other ◽  
Amino Acid Sequencing ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Spectral Maximum ◽  
Substrate Preferences ◽  
Terminal Amino ◽  
Cytochrome P 450

At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators.

Purification and characterization of three proteins isolated from the proteose peptone fraction of bovine milk

1993 ◽  
Vol 60 (2) ◽  
pp. 189-197 ◽  
Author(s):  
Esben S. Sørensen ◽  
Torben E. Petersen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Bovine Milk ◽  
Gel Chromatography ◽  
Major Protein ◽  
Terminal Amino Acid ◽  
Proteose Peptone ◽  
Sds Page ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

SummaryThree major proteins from the proteose peptone of bovine milk were purified by Sephadex G-75 gel chromatography, Q-Sepharose ion-exchange and additional Sephadex G-75 gel chromatography in the presence of urea. From their mobility in a gradient SDS-PAGE, the proteins were found to have molecular masses of 17, 28 and 60 kDa. The N-terminal amino acid sequence of the 17 kDa protein was found to be homologous with a camel whey protein. This protein has not previously been described in bovine milk. From the SDS-PAGE results, the 28 kDa protein was judged to be the major protein of proteose peptone, contributing ~ 25% of the total. The N-terminal amino acid sequence showed no homology to any known protein sequence, but the amino acid composition indicated that the 28 kDa protein is identical with the PP3 component from the proteose peptone fraction of bovine milk, or part of it. The 60 kDa protein was found to be bovine osteopontin, a very highly phosphorylated protein with an Arg-Gly-Asp sequence which mediates cell attachment.

scholarly journals Purification and characterization of truncated ribonuclease inhibitor

1991 ◽  
Vol 275 (2) ◽  
pp. 541-543 ◽  
Author(s):  
J Hofsteenge ◽  
A Vincentini ◽  
S R Stone
Keyword(s):  
Amino Acid ◽  
Kinetic Parameters ◽  
Surface Properties ◽  
Ribonuclease A ◽  
Amino Acid Residues ◽  
Ribonuclease Inhibitor ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino

A recombinant pig ribonuclease inhibitor (delta r-RI) lacking 90 or 93 N-terminal amino acid residues was isolated from a preparation of recombinant inhibitor. The kinetic parameters for the inhibition of ribonuclease A by delta r-RI were determined and found to be only slightly altered in comparison with the full-length inhibitor. The deletion did, however, affect the surface properties of RI. The results are discussed in relation to those obtained by Lee & Vallee [(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883].

scholarly journals Purification and characterization of a labile rat glutathione transferase of the Mu class

1989 ◽  
Vol 260 (3) ◽  
pp. 789-793 ◽  
Author(s):  
A Kispert ◽  
D J Meyer ◽  
E Lalor ◽  
B Coles ◽  
B Ketterer
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Glutathione Transferase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Cnbr Cleavage

A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.

scholarly journals Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum

1989 ◽  
Vol 261 (3) ◽  
pp. 973-977 ◽  
Author(s):  
L D Smith ◽  
N Budgen ◽  
S J Bungard ◽  
M J Danson ◽  
D W Hough
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Glucose Dehydrogenase ◽  
Sulfolobus Solfataricus ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Thermoplasma Acidophilum ◽  
Catalytically Active ◽  
Terminal Amino

Glucose dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme is a tetramer of polypeptide chain Mr 38,000 +/- 3000, it is catalytically active with both NAD+ and NADP+ cofactors, and it is thermostable and remarkably resistant to a variety of organic solvents. The amino acid composition was determined and compared with those of the glucose dehydrogenases from the archaebacterium Sulfolobus solfataricus and the eubacteria Bacillus subtilis and Bacillus megaterium. The N-terminal amino acid sequence of the Thermoplasma acidophilum enzyme was determined to be: (S/T)-E-Q-K-A-I-V-T-D-A-P-K-G-G-V-K-Y-T-T-I-D-M-P-E.

