Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study.
Bottom Line: Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress.Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile.Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase.
Affiliation: Laboratoire de chimie physique, UMR 8000, Université Paris Sud-CNRS, Orsay 91405, France.Show MeSH
Related in: MedlinePlus
Mentions: The trimera was expressed as fusion protein. Thus it was purified by metal chelate affinity chromatography. The above supernatant was applied to nickel affinity column after being diluted twice with buffer (0.5 M NaCl, 30 mM Na2HPO4, 20 mM imidazole and 1 mM PMSF, pH 7.4). The mixture was loaded for 1 h 30 min so that the proteins of interest effectively cling to the nickel resin. Then the column was washed with the same buffer to remove unwanted bound proteins. The proteins bound to the beads were eluted from the resin with elution buffer (0.1 M NaCl, 30 mM Na2HPO4 and 300 mM imidazole, pH 7.4). Then size exclusion chromatography was carried out to better purify the trimera. Proteins concentration was determined using a NanoDrop2000 spectrophotometer (Thermo scientific, France) and the extinction coefficient of 1.5 mg−1/mL cm. The purities of all proteins were checked by migration on 10% BisTris-NuPAGE SDS gels (Invitrogen), stained with Coomassie Brilliant Blue (Fig. S1 in Supplementary material).
Affiliation: Laboratoire de chimie physique, UMR 8000, Université Paris Sud-CNRS, Orsay 91405, France.