PURIFICATION AND CHARACTERIZATION OF LIPOXYGENASE ISOENZYMES IN GERMINATING BARLEY

Two lipoxygenase (LOX) isoenzymes were extracted from barley (Hordeum vulgare 'Robust) and purified by hydroxylapatite chromatography, yield ing LOX-1 with a pl of 5.2 and LOX-2 with a pI of 6.7. LOX-1 and LOX-2 appear to be monomeric proteins of 90 and 95 kDa, respectively. LOX-1 converted linoleic and linolenic acids into mainly 9-hydroperoxides, whereas LOX-2 produced mainly 13-hydroperoxides. Linoleic acid was fou nd to be the best substrate for both isoenzymes. K(m) values for this substrate were determined to be 1.3 x 10(-5) M for LOX-1 and 1.9 x 10( -5) M for LOX-2. LOX-2 can oxidize esterified derivatives of linoleic acid (methyl linoleate and trilinolein) more readily than LOX-1 can. L OX-2 also had more heat resistance and better stability than LOX-1 did . LOX-2 developed only after germination, and the activity of both LOX -1 and LOX-2 increased to a large extent during germination. LOX-1 was localized exclusively in the germ in sound barley. Upon germination, LOX-1 and LOX-2 developed in newly synthesized rootlet and acrospire t issue. The rootlets contained exclusively LOX-2, while the acrospire c ontained both isoenzymes.