Identification and activity of superoxide-producing protein complexes of the plasma membrane of etiolated maize seedlings subjected to low positive temperature

Kinetics of superoxide anion generation by the isolated plasma membrane was determined by the rate of formazan formation from XTT in the presence of NADPH or NADH. The plasma membrane was prepared from (control) etiolated maize seedlings grown at 25°C and from (cooled) seedlings incubated at 6°C for the last day. Membrane vesicles from the control plants possessed superoxide-producing activity, and the rate of NADH oxidation was markedly higher than that of NADPH. The low-temperature incubation of the seedlings suppressed the NADPH-dependent activity, whereas the NADH-dependent one slightly increased. The solubilized by dodecyl maltoside (DDM) plasma membranes were separated into multiprotein complexes by high-resolution clear native electrophoresis (hrCN-PAGE). The aim was to find complexes exhibiting the superoxide-producing activity sensitive to inhibition by diphenylene iodonium. Several protein complexes from the plasma membrane capable of superoxide producion in the presence of NADPH or NADH were found. The maximum diphenylene iodonium-sensitive activity was found in the high-molecular weight complex, in which proteins reacting with antibodies against C-terminal peptide of phagocytic oxidase (gp91phox) were detectable. The activity of this complex was lower in the cooled than in the control seedlings and displayed higher affinity to NADPH than to NADH. To search for the cooling-induced changes in the polypeptide content of protein complexes, the two-dimensional difference gel electrophoresis (hrCN/SDS-PAGE) was used. Control and cooled samples, whose lysine had been labeled with fluorescent dyes Cy2 and Cy3, respectively, were separated by this method in one gel. Decrease in a temperature of plant growing affected the protein content of the complex so that some new proteins appeared and several polypeptides disappeared as compared with the control. There were no significant differences between the cooled and control counterparts in the content of proteins detectable with gp91phox antibodies. Therefore, the high-molecular complex containing NADPH oxidase looses proteins under low temperature that may decrease its superoxide-producing activity.