Purification and Some Catalytic Properties of Glucose-6-Phosphate Dehydrogenase Isoforms from Barley Leaves

The cytosolic and chloroplastic isoforms of glucose-6-phosphate dehydrogenase (G₆PDH) were separated and purified from barley leaves (Hordeum vulgare L.). In etiolated leaves, only the cytosolic isoform was expressed. The molecular mass of the cytosolic enzyme, G₆PDH₁, was 112±8 kDa and that of the chloroplast enzyme, G₆PDH₂, was 136±7 kDa. The K_m values for glucose-6-phosphate and NADP were 0.133 and 0.041 mM for G₆PDH₁, and 0.275 and 0.062 mM for G₆PDH₂, respectively. The pH optimum was 8.2 for G₆PDH₁ and 7.8 for G₆PDH₂. The enzyme is absolutely specific for NADP. NADPH is a competitive inhibitor of the G₆PDH₁ in respect to glucose-6-phosphate (G₆P) and NADP (K_i = 0.050 and 0.025 mM, respectively). NADPH is a competitive inhibitor of the G₆PDH₂ in respect to NADP (K_i = 0.010 mM), but a non-competitive inhibitor in respect to the G₆P. ADP, AMP, UTP, NAD, and NADH had no effect on the activity of G₆PDH. ATP inhibited the G₆PDH₂ activity.