Purification, Properties, and Distribution of Ascorbate Peroxidase in Legume Root Nodules

Abstract

All aerobic biological systems, including N2-fixing root nodules, are subject to 02 toxicity that results from the formation of reactive intermediates such as H202 and free radicals of 02. H202 may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN Oxygen plays a critical but incompletely understood role in the metabolism of N2-fixing root nodules. Nodule function requires a delicate balance between respiratory 02 demand and the hazards of 02 toxicity. These hazards arise from the 02 sensitivity of nitrogenase and from the production of reactive intermediates such as H202 and superoxide free radicals (02*). A key defense against 02 toxicity is superoxide dismutase, an enzyme which has been detected in many aerobic organisms, including nodule host cells (21) and bacteroids (6). The superoxide dismutase reaction produces H202 which can also be damaging and must be removed through the action of catalase or peroxidase. Although catalase is present in soybean nodules (11,21), most peroxide removal probably occurs through a coupled series of oxidation-reduction reactions involving ASC3 and glutathione (5, Fig. 1 content of nodules and the activity of the first two enzymes in this system are positively correlated with nitrogenase activity and leghemoglobin content during the early stages of nodule development (5). ASC peroxidase and DHA reductase activities are not present in isolated bacteroids, thus implying these enzymes are of plant origin (5).All three of the enzymes in this system in chloroplasts have been purified and characterized (1,12,14), but very little information is available on the corresponding enzymes in root nodules. A peroxidase from soybean root nodules was characterized by Puppo et al. (20), but the effectiveness of ASC as an electron donor was not investigated. Legume root nodules reportedly contain numerous peroxidase isozymes (16), but this may be misleading because leghemoglobins are capable of very high pseudo-peroxidase activity in the presence of appropriate reductants (20).We present evidence in this report of the presence of ASC peroxidase and associated enzymes in nine species of legumes and in red alder (Alnus rubra). ASC peroxidase from soybean root nodules was purified to near homogeneity and characterized with regards to substrate specificity, substrate affinity, absorption spectra, and effects of inhibitors. The enzyme is compared with other peroxidases, especially the ASC peroxidase from spinach chloroplasts.

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

“…This agrees with what has been reported for other APX isozymes (Chen and Asada, 1989;Mittler and Zilinskas, 1991). In its substrate specificity, the mitochondrial and the peroxisomal APX seem to be different from the chloroplastic APX isozyme from pea leaves, which is highly specific for ascorbate as an electron donor (Mittler and Zilinskas, 1993), but they are more similar to the pea cytosolic APX (Mittler and Zilinskas, 1991) and to the APX I from tea leaves (Chen and Asada, 1989) and root nodules (Dalton et al, 1987). All of these APX isozymes have a broader spectrum of donor specificity than the chloroplastic APX.…”

Purification and Characterization of Pea Cytosolic Ascorbate Peroxidase

“…homodimer with a subunit Mr 29,500; the subunits do not associate via disulfide bonds (data not shown). Previous studies with partially purified cytosolic APX from tea, spinach, and legume root nodules indicated native mol wts of 57,000, 48,000, and 47,000, respectively (5,7,21). The enzyme from legume root nodules was also reported to have a subunit Mr 30,000 when assayed on SDS-PAGE (7).…”

“…The chloroplastic enzyme was found to have a higher specificity for ascorbate and a narrow pH optimum and is very labile in the absence of ascorbate. The cytosolic APX has been purified to near homogeneity from Euglena gracilis (26) and soybean root nodules (7). However, previous attempts to purify the cytosolic APX to homogeneity from pea shoots, tea, and spinach leaves were unsuccessful (5, 9, 21); therefore, there are insufficient data regarding the properties of cytosolic APX isolated from green tissue of higher plants.…”

Ascorbate is the natural substrate for plant peroxidases

“…described in the thylakoid membranes [8] but was subsequently found to exist in both soluble cytosolic and plastidic forms [9][10][11]. APX activity has been found to be present in all higher plant species examined so far [12] and has been isolated from Euglena gracilis [13], spinach [14], soybean root nodules [15], pea [ 16] and tea leaves [ 17]. Like horseradish peroxidase (H R P).…”