Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1. 1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. A lfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose a nd Reactive Blue agarose chromatography, and led to 60-fold and 150-fold pu rification for the two enzymes, respectively. The Glc6PDH I isoform account s for 17% of total activity of the enzyme in roots, and is very sensitive t o the effects of NADP(+)/NADPH ratio and dithiothreitol; the Glc6PDH 2 isof orm is less affected by reducing power and represents 83% of the total acti vity. The isoforms showed distinct pH optima, isoelectric points, K-m for g lucose-6-phosphate and a different electrophoretic mobility. The kinetic pr operties for the two enzymes were affected by ATP and metabolites. Both enz ymes are inhibited to different extents by ATP when magnesium is omitted fr om the assay mixture, whereas the addition of ATP-Mg2+ had no effect on Glc 6PDH activities. The Glc6PDH isoforms are usually present in the plastids a nd cytosol of plant cells. To verify the intracellular locations of the enz ymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location, the enzyme purified fr om the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots.