Mitochondrial and peroxisomal ascorbate peroxidase of pea leaves

The isoenzyme pattern and the substrate specificity of the membrane-bound m itochondrial and peroxisomal ascorbate peroxidases (APX; EC 1.11.1.11) from pea leaves are studied. The substrate specificity of both APXs was assayed using the electron donors ascorbate and pyrogallol, whereas a-dianisidine, hydroquinone, tetramethylbenzidine and 4-methoxy-alpha-naphthol were also assayed with mitochondrial APX (mitAPX). In leaf mitochondria, the specific activity of APX was similar with pyrogallol and ascorbate, the activity be ing inhibited by p-CMS. mitAPX showed low activity with the guaiacol peroxi dase (GPX)-type substrates, tetramethylbenzidine and 4-methoxy-alpha-naphth ol. Activity of mitAPX with hydroquinone suggest a potential role of mitAPX in the drainage of electrons from the mitochondrial electron chain at the level of ubiquinone. In peroxisomes, the APX (perAPX) specific activity was much higher with pyrogallol than with ascorbate. This perAPX was more sens itive to incubation with Triton X-100 than the mitAPX. By native PAGE the m itAPX was resolved in 6 isoenzyme bands, and the activity of the 3 main ban ds (mitAPX III, III' and IV) was inhibited by p-CMS. These 3 major isozymes were also present in mitochondrial membrane fractions. Staining for GPX ac tivity with 4-methoxy-alpha-naphthol revealed that the APX detected in mito chondria did not have the capacity to oxidize 4-MN, and therefore cannot be considered as true GPX. When intact peroxisomes and peroxisomal membranes were subjected to native PAGE, no APX activity could be detected and this w as probably due to the inactivation of perAPX. Results obtained suggest tha t pea mitochondrial APX (mitAPX) represent a distinct and novel isozyme dif ferent from those APXs of chloroplast and cytosolic origin previously repor ted. The peroxisomal APX (perAPX), however, appears to ressemble the chloro plast APXs as regards its sensitivity to Triton X-100.