Inactivation and degradation of CuZn-SOD by active oxygen species in wheat chloroplasts exposed to photooxidative stress.

Changes in CuZn-SOD activity and content in isolated wheat chloroplasts under the light, and the involvement of protease(s) and/or active oxygen species in this process were studied. Both SOD activity and content decayed with exposure time to photooxidative stress. Ascorbate, a H2O2 scavenger, prevented photooxidation-associated inactivation of SOD, while benzoate, a .OH scavenger, prevented SOD degradation. Wheat chloroplasts incubated in the dark did not hydrolyze exogenous or endogenous SOD, either H2O2-pretreated or not. Protease inhibitors did not prevent SOD degradation under photooxidative treatment, suggesting that plastid protease(s) did not participate in this process. Purified chloroplast CuZn-SOD was exposed to H2O2 and O2- or .OH-generating systems. O2- had no effect on either SOD activity or stability (estimated by native PAGE). H2O2 up to 700 microM inhibited SOD in a dose-dependent manner and induced charge/mass changes as seen by native PAGE. .OH also reduced SOD activity by inducing its fragmentation. High levels of active oxygen, as can be generated under strong stress conditions, could directly inactivate and degrade chloroplastic SOD.

Proteolytic Activity at Alkaline pH in Oat Leaves, Isolation of an Aminopeptidase.

Proteolytic activity in oat leaf extracts was measured with both azocasein and ribulose bisphosphate carboxylase (Rubisco) as substrates over a wide range of pH (3.0-9.2). With either azocasein or Rubisco activity peaks appeared at pH 4.8, 6.6, and 8.4. An aminopeptidase (AP) which hydrolyzes leucine-nitroanilide was partially purified. Purification consisted of a series of six steps which included ammonium sulfate precipitation, gel filtration, and two ionic exchange chromatographies. The enzyme was purified more than 100-fold. The apparent K(m) for leucine-nitroanilide is 0.08 millimolar at its pH optimum of 8.4. AP may be a cystein protease since it is inhibited by heavy metals and activated by 2-mercaptoethanol. Isolated chloroplasts were also able to hydrolyze leucine-nitroanilide at a pH optimum of 8.4, indicating that AP could be localized inside the photosynthetic organelles.