Purification and Characterization of Acetolactate Synthase from Barley

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branchedchain amino acids, valine, leucine, and isoleucine. ALS is the target site for several structually diverse classes of herbicides including sulfonylureas, imidazolinones. and triazolopyrimidines. We have purified ALS from etiolated barley shoots to homogeneity. The five major purification steps are ammonium sulfate fractionation, DEAE anion exchange, hydroxylapatite, Bio-Gel A gel filtration, and low pressure Mono-Q chrornatoqraphy. Approximately 170-fold purification was achieved and the yield was 0.45% of initial activity in the crude extract. Both SDS-PAGE and Western blot analysis showed a single polypeptide of ALS with an apparent molecular mass of 64 kDa. The result of nondenaturing gel electrophoresis with activity staining indicated that the molecular mass of its native form is approximately 225 to 250 kDa. The values of $K_m$ for pyruvate. pl. and optimum pH of ALS were determined to be 2.0 mM, 5.2. and 7.0. respectively Feedback inhibition studies showed that ALS is more susceptible to leucine than valine. And $IC_{50}$ value of Cadre, a class of irnidazolinones, is about $1.5\mu{M}$ for ALS.