scholarly journals A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli

1974 ◽  
Vol 139 (2) ◽  
pp. 391-398 ◽  
Author(s):  
Susan Wilkinson ◽  
Jeremy R. Knowles
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Binding Constants ◽  
Trna Synthetase ◽  
Subunit Structure ◽  
Filtration Method ◽  
E Coli

The purification of valyl-tRNA synthetase from Bacillus stearothermophilus is described. The protein was greater than 90% homogeneous on polyacrylamide-gel electrophoresis after more than 850-fold purification. It has a molecular weight of 110000, and no evidence was found for the presence of subunit structure. The properties of the purified enzyme were compared with those of purified valyl-tRNA synthetase from Escherichia coli. The thermal stability, pH-stability and dependence of activity on the temperature and pH of the assay are reported. The two enzymes recognize and charge tRNAVal from crude tRNA of the mesophile E. coli and of the thermophile B. stearothermophilus, indiscriminately. The gel-filtration method was extended to measure the binding of tRNA to synthetase directly. Binding constants for tRNAVal to valyl-tRNA synthetase from B. stearothermophilus were determined between 5° and 60°C.

scholarly journals Purification of the constituent enzyme fractions of mycobacillin synthetase

1986 ◽  
Vol 235 (1) ◽  
pp. 81-85 ◽  
Author(s):  
S K Ghosh ◽  
N K Mukhopadhyay ◽  
S Majumder ◽  
S K Bose
Keyword(s):  
Gel Electrophoresis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Sucrose Density Gradient Centrifugation ◽  
Final Purification

The final purification of the three-fraction enzyme complex mycobacillin synthetase was done by hydroxyapatite column chromatography and sucrose-density-gradient centrifugation; each of the fractions obtained migrates as a single component in SDS/polyacrylamide-gel electrophoresis and gel electrofocusing. The Mr of the enzyme fractions A, B and C by gel filtration is 260 000, 190 000 and 105 000, and that by SDS/polyacrylamide-gel electrophoresis is 252 000, 198 000 and 108 000 respectively. None of the enzyme fractions appears to possess subunit structure.

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 Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli

1974 ◽  
Vol 143 (2) ◽  
pp. 285-294 ◽  
Author(s):  
Yarlagadda S. Prasada Rao ◽  
Joseph D. Cherayil
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Chemical Modification ◽  
Gel Filtration ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
E Coli ◽  
Chemical Reagents ◽  
Mild Conditions ◽  
Trna Species

35S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H2O2, NH2OH, I2, HNO2, KMnO4 and NaIO4, under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH2OH, I2 and NaIO4 reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 2-thiocytidine (s2C) and 2-methylthio-N6-isopentenyladenosine (ms2i6A), to various extents. CNBr, HNO2 and NaHSO3 reacted with s4U, mnm5s2U and s2C, but not with ms2i6A. KMnO4 and H2O2 were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of 35S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.

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 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 properties of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli

1967 ◽  
Vol 105 (1) ◽  
pp. 17-24 ◽  
Author(s):  
R. L. Heinrikson ◽  
B S Hartley
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Distinct Species ◽  
Active Species ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Ribonuclease I ◽  
Free Strain

1. Methionyl-t-RNA synthetase (where t-RNA denotes ‘soluble’ or transfer RNA) has been purified to apparent homogeneity from a ribonuclease I-free strain of Escherichia coli. Polyacrylamide-gel electrophoresis of the final product revealed a single band. The purified enzyme catalyses the exchange of 450μmoles of pyrophosphate into ATP/mg. in 15min. at 37°. 2. Methionyl-t-RNA synthetase is specific for the l-isomer of methionine, but appears to catalyse the methionylation of two distinct species of t-RNA, both of which are specific for methionine, but only one of which may be subsequently formylated. 3. The Michaelis constant for l-methionine is 2×10−4m in the ATP–PPi exchange assay and 2×10−5m for the acylation of t-RNA. 4. Gel filtration of both crude and highly purified preparations of methionyl-t-RNA synthetase on Sephadex G-200 indicates that the active species of enzyme has a molecular weight of about 190000. The amino acid composition of the enzyme is similar to those reported for the isoleucine and tyrosine enzymes from E. coli.

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.

scholarly journals Subunit structure of Vicia graminea anti-(blood-group N) lectin

1980 ◽  
Vol 189 (1) ◽  
pp. 185-188 ◽  
Author(s):  
M J Prigent ◽  
R Bourrillon
Keyword(s):  
Blood Group ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Terminal Sequence ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Covalently Linked

The subunit of the Vicia graminea lectin with blood-group-N specificity was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in 6M-guanidinium chloride, and its molecular weights was found to be 25 000. The unique N-terminal sequence fof the first nine residues of the lectin confirmed that Vicia lectin consists of four identical chains non-covalently linked. Finally the microheterogeneity of the lectin shown by analytical isoelectric focusing is discussed.

scholarly journals Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase

1980 ◽  
Vol 191 (1) ◽  
pp. 209-219 ◽  
Author(s):  
J Hughes ◽  
G Mellows
Keyword(s):  
Escherichia Coli ◽  
Exchange Reaction ◽  
Rat Liver ◽  
Equilibrium Dialysis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Trna Synthetase ◽  
Stable Complex ◽  
E Coli ◽  
Escherichia Coli B

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9′-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9′-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.

scholarly journals Clonal analysis of thelacoperons fromKlebsiellaM5a1 and the Lac plasmid (pRE2) fromKlebsiellaV9A

1989 ◽  
Vol 54 (2) ◽  
pp. 85-91
Author(s):  
F. E. Hitchin ◽  
E. C. R. Reeve
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Clonal Analysis ◽  
Coliform Bacterium ◽  
Multicopy Plasmid ◽  
Plasmid Pbr322 ◽  
Small Segment ◽  
E Coli ◽  
The Stability

SummaryThe chromosomallacregion of the coliform bacteriumKlebsiellaM5al was cloned into the multicopy plasmid pBR322 to give pHE7 and pHE8. pHE8 contains 12·6 kb of M5al DNA, including its completelacoperon, and pHE7 contains 2·5 kb of M5al DNA and includes the completelac Ygene and a small segment oflacZ. The M5al operon has the same gene order as theEscherichia coli lacoperon. Thelacgenes of the Lac plasmid ofKlebsiellaV9A were cloned into pBR322 to give pHE1 and pHE2, of approximately 39 and 43 kb. Both plasmids were unstable in anE. coli RecA- strain, in contrast to the stability of pHE8. Polyacrylamide gel electrophoresis tests suggested that the M5a1 β-galactosidase monomer is about 5% longer, i.e. has about 50 more amino acids, than that of theE. coli Zgene. Tests made on the enzymes coded by thelacoperons of M5a1, anotherKlebsiellastrain (V9A) and its resident Lac plasmid, and several Lac+Enterobacteria, led to the conclusion that onlyEscherichia coliamong the Enterobacteria contains an activelacAgene.

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