Ascorbate Free-Radical Reduction by Glyoxysomal Membranes

Abstract

Glyoxysomal membranes from germinating castor bean (Ricinus communis L. cv Hale) endosperm contain an NADH dehydrogenase. This enzyme can utilize extraorganellar ascorbate freeradical as a substrate and can oxidize NADH at a rate which can support intraglyoxysomal demand for NAD@. NADH:ascorbate free-radical reductase was found to be membrane-associated, and the activity remained in the membrane fraction after lysis of glyoxysomes by osmotic shock, followed by pelleting of the membranes. In whole glyoxysomes, NADH:ascorbate free-radical reductase, like NADH:ferricyanide reductase and unlike NADH: cytochrome c reductase, was insensitive to trypsin and was not inactivated by Triton X-100 detergent. These results suggest that ascorbate free-radical is reduced by the same component which reduces ferricyanide in the glyoxysomal membrane redox system. NADH:ascorbate free-radical reductase comigrated with NADH:ferricyanide and cytochrome c reductases when glyoxysomal membranes were solubilized with detergent and subjected to rate-zonal centrifugation. The results suggest that ascorbate free-radical, when reduced to ascorbate by membrane redox system, could serve as a link between glyoxysomal metabolism and other cellular activities.A membrane-bound NADH dehydrogenase has been demonstrated in the glyoxysome (9, 1 1 Both 13-oxidation and the glyoxylate cycle produce NADH which does not have direct access to the mitochondrial electron transport system. Rat liver peroxisomes have been shown to be permeable to NADH through a nonspecific pore-forming protein (30). However, NADH does not appear to pass through the glyoxysomal membrane (7). This cofactor must, therefore, be reoxidized within the glyoxysome in order to maintain a constant supply of NAD+ to 13-oxidation and the glyoxylate cycle. A malate-aspartate shuttle has been proposed to serve this function (19), but the necessary movements of malate and aspartate between glyoxysomes and mitochondria have not been specifically demonstrated. We have hypothesized that intraglyoxysomal NADH could be oxidized by the membrane dehydrogenase, which could then transfer the electrons to an extraglyoxysomal acceptor (8,16).Ferricyanide or Cyt c can be used as artificial electron acceptors for the glyoxysomal membrane NADH dehydrogenase (1 1). Ascorbate free-radical may function as a cytosolic acceptor in this system because of its favorable reduction potential with respect to NADH (E'. = 0.06 V for the half reaction, dehydroascorbate --ascorbate, compared to E o = -0.32 V for the half reaction, NAD+ --NADH), and because of its apparent ubiquity in plant cells (13). Ascorbate freeradical can act as an electron acceptor and, upon reduction to ascorbate, as an electron donor which is readily oxidized by mitochondria, by a cytosolic oxidase (18), or by spontaneous oxidation. It functions as a redox buffer in both plant (13,18,22,23) and animal systems (2, 12, 25, 31) and does not readily cross membranes (27).This study was undertaken to assess the suitability of ascorbate free-ra...

Evidence for the Presence of the Ascorbate-Glutathione Cycle in Mitochondria and Peroxisomes of Pea Leaves

“…In glyoxysomal membranes, MDHAR appears to have a tuans-membrane orientation similar to that found on the outer mitochondria membranes in chromaffin vesicles (Bowditch and Donaldson, 1990;Njus and Kelley, 1993). Latency experiments of intact pea mitochondria and peroxisomes showed a high latency of MDHAR activity (about 90%) in both organelles, suggesting that the electron acceptor and donor sites of MDHAR activity are not in a cis orientation in the external side of the outer mitochondrial membrane and the cytosolic side of the peroxisomal membrane.…”

“…The inhibition of mitochondrial and peroxisomal APX by aminotriazole has also been observed in other APX isozymes (Foyer et al, 1994). In glyoxysomal membranes from castor bean endosperm, Bowditch and Donaldson (1990) demonstrated the presence of membrane-associated MDHAR activity and found that this activity was due to the 32-kD glyoxysomal membrane protein responsible for the ferricyanide reductase activity previously characterized in those membranes (Luster et al, 1988). Recently, the integral membrane polypeptides of pea leaf peroxisomes were characterized (López-Huertas et al, 1995), and the 32-kD membrane polypeptide was identified as a flavoprotein with NADH: ferricyanide reductase activity, which is also able to generate NADH-dependent 0;-radicals (LÓpez-Huertas et al, 199613).…”

“…This suggests that the MDHAR activity detected in this work in the matrix side of pea leaf peroxisomal membranes is the 32-kD membrane polypeptide previously characterized in the same membranes. It has been proposed that the trans-membrane protein MDHAR can oxidize NADH on the matrix side of the peroxisomal membrane and transfer the reducing equivalents as electrons to the acceptor monodehydroascorbate on the cytosolic side of the membrane (Luster and Donaldson, 1987;Bowditch and Donaldson, 1990). In this process molecular O, could also be an electron acceptor, with the concomitant formation of 0;-radicals (López-Huertas et al, 1996b).…”

“…The evidence reported in this work of the presente of APX and MDHAR in leaf peroxisomal membranes suggests a dual complementary function in peroxisomal metabolism of these membrane-bound antioxidant enzymes. The first function could be to reoxidize endogenous NADH to maintain a constant supply of NAD' for peroxisomal metabolism, an idea that was originally postulated for the membrane-bound NADH dehydrogenase of glyoxysomes from castor bean (Ricinus communis) endosperm (Fang et al, 1987;Luster and Donaldson, 1987;Bowditch and Donaldson, 1990). A second function of the membrane antioxidant enzymes could be protection against H,O, leaking out of peroxisomes.…”

“…The scavenging of H,O, was thought to be carried out exclusively by CAT, which is quite abundant in peroxisomes, and, therefore, the ascorbate-glutathione cycle was not expected to be localized in peroxisomes. However, an APX isoenzyme has been recently identified in membranes of pumpkin (Cucurbita pepo) glyoxysomes and peroxisomes (Yamaguchi et al, 1995) and cotton (Gossypium hirsutum) glyoxysomes (Bunkelmann and Trelease, 1996), and the presence of MDHAR activity was also detected in membranes of oilseed glyoxysomes (Bowditch and Donaldson, 1990;Bunkelmann and Trelease, 1996). Nevertheless, neither the occurrence of ascorbate and glutathione nor a mechanism for the production or regeneration of ascorbate (ascorbate-glutathione cycle) in leaf peroxisomes has been reported.…”