A DEHYDROGENASE-MEDIATED RECYCLING SYSTEM OF NADPH IN PLANT PEROXISOMES

The presence of the two NADP-dependent dehydrogenases of the pentose p hosphate pathway has been investigated in plant peroxisomes from pea ( Pisum sativum L.) leaves. Both enzymes, glucose-6-phosphate dehydrogen ase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), were present in the matrix of leaf peroxisomes, and thei r kinetic properties were studied. G6PDH and 6PGDH showed a typical Mi chaelis-Menten kinetic saturation curve, and had specific activities o f 12.4 and 29.6 mU/mg protein, respectively. The K-m values of G6PDH a nd 6PGDH for glucose 6-phosphate and for B-phosphogluconate were 107.3 and 10.2 mu M, respectively. Dithiothreitol did not inhibit G6PDH act ivity. By isoelectric focusing of peroxisomal matrices, the G6PDH acti vity was resolved into three isoforms with isoelectric points of 5.55, 5.30 and 4.85. The isoelectric point of peroxisomal 6PGDH was 5.10. I mmunoblot analyses of peroxisomal matrix with an antibody against yeas t G6PDH revealed a single cross-reactive band of 56 kDa. Post-embedmen t, EM immunogold labelling of G6PDH confirmed that this enzyme was loc alized in the peroxisomal matrices, the thylakoid membrane and matrix of chloroplasts, and the cytosol. The presence of the two oxidative en zymes of the pentose phosphate pathway in plant peroxisomes implies th at these organelles have the capacity to reduce NADP(+) to NADPH for i ts re-utilization in the peroxisomal metabolism. NADPH is particularly required for the ascorbate-glutathione cycle, which has been recently demonstrated in plant peroxisomes [Jimenez, Hernandez, del Rio and Se villa (1997) Plant Physiol. 114, 275-284] and represents an important antioxidant protection system against H2O2 generated in peroxisomes.