Physicochemical characterization and in vitro interaction with brain capillary endothelial cells of artificially monoacylated ribonucleases A
Résumé
Acylated proteins play a crucial role in cell physiology because of their increased interaction with membranes. Their isolation is difficult as a consequence of their low cellular concentration and their chemical preparation is problematic due to solubility problems. Through the use of reversed micelles, we produced tens of milligrams of acylated ribonucleases A, chosen as a model, purified them by semi-preparative high performance liquid chromatography (HPLC) and characterized them by analytical HPLC, capillary electrophoresis, mass spectrometry, peptide mapping, Edman degradation and enzyme activity. We next scrutinized the interaction with an in vitro blood-brain barrier model and demonstrated that palmitoylated and stearoylated ribonucleases A are transported from one compartment to the other across the cellular monolayer, in contrast to the native enzyme.