PURIFICATION AND CHARACTERIZATION OF A PROTOPORPHYRINOGEN-OXIDIZING ENZYME WITH PEROXIDASE-ACTIVITY AND LIGHT-DEPENDENT HERBICIDE RESISTANCE IN TOBACCO CULTURED-CELLS

The activity of protoporphyrinogen-oxidizing enzymes was found not only in crude etioplast and mitochondrial fractions but also in the soluble fraction of tobacco cell lines. Approximately 90% of the total activity was found in the soluble fraction of the SL cell line. A protoporphyrinogen-oxidizing enzyme was purified from the soluble fraction of SL by chromatography on CM-Toyopearl, hydroxyapatite, and HA-1000 columns. The purified enzyme has a molecular weight of approximately 48,000 on SDS-polyacrylamide gel electrophoresis. Apparent K-m and V-max values of the purified enzyme for protoporphyrinogen IX were 78.9 mu M and 1.3 mu mol/mg protein/min, respectively. The purified enzyme utilized uroporphyrinogen I and coproporphyrinogen I as substrates. The protoporphyrinogen-oxidizing activity of the purified enzyme was not inhibited by herbicides that inhibit protoporphyrinogen oxidase. The purified enzyme contained a heme and showed peroxidase activity toward guaiacol and pyrogallol. On the other hand, peroxidases commercially available showed the protoporphyrinogen-oxidizing activity. Based on these results, the soluble protoporphyrinogen-oxidizing enzyme in tobacco cultured cells seemed to be a kind of peroxidase. The soluble protoporphyrinogen-oxidizing enzyme with herbicide resistance may play an important role in the oxidation of protoporphyrinogen IX which accumulates out of the site of heme and chlorophyll biosynthesis in the herbicide-treated plants. (C) 1994 Academic Press, Inc.