The Purification and Characterization of Escherichia coli Enolase

Enolase has been purified from aqueous extracts of Escherichia coli acetone powder by ( a ) heat treatment, ( b ) fractionation with acetone, ( c ) TEAE-cellulose chromatography, ( d ) Sephadex G-100 chromatography, and ( e ) crystallization. The purified, crystalline enzyme migrates as a single ban...

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Bibliographic Details
Published in:The Journal of biological chemistry 1971-11, Vol.246 (22), p.6797-6802
Main Authors: Spring, T G, Wold, F
Format: Article
Language:English
Subjects:
Acetone
Amino Acids - analysis
Binding Sites
Catalysis
Chemical Precipitation
Chromatography, Gel
Chromatography, Ion Exchange
Crystallization
Detergents
Electrophoresis, Disc
Escherichia coli - enzymology
Fluorine
Hot Temperature
Hydro-Lyases
Hydrogen-Ion Concentration
Kinetics
Macromolecular Substances
Magnesium
Methods
Molecular Weight
Peptides - analysis
Phosphopyruvate Hydratase - analysis
Phosphopyruvate Hydratase - antagonists & inhibitors
Phosphopyruvate Hydratase - isolation & purification
Species Specificity
Sulfuric Acids
Ultracentrifugation
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Summary:Enolase has been purified from aqueous extracts of Escherichia coli acetone powder by ( a ) heat treatment, ( b ) fractionation with acetone, ( c ) TEAE-cellulose chromatography, ( d ) Sephadex G-100 chromatography, and ( e ) crystallization. The purified, crystalline enzyme migrates as a single band in disc gel electrophoresis and is homogeneous by ultracentrifugal analysis. The molecular weight of the enzyme is approximately 90,000, as determined by sedimentation velocity and sedimentation equilibrium experiments. The subunit molecular weight estimated by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate is 46,000, suggesting that the enzyme is composed of two subunits of equal size. Functionally there are many similarities between E. coli enolase and other enolases studied. Thus, the dependence on Mg 2+ for activity and the inhibition by fluoride in the presence of phosphate are quantitatively very similar for all enolases. Other catalytic parameters ( K m , V max , and pH optimum) are also similar, but minor quantitative distinction indicates that E. coli enolase is more closely related to yeast enolase than to enolases from vertebrate muscle.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)45916-4