[Aging of the photosynthetic apparatus : III. Variations and characteristics of o-diphenoloxidase activity (Polyphenoloxidase) during aging in vitro of isolated spinach chloroplasts]

Abstract

The variations and characteristics of o-diphenoloxidase activity (O-diphenol-O2-oxidoreductase EC 1.10.3.1) were examined in aging, isolated spinach chloroplasts to determine whether this activity, measured in the presence of 4-methylcatechol as substrate, could be responsible for the inhibition of O2 evolution during aging of these organelles in dark and light.The rate of the Hill reaction (oxygen evolution and the corresponding photoreduction of ferricyanide) during aging in the dark was inhibited at pH 8.0 and stimulated at pH 6.5. This difference did not depend on the nature of the buffer used (Tris-HCl or phosphate). Furthermore, the pH optimum for the ferricyanide-Hill reaction was shifted to lower values (from pH 8.0 to 6.5) on aging of chloroplasts. This phenomenon is probably due to uncoupling during aging. In the light, the Hill reaction was markedly inhibited. However, the ratio μmoles O2 evolved/μmoles ferricyanide reduced diminished slowly in darkness and rapidly when the chloroplasts were aged in the light.Aging of chloroplasts in darkness was accompanied by a slow decrease in the latent period which precedes the initiation of the oxidation, followed by an increase in O-diphenoloxidase activity. Light-aged chloroplasts showed an initial stimulation and then a smaller increase in enzyme activity compared with that of the dark-aged chloroplasts. This latter phenomenon was probably due to secondary reactions caused by photo-inactivation. Under light conditions, the latent period decreased rapidly and disappeared after one hour.This latent period varied considerably with the season and was reduced or obliterated by treatments with light, fatty acids, Triton-X, hypotonic medium and increasing concentrations of substrate: that is by treatments which generally enhance chloroplast swelling. Thus it appears that the latent period is not a characteristic of O-diphenoloxidase but depends on the integrity of chloroplast structure.The enzyme activity was characterized by a stoichiometry of about 1 μmoles O2 consumed per 1.2 μmoles substrate oxidized, indicating that oxidation was probably proceeding further than conversion of O-diphenol to O-diquinone. The latter compound could be used as a Hill oxidant and it permitted measurement of O2 evolution in the same reaction mixture in the presence of light. Under these experimental conditions, O2 evolution (a DCMU sensitive reaction) was first stimulated in dark-aged chloroplasts and rapidly inhibited in light-aged chloroplasts.At appropriate concentrations, KCN, a potent inhibitor of oxidases, enhanced O2 evolution, suggesting that O-diphenoloxidase activity interferes with O2 evolution. This possibility is discussed in view of our previous findings on chloroplast aging in vitro.