EVIDENCE FOR THE PRESENCE OF THE ASCORBATE-GLUTATHIONE CYCLE IN MITOCHONDRIA AND PEROXISOMES OF PEA LEAVES

The presence of the enzymes of the ascorbate-glutathione cycle was inv estigated in mitochondria and peroxisomes purified from pea (Pisum sat ivum L.) leaves. All four enzymes, ascorbate peroxidase (APX; EC 1.11. 1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate r eductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), were pr esent in mitochondria and peroxisomes, as well as in the antioxidants ascorbate and glutathione. The activity of the ascorbate-glutathione c ycle enzymes was higher in mitochondria than in peroxisomes, except fo r APX, which was more active in peroxisomes than in mitochondria. Inta ct mitochondria and peroxisomes had no latent APX activity, and this r emained in the membrane fraction after solubilization assays with 0.2 M KCI. Monodehydroascorbate reductase was highly latent in intact mito chondria and peroxisomes and was membrane-bound, suggesting that the e lectron acceptor and donor sites of this redox protein are not on the external side of the mitochondrial and peroxisomal membranes. Dehydroa scorbate reductase was found mainly in the soluble peroxisomal and mit ochondrial fractions. Glutathione reductase had a high latency in mito chondria and peroxisomes and was present in the soluble fractions of b oth organelles. In intact peroxisomes and mitochondria, the presence o f reduced ascorbate and glutathione and the oxidized forms of ascorbat e and glutathione were demonstrated by high-performance liquid chromat ography analysis. The ascorbate-glutathione cycle of mitochondria and peroxisomes could represent an important antioxidant protection system against H2O2 generated in both plant organelles.