The purification and characterization of fatty acid hydroperoxide lyase in sunflower.
Two fatty acid hydroperoxide lyases (HPO lyase I and II) were purified to apparent homogeneity from etiolated hypocotyls of sunflower (Helianthus annuus L.) by a combination of ion-exchange, hydrophobic interaction, and gel filtration chromatography. The two HPO lyases were separated during the hydrophobic interaction chromatography step, with HPO lyase I more hydrophilic than HPO lyase II. The estimated M(r) of both native HPO lyases was determined by gel filtration to be 200,000 and SDS-PAGE in the presence of 100 mM dithiothreitol showed that the enzyme was composed of a single 53 kDa peptide, suggesting that the enzyme exists as a tetramer in vivo. HPO lyase was also abundant in the cotyledons and green leaves. HPO lyases I and II from hypocotyl metabolized 13-hydroperoxylinoleic acid and 13-hydroperoxylinolenic acid to the same extent, but the green leaf enzyme was more than ten-fold more active with 13-hydroperoxylinolenic acid than 13-hydroperoxylinoleic acid. A difference spectrum between CO-bound and CO-unbound dithionite-reduced HPO lyase I showed an absorbance maximum at 452 nm, indicating that it was a cytochrome P450-type enzyme. The activities of HPO lyase I and II were significantly inhibited by nordihydroguaiaretic acid, sulfhydryl reagents, and piperonylbutoxide, which is a cytochrome P450 inhibitor.
Itoh
A
A
Northern Crop Science Laboratory, U.S. Department of Agriculture, Fargo, ND 58105, USA.
Vick
B A
BA
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006368:Helianthus / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011487:Protein Conformation; D012995:Solubility; D013053:Spectrophotometry; D013379:Substrate Specificity; D014018:Tissue Distribution