A Novel Fibrinogen-clotting Enzyme, TL-BJ, from the Venom of the Snake Bothrops jararaca: Purification and Characterization

2000 ◽  
Vol 83 (03) ◽  
pp. 438-444 ◽  
Author(s):  
Claudio Sampaio ◽  
Reinhard Mentele ◽  
Antonio Camargo ◽  
Edwin Fink ◽  
Solange Serrano
Keyword(s):  
Amino Acid Sequences ◽  
Amidolytic Activity ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Bothrops Jararaca ◽  
Sds Page ◽  
Competitive Inhibitors ◽  
Molecular Masses ◽  
Terminal Amino ◽  
Serine Endopeptidases

SummaryThree chromatographically distinct forms of a novel fibrinogenclotting serine endopeptidase, TL-BJ 1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-ArgpNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.

scholarly journals Purification and characterization of acidic glutathione S-transferase 6 from human brain

1991 ◽  
Vol 274 (2) ◽  
pp. 405-408 ◽  
Author(s):  
T Suzuki ◽  
D C Shaw ◽  
P G Board
Keyword(s):  
Human Brain ◽  
Minor Component ◽  
Terminal Sequence ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
A Minor ◽  
Additional Member

An acidic glutathione S-transferase (GST) isoenzyme termed GST6 has been isolated from human brain, characterized and compared with other isoenzymes. The N-terminal amino acid sequence of GST6 was found to be identical with that of GST4 previously purified from human muscle. GST6 cross-reacted with antibody raised against GST4, but not with antisera raised against GST1, GST2 or GST3. The subunit Mr and pI of GST6 were found to be different from those of GST4. The present results indicate that GST6 is another member of the Mu evolutionary class which in man also includes GST1, GST4 and GST5. A minor component that co-purified with GST6 was shown to have an N-terminal sequence similar to, but not identical with, that of GST3. This isoenzyme may be an additional member of the Pi evolutionary class.

Purification and characterization of a Baeyer–Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways

2000 ◽  
Vol 347 (3) ◽  
pp. 693-701 ◽  
Author(s):  
Mariët J. VAN DER WERF
Keyword(s):  
Rhodococcus Erythropolis ◽  
Degradation Pathways ◽  
Rearrangement Product ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Prosthetic Group ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMKMO) is a monomeric enzyme of molecular mass 60 kDa. It contains 1 mol of FAD/monomer as the prosthetic group. The N-terminal amino acid sequence showed homology with many other NADPH-dependent and FAD-containing (Type 1) BVMOs. Maximal enzyme activity was measured at pH 9 and 35 °C. MMKMO has a broad substrate specificity, catalysing the lactonization of a large number of monocyclic monoterpene ketones and substituted cyclohexanones. The natural substrates 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone were converted stoichiometrically into 3-isopropenyl-6-oxoheptanoate (the spontaneous rearrangement product of the lactone formed by MMKMO), 4-isopropenyl-7-methyl-2-oxo-oxepanone and 7-isopropyl-4-methyl-2-oxo-oxepanone respectively. The MMKMO-catalysed conversion of iso-dihydrocarvone showed an opposite regioselectivity to that of dihydrocarvone; in this case, 6-isopropenyl-3-methyl-2-oxo-oxepanone was formed as the product. MMKMO converted all enantiomers of the natural substrates with almost equal efficiency. MMKMO is involved in the conversion of the monocyclic monoterpene ketone intermediates formed in the degradation pathways of all stereoisomers of three different monocyclic monoterpenes, i.e. limonene, (dihydro)carveol and menthol.

Purification and Characterization of Three Thermostable Endochitinases of a Noble Bacillus Strain, MH-1, Isolated from Chitin-Containing Compost

1998 ◽  
Vol 64 (9) ◽  
pp. 3397-3402 ◽  
Author(s):  
Kenji Sakai ◽  
Akira Yokota ◽  
Hajime Kurokawa ◽  
Mamoru Wakayama ◽  
Mitsuaki Moriguchi
Keyword(s):  
Culture Fluid ◽  
Amino Acid Sequences ◽  
Diaminopimelic Acid ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Molecular Masses ◽  
Bacterium Strain ◽  
Terminal Amino ◽  
Temperature Optima

ABSTRACT A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin-containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2,6-O-dimethyl)-β-cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genusBacillus. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized fromBacillus sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75°C) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)6], chitinases L and S produced (GlcNAc)2 at the highest rate while chitinase M produced (GlcNAc)3 at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)2. Chitinase L produced (GlcNAc)2 and (GlcNAc)3 in most abundance from 66 and 11% partially acetylated chitosan. Thep-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)2, and those of chitinases M and S were highest toward pNP-(GlcNAc)3. All three enzymes were inert to pNP-GlcNAc. AgCl, HgCl2, and (GlcNAc)2inhibited the activities of all three enzymes, while MnCl2and CaCl2 slightly activated all of the enzymes.

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