Glucose 6-Phosphate Dehydrogenase Isozymes of Maize Leaves

Abstract

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (ZC mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP' to take advantage of the corresponding molcular weight inrase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molculr weight 209,000 in the presence of NADPr. The association behavior ofenzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves. Glucose 6-phosphate dehydrogenase of dark-grown maize leaves iso-electric point (pI) 43 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K. of NADP+ being 2.5-fold higher for pI 43 form. Free ATP (K. = 0.64 millimolar) and ADP (K,,= 1.13 millimolar) act as competitive inhibitors with respect to NADPF in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.