scholarly journals The purification of shikimate dehydrogenase from Escherichia coli

1985 ◽  
Vol 226 (1) ◽  
pp. 217-223 ◽  
Author(s):  
S Chaudhuri ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Shikimate Dehydrogenase ◽  
E Coli ◽  
Gel Permeation

A procedure was developed for the purification of shikimate dehydrogenase from Escherichia coli. Homogeneous enzyme with specific activity 1100 units/mg of protein was obtained in 21% overall yield. The subunit Mr estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 32 000. The native Mr, estimated by gel-permeation chromatography on a TSK G2000SW column, was also 32 000. E. coli shikimate dehydrogenase is therefore a monomeric NADP-linked dehydrogenase.

scholarly journals Purification and characterization of 3-dehydroquinase from Escherichia coli

1986 ◽  
Vol 239 (3) ◽  
pp. 699-704 ◽  
Author(s):  
S Chaudhuri ◽  
J M Lambert ◽  
L A McColl ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.

scholarly journals Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

1983 ◽  
Vol 213 (1) ◽  
pp. 187-191 ◽  
Author(s):  
A Lewendon ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Purification Step ◽  
Phosphate Synthase

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

scholarly journals The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

1977 ◽  
Vol 167 (2) ◽  
pp. 509-512 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Partial Specific Volume ◽  
Triton X

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.

scholarly journals Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12

1981 ◽  
Vol 193 (3) ◽  
pp. 861-867 ◽  
Author(s):  
R H Jackson ◽  
A Cornish-Bowden ◽  
J A Cole
Keyword(s):  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soret Band ◽  
Horse Heart Cytochrome ◽  
E Coli ◽  
Highly Active ◽  
Covalently Bound

A substantially improved purification of Escherichia coli NADH-dependent nitrite reductase was obtained by purifying it in presence of 1 mM-NO2- and 10 microM-FAD. The enzyme was obtained in 20% yield with a maximum specific activity of 1.04 kat . kg-1: more than 95% of this sample subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis migrated as a single band of protein. This highly active enzyme contained one non-covalently bound FAD molecule, and, probably, 5 Fe atoms and 4 acid-labile S atoms per subunit. No FMN, covalently bound flavin or Mo was detected. The spectrum of the enzyme shows absorption maxima at 386, 455, 530 and about 575 nm with a shoulder at 480–490 nm. The Soret-band/alpha-band absorbance ratio is about 4:1. These spectral features are characteristic of sirohaem, apart from the maximum at 455nm, which is attributed to flavin. The enzyme also catalyses the NADH-dependent reduction of horse heart cytochrome c, 2,6-dichlorophenol-indophenol and K3Fe(CN)6. The presence of sirohaem in E. coli nitrite reductase explains the apparent identity of the cysG and nirB gene of E. coli and inability of hemA mutants to reduce nitrite.

scholarly journals The purification and properties of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli and Proteus mirabilis

1971 ◽  
Vol 123 (4) ◽  
pp. 493-500 ◽  
Author(s):  
J. W. Dale ◽  
J. T. Smith
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Resistance Factor ◽  
Ph Optimum ◽  
E Coli ◽  
R Factor ◽  
Purification And Properties

1. The β-lactamase specified by the R-1818 resistance factor in Escherichia coli was purified 300-fold; the resulting preparation gave a single peak on Sephadex G-100 and a single band on polyacrylamide-gel electrophoresis. 2. The β-lactamase specified by the same R-factor in Proteus mirabilis was purified over 2000-fold, but was still far from pure. The specific activity of this preparation was one-fifth that of the purified enzyme from E. coli. 3. The two enzymes were shown to be identical as regards substrate specificity, pH optimum, Km values and molecular weight. 4. It is suggested that the low β-lactamase activity of extracts of P. mirabilis (R-1818), about 5% of that from E. coli (R-1818) in crude extracts, could be due to inefficient transcription of the R-factor DNA by Proteus RNA polymerase.

scholarly journals The composition and the structure of bacterioferritin of Escherichia coli

1981 ◽  
Vol 197 (1) ◽  
pp. 171-175 ◽  
Author(s):  
J Yariv ◽  
A J Kalb ◽  
R Sperling ◽  
E R Bauminger ◽  
S G Cohen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Electron Microscope ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Iron Compound ◽  
Protein Subunit ◽  
Electron Dense Core

Bacterioferritin isolated from Escherichia coli is of two kinds: a protein containing a polynuclear iron compound, the bacterioferritin proper and a protein free of the polynuclear iron compound, the apo-bacterioferritin. Bacterioferritin of both kinds is characterized by absorption maxima at 417,530 and 560 nm, contributed by protohaem IX. Single crystals of bacterioferritin of the space group I432 suggest that the molecule is made up of 24 identical subunits related by a cubic point symmetry. The molecular weight of the protein subunit, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, is 15000. In the electron microscope the bacterioferritin molecule appears to be a sphere of 9.5 nm (95 A) diameter composed of a negatively staining outer shell and an inner electron-dense core of 6 nm (60 A) diameter.

scholarly journals Characterization of an Escherichia coli O157:H7 Plasmid O157 Deletion Mutant and Its Survival and Persistence in Cattle

2007 ◽  
Vol 73 (7) ◽  
pp. 2037-2047 ◽  
Author(s):  
Ji Youn Lim ◽  
Haiqing Sheng ◽  
Keun Seok Seo ◽  
Yong Ho Park ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Differentially Expressed ◽  
Sequence Matching ◽  
Two Dimensional Gel Electrophoresis ◽  
E Coli ◽  
Cell Lysates

ABSTRACT Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic ΔpO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the ΔpO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the ΔpO157 mutant. The ΔpO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the ΔpO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the ΔpO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed ∼50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.

Purification and characterization of an exoglucanase from Streptomyces flavogriseus

1984 ◽  
Vol 30 (9) ◽  
pp. 1171-1178 ◽  
Author(s):  
C. R. Mackenzie ◽  
D. Bilous ◽  
K. G. Johnson
Keyword(s):  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Extracellular Enzymes ◽  
Main Product ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

Streptomyces flavogriseus, a mesophilic actinomycete, produces high levels of extracellular enzymes capable of hydrolyzing cellulose and xylan. One such enzyme, an exoglucanase, has been purified to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel permeation chromatography on Bio-Gel P-60, preparative isoelectric focusing, and concanavalin A affinity chromatography. This purification sequence disclosed the presence of several distinct endoglucanase and xylanase fractions. Homogeneity of the purified enzyme was demonstrated by analytical isoelectric focusing and sodium dodecyl sulphate – polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 45 000 and an isoelectric point of 4.15. The enzyme demonstrated negligible activity with carboxymethylcellulose as the substrate. It was able to extensively hydrolyse acid-swollen cellulose; the main product of enzyme action was cellobiose.

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