Previous studies conducted in cytosolic extracts of the freshwater hydrozoan Hydra vulgaris led to the finding of an abundant 11(R)-lipoxygenase catalyzing the peroxidation of polyunsaturated fatty acid (PUFAs) on the tenth carbon atom from the aliphatic end (omega 10 peroxidation). Here we describe experiments aimed at identifying the actual metabolites generated in vivo by such enzymic activity, Homogenates of H. vulgaris polyps were analyzed by HPLC. This showed the presence of three major components chromatographically identical to three metabolites obtained when incubating the homogenates with exogenous alpha-linolenic acid (alpha-LA). The presence, in extracts of polyps prelabelled with [C-14]-alpha-linolenic acid, of radioactive metabolites displaying the same chromatographic properties, substantiated the hypothesis that the natural products isolated in vivo are derived from alpha-LA. Gas chromatographic analyses revealed that this was the most abundant PUFA in both free and phosphoglyceride-bound fatty acid pools. [H-1]-NMR analysis of the endogenous substances, carried out in comparison with products obtained from exogenously incubated alpha-LA, indicated that their structures were those of 9-hydroxy-, 9-hydroperoxy- and 9-keto-octadeca-10E-12Z-15Z-trienoic acids (9-alpha-HOTrE, -HPOTrE and -KOTrE). Hydra homogenates transformed 9-alpha-HPOTrE partly into 9-alpha-HOTrE and partly into 9-alpha-KOTrE. Chiral phase HPLC conducted on 9-alpha-HOTrE established that this metabolite was composed mostly of the R enantiomer. These observations, and the finding that the presence of exogenous arachidonic acid in incubated homogenates significantly reduces the production of alpha-LA metabolites, provide strong evidence that these compounds are produced by an enzymic activity identical to the previously-described H. vulgaris (R)-omega 10-lipoxygenase. Further experiments suggested that alpha-LA, acting as the native substrate for this enzyme, is mainly esterified on the 2 position of Hydra phosphoglycerides, and that the production of the alpha-LA metabolites described here for the first time from natural sources, can be potentially enhanced in vivo by stimuli activating phospholipase A(2).
HYDRA-VULGARIS OMEGA-10-LIPOXYGENASE IS USED IN-VIVO TO SYNTHESIZE NEW ALPHA-LINOLENIC ACID METABOLITES
Gianfrani C;Di Marzo V;De Petrocellis L;
1995
Abstract
Previous studies conducted in cytosolic extracts of the freshwater hydrozoan Hydra vulgaris led to the finding of an abundant 11(R)-lipoxygenase catalyzing the peroxidation of polyunsaturated fatty acid (PUFAs) on the tenth carbon atom from the aliphatic end (omega 10 peroxidation). Here we describe experiments aimed at identifying the actual metabolites generated in vivo by such enzymic activity, Homogenates of H. vulgaris polyps were analyzed by HPLC. This showed the presence of three major components chromatographically identical to three metabolites obtained when incubating the homogenates with exogenous alpha-linolenic acid (alpha-LA). The presence, in extracts of polyps prelabelled with [C-14]-alpha-linolenic acid, of radioactive metabolites displaying the same chromatographic properties, substantiated the hypothesis that the natural products isolated in vivo are derived from alpha-LA. Gas chromatographic analyses revealed that this was the most abundant PUFA in both free and phosphoglyceride-bound fatty acid pools. [H-1]-NMR analysis of the endogenous substances, carried out in comparison with products obtained from exogenously incubated alpha-LA, indicated that their structures were those of 9-hydroxy-, 9-hydroperoxy- and 9-keto-octadeca-10E-12Z-15Z-trienoic acids (9-alpha-HOTrE, -HPOTrE and -KOTrE). Hydra homogenates transformed 9-alpha-HPOTrE partly into 9-alpha-HOTrE and partly into 9-alpha-KOTrE. Chiral phase HPLC conducted on 9-alpha-HOTrE established that this metabolite was composed mostly of the R enantiomer. These observations, and the finding that the presence of exogenous arachidonic acid in incubated homogenates significantly reduces the production of alpha-LA metabolites, provide strong evidence that these compounds are produced by an enzymic activity identical to the previously-described H. vulgaris (R)-omega 10-lipoxygenase. Further experiments suggested that alpha-LA, acting as the native substrate for this enzyme, is mainly esterified on the 2 position of Hydra phosphoglycerides, and that the production of the alpha-LA metabolites described here for the first time from natural sources, can be potentially enhanced in vivo by stimuli activating phospholipase A(2).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
