Figure 4

Time course of turnover of 13-HOD by glyoxysomes from etiolated cucumber. β-oxidation assays were prepared as described in the material and method section. HPLC-traces after different reaction times were detected at 260 nm (A) and 234 nm (B). Absorption signals of the individual intermediates are indicated by open (234 nm, conjugated double bond) or closed (260 nm, CoA-ester) circles and open squares (280 nm, keto-diene system). Besides 13-HOD-CoA (retention time ~ 100 min) acyl-CoAs bearing the conjugated hydroxy-diene system after one (retention time ~ 96 min) and two (retention time ~ 92 min) cycles of β-oxidation are detected and have been validated by MS. The intermediate appearing after 120 min of reaction (retention time ~ 89 min, absorption at 234 and 260 nm) is very likely the 13-HOD-intermediate after three cycles of β-oxidation. Intermediates lacking the 234 nm absorption coelute with authentic acyl-CoA standards with chain length between 4 and 12 carbons. Intermediates lacking the 260 nm absorbtion are presumed to be 13-HOD after one (retention time ~ 111 min) or two (retention time ~ 100 min) rounds of β-oxidation.