STUDIES ON LINOLEIC-ACID 8R-DIOXYGENASE AND HYDROPEROXIDE ISOMERASE OF THE FUNGUS GAEUMANNOMYCES-GRAMINIS

Linoleic acid is sequentially converted to 7S,8S-dihydroxy-9Z, 12Z-oct adecadienoic acid by the 8R-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis, which is a common pathogen of whe at. The objective of this study was to separate and characterize the t wo enzyme activities. The isomerase activity was found mainly in the m icrosomal fraction of the mycelia and the 8R-dioxygenase in the cytoso l. The 8R-dioxygenase could be partially purified by ammonium sulfate precipitation, gel filtration, ion exchange chromatography or isoelect ric focusing. The 8R-dioxygenase was unstable during purification, but it could be stabilized by glutathione, glutathione peroxidase and eth ylenediaminetetraacetic acid. Several protease inhibitors reduced the enzyme activity. Gel filtration with Sephacryl S-300 showed that most 8R-dioxygenase activity was eluted with the front with little retentio n. Isoelectric focusing in the presence of ethylene glycol (20%) indic ated an isoelectric point of pi 6.1-6.3. The enzyme was retained on st rong anion exchange columns at pH 7.4 and could be eluted with 0.3-0.5 M NaCl Incubation of the enzyme with 0.1 mM linoleic acid led to part ial inactivation, which may indicate product inhibition. Paracetamol a nd the lipoxygenase inhibitor ICI 230,487 at 30 mu M inhibited the 8R- dioxygenase by 44 and 58%, respectively. 8R-hydroperoxy-9Z,12Z-octadec adienoic acid was isolated from incubations of linoleic acid with the partially purified enzyme or with the cytosol in the presence of p-hyd roxymercuribenzoate. The hydroperoxide was rapidly converted by the hy droperoxide isomerase in the microsomal fractions to 7S,8S-dihydroxy-9 Z,12Z-octadecadienoic acid. The isomerase was neither inhibited by car bon monoxide nor by ketoconazole (100 mu M) The isomerase was active o ver a broad pH range. It could be separated from the 8R-dioxygenase by differential centrifugation, by ammonium sulfate precipitation and by gel filtration. We conclude that the linoleic acid 8R-dioxygenase and the hydroperoxide isomerase are two separate enzymes.