Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12

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Abstract
Acetohydroxyacid synthase from E. coli K-12 was purified to near homogeneity. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of 2 polypeptides, 60,000 MW and 9500 MW. These 2 polypeptides were present in constant proportion to each other and to enzyme activity. The molar ratio of the 2 polypeptides (MW 9500:60,000), estimated from stained polyacrylamide gels was 1. Antisera raised against the 60,000 MW polypeptide precipitated both the 60,000 and the 9500 MW polypeptides from extracts of cells labeled with [35S]methionine. The addition of sodium dodecyl sulfate before immunoprecipitation eliminated the smaller polypeptide and only the larger one was recovered. The hydrodynamic properties of the native enzyme confirmed a previous report that the largest enzymatically active species is .apprx. 200,000; this species contains both the 60,000 MW and 9500 Mw polypeptides.