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Acetolactate Synthase from Barley (Hordeum vulgare L.): Purification and Partial Characterization

Abstract Acetolactate synthase (EC 4.1.3.18; ALS) has been extracted from etiolated barley shoots (Hordeum vulgare L.) and purified to near homogeneity. Purification was made possible by a fivestep procedure using hydrophobic interaction, gel filtration, anion-exchange and hydroxylapatite chromatography, the last two steps performed with an HPLC- and FPLC-system, respectively. A 300-fold purification was achieved representing 13% of the initial activity in the crude extract; only small amounts of pure acetolactate synthase could be isolated. Although the enzyme was found labile during the chromatographic steps, purified ALS maintained its activity for several hours and could be stored at 70 K for weeks with a 15-30% loss. The apparent molecular weights of the enzymatically active species as determined by gel filtration were about 440 kDa and 200 kDa, respectively. We assume these species are no isozymes but different polymeric forms of a basic unit of ALS. SDS-PAGE analysis showed one polypeptide with an apparent molecular weight of 58 kDa. Preliminary enzymatic characterization of the purified enzyme confirms a marked synergism in the feedback control by branched-chain amino acids. The combination of valine plus leucine exhibited the most co-operative inhibition.