Purification and some catalytic properties of glucose-6-phosphate dehydrogenase isoforms from barley leaves

The cytosolic and chloroplastic isoforms of glucose-6-phosphate dehydrogena se (G(6)PDH) were separated and purified from barley leaves (Hordeum vulgar e L.). In etiolated leaves, only the cytosolic isoform was expressed. The m olecular mass of the cytosolic enzyme, G(6)PDH(1), was 112 +/-8 kDa and tha t of the chloroplast enzyme, G(6)PDH(2), was 136 +/-7 kDa. The K-m values f or glucose-6-phosphate and NADP were 0.133 and 0.041 mM for G(6)PDH(1), and 0.275 and 0.062 mM for G(6)PDH(2), respectively. The pH optimum was 8.2 fo r G(6)PDH(1) and 7.8 for G(6)PDH(2). The enzyme is absolutely specific for NADP. NADPH is a competitive inhibitor of the G(6)PDH(1) in respect to gluc ose-6-phosphate (G(6)P) and NADP (K-i = 0.050 and 0.025 mM, respectively). NADPH is a competitive inhibitor of the G(6)PDH(2) in respect to NADP (K-i = 0.010 mM), but a non-competitive inhibitor in respect to the G(6)P. ADP, AW, UTP, NAD, and NADH had no effect on the activity of G(6)PDH. ATP inhibi ted the G(6)PDH(2) activity.