Interestingly, www.selleckchem.com/products/CP-690550.html in tobacco protoplasts co expressing OLE RFP and HPLF1 2 YFP chimeric proteins, the amount of YFP fluorescence associated with LD showed a significant increase. A precise quantification of this change in fluorescence distribution appeared diffi cult since each cell can express a different amount of pro tein within the same population. Therefore, we counted the LD detected in several tobacco protoplasts expressing HPLF1 2 YFP or co expressing HPLF1 2 YFP and oleosin RFP. In the protoplasts expressing both the chimeric pro teins the number of LD detected was three four times greater than that found in protoplasts expressing HPLF YFP alone. A representative image of HPLF1 YFP fluores cence distribution in the presence and absence of oleosin is shown in Fig. 6.

Purified seed lipid bodies can activate HPLF In a previous work, we showed that recombinant HPLF purified to homogeneity from E. coli cultures Inhibitors,Modulators,Libraries is active in the absence of detergent. Nevertheless, the spe cific activity of the detergent free protein is greatly reduced in comparison with the activity recorded with the enzyme solubilised in a detergent containing buffer, Inhibitors,Modulators,Libraries or after treat ment of the detergent free protein with detergent micelles. To verify if purified seed lipid bodies could induce the Inhibitors,Modulators,Libraries conformational changes required for HPLF activation, the enzyme was purified to homogeneity by immobilised Inhibitors,Modulators,Libraries metal affinity chromatography. Sedimentation analyses on linear sucrose gradients were than compared of the native detergent free HPLF with the same enzyme solubilised in the presence of seed lipid bodies or 5 mM Emulphogene.

Inhibitors,Modulators,Libraries As shown in Fig. 7B and 7C, HPLF sol ubilised in the presence of lipid bodies or detergent peaked at the same fractions, thus showing the same sedimentation constant. In contrast, the native detergent free HPLF showed a dif ferent sedimentation constant. Furthermore, the different fractions recovered from sucrose gradients after HPLF solubilisation in the presence of lipid bodies, were separated by SDS PAGE and stained by Coomassie blue. Our results indi cated that oleosin and HPLF peaked at the same fractions, thus confirming the association between HPLF and lipid bodies. Finally, we determined the Km and kcat of purified HPLF with 13 HPOT, the preferred substrate of the enzyme, in the presence and absence of purified lipid bodies. A com parison of Figs.

7F and 7F shows clearly that the kinetics of the interaction between the preferred substrate Enzalutamide solubility 13 HPOT and HPLF is dramatically affected by the presence of lipid bodies. The kcat was increased 11 fold in the pres ence of lipid bodies, which was very similar to the fold increase observed using synthetic detergent micelle. the kcat value of 724 s 1 indicates that HPLF was fully acti vated by lipid bodies.