title: Purification and characterization of acetohydroxyacid reductoisomerase from spinach chloroplasts.
abstract: Acetohydroxyacid reductoisomerase was purified over 400-fold to a specific activity of 62 nkat.mg-1, with 2-aceto-2-hydroxybutyrate as substrate, from the stroma of spinach leaf chloroplasts. The enzyme was not intrinsically membrane bound. The native enzyme was a tetramer with a subunit Mr of 59,000. The activity was optimum between pH 7.5 and 8.5. The apparent Km for 2-acetolactate was 25 microM and for 2-aceto-2-hydroxybutyrate was 37 microM. The enzyme required Mg2+ and the Vmax. was attained at physiological Mg2+ concentrations. NADP+ competitively inhibited the reaction when NADPH was the varied substrate. The native enzyme eluted from Mono-Q ion-exchange resins as three distinct peaks of activity. This elution pattern was preserved when the peaks were combined, dialysed and re-chromatographed. Each form exhibited identical Mr of 59,000 after SDS/polyacrylamide gel electrophoresis (PAGE), whereas they were easily distinguishable from each other after PAGE under non-denaturing conditions. These results provide evidence for the existence of multiple forms of acetohydroxyacid reductoisomerase in chloroplasts isolated from spinach leaves.
authors.affiliation: Laboratoire de Physiologie Cellulaire Végétale (Unité mixte CNRS Rhône-Poulenc Agrochimie, UM 41), Lyon, France.
authors.forename: R
authors.initials: R
authors.lastname: Dumas
authors.forename: J
authors.initials: J
authors.lastname: Joyard
authors.forename: R
authors.initials: R
authors.lastname: Douce
mesh_terms: D015072:2-Acetolactate Mutase / Q000302:isolation & purification*; D002736:Chloroplasts / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006885:Hydroxybutyrates / Q000378:metabolism; D007535:Isomerases / Q000302:isolation & purification*; D007773:Lactates / Q000378:metabolism; D008274:Magnesium; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism
chemical_list: D006885:Hydroxybutyrates; D007773:Lactates; C027745:alpha-aceto-alpha-hydroxybutyrate; D009249:NADP; C006359:alpha-acetolactate; D007535:Isomerases; D015072:2-Acetolactate Mutase; D008274:Magnesium