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 micromol 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 pI value 7.2. The native Mr was 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 microM), and a forward:reverse Vmax ratio 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 ALDc concentration and (b) the latter stages of COS maturation and germination are accompanied by marked reductions in ALD activity and ALDc concentration. The most significant elevation in ALDc concentration occurred over the first 4 days of germination when COS initiates the gluconeogenic conversion of P-enolpyruvate and triose-P, derived from reserve triacylglycerols, into the sucrose required to support early seedling growth.
Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
R J
RJ
Hodgson
W C
WC
Plaxton
D000596:Amino Acids / Q000032:analysis; D002367:Ricinus communis / Q000196:embryology / Q000201:enzymology*; D002412:Cations; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000302:isolation & purification*; D018525:Germination; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000737:chemistry; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010947:Plants, Toxic; D012639:Seeds / Q000196:embryology / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D002412:Cations; D007106:Immune Sera; D005634:Fructose-Bisphosphate Aldolase