CHARACTERIZATION OF ADPGLUCOSE PYROPHOSPHORYLASE FROM A STARCH-DEFICIENT MUTANT OF ARABIDOPSIS-THALIANA (L)

A starch-deficient mutant of Arabidopsis thaliana (L.) has only approximately 5% of ADPglucose pyrophosphorylase activity but accumulates approximately 40% of starch as the wild-type. ADPglucose pyrophosphorylase from the mutant and wild-type leaves was partially purified and characterized. The activity of each enzyme was inhibited by an antibody raised against purified spinach-leaf ADPglucose pyrophosphorylase. When analyzed by Western-blot hybridization after SDS-PAGE, the partially purified mutant enzyme was deficient in the larger of the two subunits observed in the wild-type enzyme. However, by Western-blot hybridization after native PAGE, the mutant enzyme demonstrated two equally stained cross-reactive bands instead of a single band for the wild-type enzyme. Of the mutant enzyme activity, > 95% was found in the lower band. The mass of the native enzyme from either wild-type or mutant was approximately 210 kD, as determined by gel filtration, and those of the subunits were approximately 54 and 48 kD for the wild-type enzyme and 48 kD for the mutant enzyme. The mutant ADPglucose phyrophosphorlase, just as the wild-type enzyme, was activated allosterically by 3-P-glycerate and inhibited by P(i). However, the mutant enzyme required higher concentrations of 3-P-glycerate for maximal activation and was more sensitive to inhibition by P(i) than the wild-type enzyme. In the presence of saturating concentrations of 3-P-glycerate, the K(m) values for the mutant enzyme for ATP, Glc-1-P, and Mg2+ were approximately 6-, approximately 5-, and approximately 2-fold higher, respectively, than those of the wild-type enzyme. Changes in the kinetics of the mutant enzyme may be due to a deficiency of one of the two subunits, and/or modification of the remaining subunit.