Biochemistry of the cytosolic isozymes of fructose-1,6-bisphosphatase and fructose-1,6-bisphosphate aldolase from germinated castor oil seeds

Cytosolic fructose-1,6-bisphosphatase (FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar}) is the first regulated step in the gluconeogenic conversion of phosphoenolpyruvate (PEP) to hexose-phosphates in germinating castor oil seed (COS). FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} was purified 3000-fold from COS endosperm to a specific activity of 72 units/mg protein and electrophoretic homogeneity. Polyacrylamide gel electrophoresis and gel filtration indicated that FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} exists as a heterotetramer composed of 41- and 39-kDa subunits. Experiments on the effect of polyethylene glycol (PEG) on the structure and activity of FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} in dilute solution revealed that PEG: (i) stabilized the active 140-kDa heterotetrameric structure, (ii) led to a 3-fold decrease in the enzyme's {dollar}K{bsol}sb{lcub}m{rcub}{dollar} (fructose-1,6-bisphosphate (F-1,6-P{dollar}{bsol}sb2{dollar})), (iii) increased substrate inhibition, and (iv) enhanced intrinsic fluorescence.; Cytosolic F-1,6-P{dollar}{bsol}sb2{dollar} aldolase (Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar}) co-purified with FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} to near homogeneity and was homogeneous following separation from FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} during hydrophobic chromatography. Co-purification of Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} and FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} and an observed decrease in COS Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar}'s intrinsic fluorescence in the presence of FBPase suggested a specific interaction between the two sequential enzymes. However, analytical multi-angle laser light scattering (MALLS), gel filtration, protease protection and 'far western' analyses did not detect formation of a high molecular weight complex when the purified enzymes were recombined at their estimated in vivo concentrations in the COS cytosol. The 40 kDa Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} subunit cross-reacted with anti-(carrot Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar}) IgG. Gel filtration and MALLS indicated the enzyme is 160-175 kDa homotetramer. Specific activities of 2.50 and 3.23 units/mg protein for cleavage and synthesis of F-1,6-P{dollar}{bsol}sb2{dollar} (respectively), give this enzyme unusually high relative glycolytic activity. Time-course studies indicated a close correlation between total Ald activity and Ald{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} concentration during COS development and germination. The results suggest that the synthesis of ALD{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} is highly regulated in germinated COS, and that this regulation follows a preset developmental program.; Quantitative changes in COS FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} intrinsic fluorescence due to ligand binding were used to elucidate a model for the regulation of FBPase{dollar}{bsol}sb{lcub}{bsol}rm c{rcub}{dollar} by its ligands. It is hypothesized that a pronounced allosteric transition mediated by AMP binding increases access of F-1,6-P{dollar}{bsol}sb2{dollar} and F-2,6-P{dollar}{bsol}sb2{dollar} to a common inhibitory binding site that overlaps with the enzyme's catalytic site.