The citrate-stimulated starch synthase of starchy maize kernels: Purification and properties

Chromatography of maize kernel extracts on DEAE-cellulose resolves two fractions of starch synthase activity, one of which (starch synthase 1) is capable of synthesizing α-glucan in the absence of exogenous primer and the presence of 0.5 m citrate (J. L. Ozbun, J. S. Hawker, and J. Preiss, Plant Physiol. (1971) 48, 765–769). This starch synthase has been purified 200-fold from developing kernels of normal maize, and shown to have no detectable activities of branching enzyme, amylase, pullulanase, phosphorylase, and D enzyme. The preparation, however, was not electrophoretically homogeneous. This preparation had a K_m value of 0.033 mm for ADPglucose in the presence of 0.5 m citrate. The reaction in the presence of citrate was stimulated 10-fold by the addition of excess purified branching enzyme. This stimulation is higher than those reported previously (C. D. Boyer and J. Preiss, Plant Physiol. (1979) 64, 1039–1042) but is consistent with the predicted effects of removal of amylase activity. The effects of salts other than citrate on activity in the absence of exogenous primer were small, but the stimulation could be restored by the addition of excess purified branching enzyme. Citrate increased the affinity of the enzyme for the endogenous primer present to such a level that no effect of exogenous primer on reaction rate could be observed in the presence of 0.5 m citrate. Analysis of the glucan/iodine complex and the enzymatic breakdown products patterns from the products of the starch synthase reaction indicates a high degree of linearity. The results obtained are discussed in relation to the biosynthesis of starch in vivo.