Abstract
For the first time, HMG-CoA reductase, the membrane protein responsible for cholesterol synthesis, was incorporated into a lipid membrane consisting of DOPC:Chol:SM at a 1:1:1 molar ratio, which mimics the lipid rafts of cell membranes. The membrane containing the protein was generated in the form of either a proteoliposomes or a film obtained by spreading the proteoliposomes at the air-water interface to prepare a protein-rich and stable lipid layer over time. The lipid vesicle parameters were characterized using dynamic light scattering (DLS) and fluorescence microscopy. The incorporation of HMG-CoA reductase was reflected in the increased size of the proteoliposomes compared to that of the empty liposomes of model rafts. Enzyme reconstitution was confirmed by measuring the activity of NADPH, which participates in the catalytic process. The thin lipid raft films formed by spreading liposomes and proteoliposomes at the air-water interface were investigated using the Langmuir technique. The activities of the HMG-CoA reductase films were preserved over time, and the two lipid raft systems, nanoparticles and films, were exposed to solutions of fluvastatin, a HMG-CoA reductase inhibitor commonly used in the treatment of hypercholesterolemia. Both lipid raft systems constructed were useful membrane models for the determination of reductase activity and for monitoring the statin inhibitory effects and may be used for investigating other integral membrane proteins during exposure to inhibitors/activators considered to be potential drugs.