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.

scholarly journals Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A.

1988 ◽  
Vol 168 (2) ◽  
pp. 507-525 ◽  
Author(s):  
M Achtman ◽  
M Neibert ◽  
B A Crowe ◽  
W Strittmatter ◽  
B Kusecek ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
The Other ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Electrophoretic Variants ◽  
C Protein ◽  
Neisseria Gonorrhoea ◽  
Terminal Amino ◽  
Protein Variants

Methods published for the purification of P.II proteins from Neisseria gonorrhoea have been modified to allow the purification of class 5 proteins from Neisseria meningitidis serogroup A bacteria. The five class 5 protein electrophoretic variants detected within an epidemic in the Gambia (a, b, c, d, and e) and three other variants (f, g, and h) found within other isolates of the same clone in West Africa have been purified with yields of 6-28 mg. The NH2-terminal amino acid sequence for variant c differs from those of the other class 5 proteins, whereas the latter are very similar to the sequence predicted for two class 5 proteins from DNA analyses of serogroup C meningococci and determined for 8 P.II proteins from gonococci. Numerous other regulatory, chemical, and serological differences were found between the c protein and the other class 5 proteins such that we recommend that the class 5 proteins be subdivided into two subclasses. mAbs have been isolated that distinguish between these two protein subclasses and Western blotting with these antibodies enabled us to conclude that both protein subclasses were found in bacteria isolated from different epidemics and pandemics of the last 50 yr.

Purification and Characterization of Hepatic P-450Iie1 from Acetone-Treated Mice

1993 ◽  
Vol 9 (3) ◽  
pp. 539-546 ◽  
Author(s):  
Vincenzo Longo ◽  
Silvia Menicagli ◽  
Michael Minks ◽  
Annalisa Santucci ◽  
Pier Giovanni Gervasi
Keyword(s):  
Polyclonal Antibodies ◽  
High Spin State ◽  
Purification And Characterization ◽  
Turnover Number ◽  
High Turnover ◽  
Hepatic Microsomes ◽  
Aminoacid Sequence ◽  
Reconstituted System ◽  
Cytochrome P 450

A new cytochrome P-450 isozyme (Mr = 52,000) was purified to apparent electrophoretic homogeneity from hepatic microsomes of mice treated with acetone and its biochemical, spectral, and immunological properties characterized. Several criteria indicated that the purified cytochrome was distinct from the known mouse P-450 isozymes. The absolute spectrum of its oxidized form indicated that it was in the high spin state. In a reconstituted system, it showed low catalytic activities towards 7-ethoxycoumarin, aminopyrine, and coumarin, whereas it catalyzed the oxidation of aniline, acetone, dimelhylnitrosoamine with high turnover number. The mouse enzyme was immunoreactive with polyclonal antibodies against rat P-45011E1 and exhibited an NH2-terminal aminoacid sequence with a high homology to that of rat-P-450IIEI. Based upon the above catalytic, spectral, immunological and structural properties, the purified mouse P-450 appears to be the ortholog of previously described P-450IIE1 (s) of other species.

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.

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.

Combining LC-MS/MS, PMF and N-terminal amino acid sequencing for multiplexed characterization of a bacterial surfactant glycoprotein biosynthesized by Acinetobacter radioresistens S13

RSC Advances ◽  
2014 ◽  
Vol 4 (21) ◽  
pp. 10918-10927 ◽  
Author(s):  
Marta Riva Violetta ◽  
Roberto Mazzoli ◽  
Cristina Barello ◽  
Paolo Fattori ◽  
Maria G. Giuffrida ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Glycosylation ◽  
Experimental Results ◽  
Bacterial Protein ◽  
Amino Acid Sequencing ◽  
Terminal Amino Acid ◽  
Acinetobacter Radioresistens ◽  
Terminal Amino ◽  
The One

The present study has highlighted the mechanisms of bacterial protein glycosylation. Experimental results underline that the consensus sequon can be different from the one found in Eukarya.

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.

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