Copurification of Cytosolic Fructose-1,6-bisphosphatase and Cytosolic Aldolase from Endosperm of Germinating Castor Oil Seeds

Abstract

The cytosolic isozymes of fructose-1,6-bisphosphatase (FBPase c) and aldolase (ALD c) from germinating castor oil seed endosperm (COS) ( Ricinus communis L.; cv Hale) were purified to homogeneity and final specific activities 49 and 2.8 (μmol product produced/min)/mg protein, respectively. Nondenaturing polyacrylamide gel electrophoresis of the final FBPase c preparation resolved a single protein-staining band which comigrated with FBPase activity. Two protein-staining bands of 41 and 39 kDa that occurred in an approximate 1:1 ratio were observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final FBPase c preparation. Rabbit anti-(FBPase c) immune serum immunoprecipitated the activities of FBPase c, but not that of the plastidic isozyme of FBPase from germinated COS. Immunoblot analysis utilizing affinity purified anti-(COS FBPase c) immunoglobulin G established that the 39-kDa subunit of FBPase c did not arise via proteolytic cleavage of the 41-kDa subunit during tissue extraction and enzyme purification. However, FBPase c was susceptible to degradation by endogenous protease(s) during incubation of an acidic (pH 5.9) clarified COS extract at 25°C. This proteolysis caused the production of a 32-kDa antigenic polypeptide and resulted in FBPase inactivation. Gel filtration indicated that purified FBPase c exists in at least 8 different oligomeric forms ranging in size from >2 million to <34 kDa. The majority of FBPase c, however, eluted as a 143-kDa heterotetramer. Sodium dodecyl sulfate gel electrophoresis of the final ALD c preparation yielded a single 40-kDa protein-staining polypeptide that cross-reacted with anti-(carrot ALD c) IgG. FBPase c copurified with ALD c through polyethylene glycol fractionation, Q-Sepharose, and phosphocellulose chromatographies, and the intensity of the fluorescence emission spectrum of ALD c was greatly reduced in the presence of COS FBPase c, but not rabbit muscle FBPase. These findings suggest that these two metabolically sequential enzymes might specifically interact in the cytosol of the highly gluconeogenic germinating COS. Our results also demonstrate that endogenous nonspecific acid phosphatase activity can interfere with the spectrophotometric assay for FBPase and can thus result in overestimations of FBPase activity in impure plant extracts.