Hydroperoxide Lyase and Other Hydroperoxide-Metabolizing Activity in Tissues of Soybean, Glycine max

Abstract

Hydroperoxide lyase (HPLS) activity in soybean (Glycine max) seed/seedlings, leaves, and chloroplasts of leaves required detergent solubilization for maximum in vitro activity. On a per milligram of protein basis, more HPLS activity was found in leaves, especially chloroplasts, than in seeds or seedlings. The total yield of hexanal from 13(S)-hydroperoxy-cis-9,trans-1 1-octadecadienoic acid (13S-HPOD) from leaf or chloroplast preparations was 58 and 66 to 85%, respectively. Because of significant competing hydroperoxide-metabolizing activities from other enzymes in seed/seedling preparations, the hexanal yields from this source were lower (36-56%). Some of the products identified from the seed or seedling preparations indicated that the competing activity was mainly due to both a hydroperoxide peroxygenase and reactions catalyzed by lipoxygenase. Different HPLS isozyme compositions in the seed/seedling versus the leaf/chloroplast preparations were indicated by differences in the activity as a function of pH, the Km values, relative V .. with 13S-HPOD and 13(S)-hydroperoxy-cis-9,trans-1 ,cis-15-octadecatrienoic acid (13S-HPOT), and the specificity with different substrates. With regard to the latter, both seed/seedling and chloroplast HPLS utilized the 13S-HPOD and 13S-HPOT substrates, but only seeds/ seedlings were capable of metabolizing 9(S)-hydroperoxy-trans-10,cis-12-octadecadienoic acid into 9-oxononanoic acid, isomenc nonenals, and 4-hydroxynonenal. From 13S-HPOD and 13S-HPOT, the products were identified as 12-oxo-cis-9-dodecenoic acid, as well as hexanal from 13S-HPOD and cis-3-hexenal from 13S-HPOT. In seed preparations, there was partial isomerization of the cis-3 or cis-9 into trans-2 or trans-10 double bonds, respectively.HPLS', an enzyme that cleaves hydroperoxides of polyunsaturated fatty acids into both aldehyde and w-oxoacid fragments, occurs widely in plants. Depending on substrate specificity of HPLS isoenzymes for isomeric hydroperoxides of linoleic or linolenic acids, the enzyme produces either green/ ' Abbreviations: HPLS, hydroperoxide lyase; CP-HPLC, chiralphase HPLC; El, electron impact; OTMS, trimethylsilyloxy; rac-HPOD, racemic cis,trans-and trans,trans-diene linoleic acid hydroperoxides; rac-HPOT, racemic cis,cis,trans-triene linolenic acid hydroperoxides; 9S-HPOD, 9(S)-hydroperoxy-trans-10,cis-12-octadecadienoic acid; 13S-HPOD, 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid; 12S-HPOT, 12(S)-hydroperoxy-cis-9,trans-13,cis-1 5-octadecatrienoic acid; 1 3S-HPOT, 1 3(S)-hydroperoxy-cis-9,trans-11 ,cis-5-octadecatrienoic acid; SP-HPLC, straight-phase HPLC; Triton X-1 OOR, Triton X-100 reduced; m/z, mass to charge ratio. beany/grassy odors (hexanal or cis-3-hexenal) from 1 3-hydroperoxides or cucumber/pear odors (cis-3-nonenal or cis-3,cis-6 nonadienal) from 9-hydroperoxides (reviewed in refs. 4 and 15). The corresponding w-oxoacid fragments, 12-oxo-cis-9-dodecenoic acid and 9-oxononanoic acid, have no known function, except the former isomerizes into 12-oxo-trans-10-dodecenoi...

Developmental Change in C6-Aldehyde Formation by Soybean Leaves

“…A good example of apparent LAH limitation to C6-aldehyde formation is found in mature soybean seeds. The addition of FFA substrates greatly increases C6-aldehyde formation by soybean seed meal (23) and seed extracts (8). In our current work, although the limiting action of LAH was not directly investigated, the possibility cannot be excluded with the young leaves.…”

“…Results showed that in soybean leaves (regardless of age), there was little difference in LOX activity with 18:2 or 18:3, but based on spectrophotometric analyses, HPL preferentially utilized 13-HPOT, although C6-aldehyde formation was far higher in the presence of 13-HPOD than in the presence of 13-HPOT (Table I) a These results are apparently underestimated because of a comparatively lower partition of trans-2-hexenal and cis-3-hexenal from aqueous solution into headspace and rapid depletion of cis-3-hexenal at longer times (8).…”

“…Thus, it appears that much of the product of soybean leaf LOXs does not lead to C6-aldehyde formation, probably due to formation of LOX products that are not substrates for HPL and/or possible competition of some of leaf LOX isozymes with HPL, as is apparently the case with soybean embryo LOX 3 (12, 23). Gardner et al (8) recently showed that soybean leaf chloroplast HPL has little reactivity with 9-HPOT (which may be a leaf LOX product) as substrate.…”

“…Soybean leaves at the third, sixth, and eighth positions, collected at the same time as the disks, were homogenized with a mortar and pestle in a total 0.5-mL Hepes buffer (50 mM, pH 7.5) containing 0.5% Triton X-100R at 0°C (8). Subsequently, the homogenate was centrifuged at 13,600g for 15 min to obtain a supernatant containing the enzymes.…”

“…HPL activity and substrate specificity were detected by NADH oxidation at 340 nm in the presence of yeast alcohol dehydrogenase and 13-HPOT (21), and the decrease in absorbance of conjugated diene substrate was measured at 235 nm (8). Formation of C6-aldehydes by leaf extract supernatants in the presence of 1 mm 18:3, 1 mi 13-HPOT, or 1 mM 13-HPOD was analyzed in 1.8-mL screwtop vials based on the method described by Hildebrand et al (12).…”