The supernatant was centrifuged again at 5000 g to separate the crude plastidial fraction from Nutlin-3a Sigma the other proteins. The fraction A was resus pended in sucrose buffer and layered onto a three steps sucrose gradient consisting of 1. 45, 0. 84, 0. 45 M sucrose and centrifuged at 100,000 g for 1 hr at 4 C as previ ously described. After centrifugation intact plastids were recovered at the interface between 1. 45 and 0. 80 M sucrose, diluted with 100 mM Tris HCl, pH 8. 0 and cen trifuged again at 10000 g for 10 min at 4 C. The pellet was resuspended in SDS PAGE sample buffer. Fraction B was used to separate lipid bodies, micro somes and cytosol fractions by two layer flotation as above described. After centrifugation at 100000 g for 1 h at 4 C, the following fractions were recovered the LB fraction from the top of the gradient.
the cytosolic protein fraction was resuspended in SDS PAGE sample buffer, whereas the proteins from the LB and cytosolic protein fraction were precipitated with trichloroacetic acid and resuspended in SDS PAGE sample Inhibitors,Modulators,Libraries buffer. HPLF purification, kinetic analyses and purification of seed lipid bodies Recombinant HPLF was purified to homogeneity from E. coli Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries BL21 cells by immobilised metal affinity chro matography as described previously. Steady state kinetic data were collected using Shimadzu kinetics software. Activity was determined at 25 C in a standard assay containing 100 mM sodium phosphate buffer, pH 6. 5 by monitoring the disappearance of sub strate at 234 nm. Substrate was diluted from a 20 mM stock that was stored at 70 C in ethanol.
the exact con centration after dilution was determined using a molar extinction of 25 mM 1. cm 1. Km and kcat for 13 HPOT were calculated by fitting the data sets to a one site saturation model for simple ligand Inhibitors,Modulators,Libraries binding using SigmaPlot 8. Lipid bodies were isolated from water melon seeds, by two layer flotation as previously reported, further purified by two sequential washings with 2. 0 M NaCl and finally resuspended in 150 mM Tris HCl, pH 7. 5, containing 0. 6 M sucrose. Rate zonal sucrose gradients Different aliquots of purified HPLF were incubated with 100 mM sodium phosphate buffer pH 6. 5, purified lipid bodies or 5 mM Emulphogene for 15 min at 25 C and than loaded onto linear 5 to 20% sucrose gradients and centrifuged at 150000 g for 20 h.
After cen trifugation, 1 ml fractions were collected from the bottom of the tube and the sucrose concentration determined. Proteins from each aliquot were precipitated with trichlo roacetic acid and resuspended in SDS PAGE sample buffer. Western blot analyses were performed according to the ECL protocol and Inhibitors,Modulators,Libraries a 1 4000 scientific research dilution of an anti His antiserum. Background Fruit bearing crop species are an important component of the human diet providing nutrition, dietary diversity and pleasure.