Purification and Characterization of Cytosolic Fructose-1,6-bisphosphate Aldolase from Endosperm of Germinated Castor Oil Seeds

Cytosolic fructose-1,6-bisphosphate (FBP) aldolase (ALDc) from the endosperm of 4- to 5-day germinated castor oil seeds (COS) has been purified 83-fold to electrophoretic homogeneity and a final specific activity of 2.5 μmol FBP cleaved/min/mg protein. SDS–PAGE and denaturing isoelectric focusing of the final preparation revealed a single protein-staining band of 40 kDa and pIvalue 7.2. The nativeMrwas determined by gel-filtration chromatography and multiangle laser light scattering to be 160–175 kDa, indicating that the enzyme is homotetrameric. The enzyme (a) is a class I aldolase, since EDTA or Mg2+had no effect on its activity; and (b) was relatively heat stable and had an activation energy of 100 kJ/mol. It exhibited a broad pH-activity optima of 7.2, a relatively high affinity for FBP (Km= 0.16 μM), and a forward:reverseVmaxratio of 0.77. Rabbit anti-(COS ALDc) antibodies cross-reacted with COS ALDc, but not with the corresponding plastidic isozyme. Time-course studies revealed that (a) the increase in total ALD activity that occurs during COS development and early germination coincides with an increase in ALDcconcentration and (b) the latter stages of COS maturation and germination are accompanied by marked reductions in ALD activity and ALDcconcentration. The most significant elevation in ALDcconcentration occurred over the first 4 days of germination when COS initiates the gluconeogenic conversion ofP-enolpyruvate and triose-P, derived from reserve triacylglycerols, into the sucrose required to support early seedling growth.