Purification and characterization of a protoporphyrinogen-oxidizing enzyme with peroxidase activity and light-dependent herbicide resistance in tobacco cultured cells
Yamato, S. | Katagiri, M. | Ohkawa, H.
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 Km and Vmax values of the purified enzyme for protoporphyrinogen IX were 78.9 micromolar and 1.3 micromoles/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.
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