Lamellar superoxide dismutase of isolated chloroplasts

Abstract

Photooxidation of hydroxylamine to nitrite by spinach (Spinacia oleracea L. and sugarbeet (Beta vulgaris L.) chloroplast lamellae in the presence of autoxidable electron acceptors is inhibited by either solubilized or membrane-bound superoxide dismutase (SOD). This inhibition is reversed by KCN. The rates of hydroxylamine photooxidation by chloroplast lamellae, a reaction which is apparently driven by the superoxide free-radical ion, was used for quantitating the amount of SOD bound to chloroplast lamellae, as compared to a soluble enzyme of defined concentration. After digitonin fragmentation of chloroplast lamellae, ca. 80% of the SOD activity is associated with subchloroplast particles sedimenting after 2 h centrifugation at 200 000 x g. Less than 10% of the SOD activity is associated with particles sedimenting after centrifugation for 30 min at 20 000 x g. 5-10% of the cyanide-sensitive SOD is recovered in the soluble fraction of the subchloroplast-free supernatant after centrifugation at 200 000 x g for 2 h.

Deactivation of superoxide dismutase on EDTA‐treated chloroplasts

“…To corroborate this interpretation, another assay for O~-was applied; the oxidation of hydroxylamine to nitrite [ 17]. Table 1 shows the rates of nitrite production for control and EDTA-treated chloroplasts, respectively, under continuous illumination.…”

“…The chloroplasts were illuminated for 5 min. Afterwards they were precipitated by heat and the nitrite was assayed in the supernatant by the method of Elstner and Heupel [17]. ing during reduction of 02 to O~-.…”

“…For the measurements a 2 cm cuvette was used which contained 15 ml of the following reaction mixture: 10 mM KCI, 30 ~tM cresol red and 67/IM benzylviologen as artificial electron acceptor. The formation and determination of nitrite was assayed using the procedure of Elstner et al [ 14,17]. Superoxide dismutase was purchased from Miles-Seravac.…”

Superoxide formation in pea chloroplasts by a dioxathiadiaza‐2,5‐pentalene derivative, a new lipophilic

“…Addition of cyanide increased the rate of formation of DMPO-OOH, in both presence and absence of HEP II ( fig.5a,c). This is probably due to an inhibition of the endogenous SOD activity [ 11,121. The effect of cyanide was not due to an effect on the rate of photosynthetic electron flow (not shown).…”

“…Hz02 is generated by the dismutation of superoxide, catalysed by the enzyme SOD associated with PS I [11,12].…”