Figure 4

Analysis of LOX and HPL activities at 234 nm by spectrophotometry. Total proteins were isolated from yeast cells expressing Nb-9-LOX (A), Cm-9/13-HPL (B), and Cl-13-HPL (C) which were harvested at time points corresponding to 0 (-■-), 4 (-●-), 8 (-▲-), and 24 (-♦-) hours after galactose induction. Nb-9-LOX activity was measured at pH 7.0 using linoleic acid as a substrate, Cm-9/13-HPL activity was measured at pH 7.0 using 9(S)-HPOD as a substrate, and Cl-13-HPL activity was measured at pH 6.0 using 13(S)-HPOD as a substrate. LOX activity was measured at room temperature by the formation of the conjugated diene at 234 nm, while HPL activity was measured by the decrease of A₂₃₄ due to cleavage of the substrate. (D) Reaction of linoleic acid with yeast cell extract of Nb-9-LOX (-Δ-), followed by addition of yeast cell extract of Cl-13-HPL (-□-) or yeast cell extract of Cm-9/13-HPL (-○-) at 90 seconds (arrow).