Figure 7

Effect of detergent or lipid bodies on the enzymatic activity of HPLF. (A): SDS-PAGE gel electrophoresis of HPLF after purification by IMAC; (B-D): Sedimentation analyses. Purified HPLF was incubated in the presence of purified seed lipid bodies (B), 5 mM Emulphogene detergent (C) or 100 mM sodium phosphate buffer, pH 6.5 (D) and loaded onto linear 5–20% sucrose gradients. After centrifugation the gradients were fractionated and analysed by SDS-PAGE and Western-blot analyses using a specific His-tag antiserum. Numbering refers to the 14 fractions collected from the bottom of the gradients. (E, F): Kinetic analysis of HPLF in the presence and absence of lipid bodies. HPLF (1.8 pmol) diluted in 100 mM sodium phosphate buffer, pH 6.5 alone (E), or in the same buffer containing 0.3 M sucrose and lipid bodies (F) was assayed with 13-HPOT (0–640 μM) under the standard assay conditions (See Materials and Methods). (G): Fold-activation of HPLF by lipid bodies as a function of 13-HPOT concentration. Fold-activation is defined as the ratio of the activity in the presence of lipid bodies/activity in the absence of lipid bodies, determined from the kinetic plots in (E) and (F).