Light-Dependent Reduction of Hydrogen Peroxide by Ruptured Pea Chloroplasts

Abstract

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H202-dependent oxidation of ascorbate and ascorbate-dependent reduction of H202. The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This To date, detailed studies on the mechanism(s) of 02 evolution coupled to H202 reduction have not been presented. Here, we report such studies using ruptured chloroplasts. Reaction 5 is also consistent with catalase activity. Although catalase is predominantly a peroxisomal enzyme (13), residual catalase activity is invariably associated with isolated chloroplast preparations (1). Accordingly, evidence is presented which excludes the possibility that catalase activity is involved in the operation of reaction 5 in ruptured chloroplasts, in the light. MATERIALS AND METHODSChemicals. Chemicals were obtained from the sources described previously (16). H202 and t-butyl hydroperoxide concentrations were determined by titration with standardized KMnO4. The GSH contained 6% GSSG (16).Chloroplast Preparation. Unwashed, sonicated, or osmotically shocked chloroplasts were prepared as described previously (15, 16) from pea seedlings (Pisum sativum cv. Massey Gem). In the studies reported here, the unwashed chloroplasts were repeatedly washed (4 times) with a washing medium (16)

Purification and Characterization of Pea Cytosolic Ascorbate Peroxidase

“…The isolated APX was found to catalyze the oxidation of pyrogallol and guaiacol; however, when H202 concentrations were low, the enzyme had a higher activity with ascorbate as an electron donor. In agreement with previous studies (16), no activity was detected when ruptured chloroplasts were assayed with either pyrogallol or guaiacol as electron donor (Table III). In contrast to the purified APX, HRP displays a distinct preference for pyrogallol and guaiacol as electron donors (Table III).…”

“…A universal buffer (30) was used to assay the activity in the pH range of 4 to 10. The broad pH optimum (5-8) for activity of the purified cytosolic APX enzyme is shown in Figure 5; in contrast, APX activity, derived from ruptured pea chloroplasts, had a different pH optimum of about pH 8, also reported by Jablonski and Anderson (16 ,ug of HRP (Fig. 6).…”

“…7). (10,16) and thus attributed to the chloroplastic isoform. Using a different procedure for intact chloroplast isolation, wherein at least 90% of the chloroplasts was determined to be intact, we have found that only 40% of total APX activity was confined to chloroplasts.…”

“…Chloroplastic APX is very labile in the absence of ascorbate (16,21). The stability of the purified pea cytosolic APX was, therefore, compared with that of HRP and APX activity obtained after rupture of intact chloroplasts from pea.…”

The response of sensitive and tolerant clones of Populus tremuloides to dynamic ozone exposure under controlled environmental conditions

“…Continuous efflux of AA from the cytosol to the apoplast was found in studies of many species (Castillo & Greppin, 1988 ;Luwe et al, 1993 ;Luwe, 1996) and a rapid decrease of AA in the apoplast is evidence of a direct reaction between AA and free radicals. Ascorbic acid, glutathione (GSH), and the enzymes ascorbate peroxidase, glutathione reductase and superoxide dismutase are also known antioxidants which scavenge and remove toxic products before cellular damage occurs in the chloroplast (Jablonski & Anderson, 1982 ;Sakaki et al, 1983 ;Halliwell & Gutteridge, 1989). The difference between two aspen clones in response to O $ can be considered a biochemical characteristic internal to the leaf.…”