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 onl y in crude etioplast and mitochondrial fractions but also in the solub le fraction of tobacco cell lines. Approximately 90% of the total acti vity was found in the soluble fraction of the SL cell line. A protopor phyrinogen-oxidizing enzyme was purified from the soluble fraction of SL by chromatography on CM-Toyopearl, hydroxyapatite, and HA-1000 colu mns. The purified enzyme has a molecular weight of approximately 48,00 0 on SDS-polyacrylamide gel electrophoresis. Apparent K-m and V-max va lues of the purified enzyme for protoporphyrinogen IX were 78.9 mu M a nd 1.3 mu mol/mg protein/min, respectively. The purified enzyme utiliz ed uroporphyrinogen I and coproporphyrinogen I as substrates. The prot oporphyrinogen-oxidizing activity of the purified enzyme was not inhib ited by herbicides that inhibit protoporphyrinogen oxidase. The purifi ed enzyme contained a heme and showed peroxidase activity toward guaia col and pyrogallol. On the other hand, peroxidases commercially availa ble showed the protoporphyrinogen-oxidizing activity. Based on these r esults, the soluble protoporphyrinogen-oxidizing enzyme in tobacco cul tured cells seemed to be a kind of peroxidase. The soluble protoporphy rinogen-oxidizing enzyme with herbicide resistance may play an importa nt role in the oxidation of protoporphyrinogen IX which accumulates ou t of the site of heme and chlorophyll biosynthesis in the herbicide-tr eated plants. (C) 1994 Academic Press, Inc.