Purification and comparative properties of the cytosolic isocitrate dehydrogenases (NADP) from pea (Pisum sativum) roots and green leaves.
The cytosolic isocitrate dehydrogenases (NADP-IDH) were purified to homogeneity from pea roots and green leaves with a high yield by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephacryl S-200 gel filtration, Matrex red-A affinity chromatography and phenyl-Superose HR 5/5 HPLC. Both isoenzymes were dimeric proteins, consisting of two apparently identical 41-kDa subunits, having similar secondary structures with an alpha-helical content of 20% and a beta-pleated sheet content of 43%. Similarly immunoassays suggested that the two isoenzymes were closely related in terms of antigenic determinants. However, the two proteins were distinguishable by their electrophoretic mobilities and amino acid compositions. The profiles of the two isoenzymes as a function of pH were similar and exhibited a broad pH optimum from 8.5 to 9.0 with Mg2+ as cofactor and 8.0 to 8.5 when Mn2+ was used. Compared to the root isoenzyme, the leaf NADP-IDH appeared to be more heat-labile. However, these isoenzymes exhibited similar behavior for thermal denaturation in the presence of bovine serum albumin and were stabilized upon addition of substrate, metal and coenzyme. Their values of activation energy were estimated as 47 kJ/mol. When using Mn2+ as cofactor, the two isoenzymes displayed identical Km(Mn2+), Km(DL-isocitrate) and Km(NADP) values, which were calculated to be 2.1 microM, 5.7 microM and 2.7 microM respectively. With Mg2+ as cofactor, their Km(Mg2+) K(DL-isocitrate)m and Km(NADP) values were also not statistically different, being 34.0 microM, 15.2 microM and 2.6 microM for the root NADP-IDH, and 29.0 microM, 20.3 microM and 3.1 microM for the leaf isoenzyme. From the above data it can be concluded that although the cytosolic NADP-IDH in pea roots and leaves are organ-specific isozymes, their similar physicochemical and kinetic properties suggest that the two isozymes might be involved in identical metabolic functions.
D000596:Amino Acids / Q000032:analysis; D002848:Chromatography, DEAE-Cellulose; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010946:Plants, Medicinal; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013696:Temperature
D000596:Amino Acids; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)