Abstract
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 Mg2+ for activity and the inhibition by fluoride in the presence of phosphate are quantitatively very similar for all enolases. Other catalytic parameters (Km, Vmax, 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.
Highlights
The molecular weight of E. coli enolase was determined by sedimentation-diffusion measurements and by sedimentation equilibrium
When the method was applied to E. coli enolase, with bovine catalase and rabbit muscle aldolase as reference proteins a subunit molecular weight of 46,000 f 1,000 was calculated as the average of three duplicate gels
The results indicate that E. coli enolase is very similar to the other enolases in both physical and catalytic properties
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. There are many similarities between E. coli enolase and other enolases studied. Other catalytic parameters (I&, VIllS.X,and pH optimum) are similar, but minor quantitative distinction indicates that E. coli enolase is more closely related to yeast enolase than to enolases from vertebrate muscle. Most of the well characterized enolases are from vertebrate muscle, considerable work has been done on yeast enolase (for a recent review, see Reference I). Very similar subunits in enolases from as widely different sources as yeast, fish, and mammals. Little work has been done on bacterial enolases [2, 3], and this study was undertaken to purify and characterize the enzyme from one bacterial species, Escherichia coli. Preliminary work with E. coli enolase [3], suggested that E. co& would be a reasonable source of the enzyme. E. coli is commercially available in quantities sufficient for large scale purification
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