Physicochemical characterization and in vitro interaction with brain capillary endothelial cells of artificially monoacylated ribonucleases A |
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| Authors: | Chopineau Joël Robert Stéphane Fenart Laurence Cecchelli Roméo Lagoutte Bernard Paitier Stéphanie Dehouck Marie-Pierre Domurado Dominique |
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| Affiliation: | (1) Laboratoire de Technologie Enzymatique, UPRESA 6022 CNRS, Université de Technologie de Compiègne, BP 20529, F-60205 Compiègne Cedex, France;(2) SERLIA, U 325 INSERM, Institut Pasteur de Lille, BP 245, F-59019 Lille Cedex, France;(3) Service de Bioénergétique, URA 1290 CNRS, Département de Biologie Cellulaire et Moléculaire, Centre d'études de Saclay, F-91191 Gif-sur-Yvette Cedex, France;(4) Groupe de Pharmacocinétique des Conjugués Macromoléculaires, Centre de Recherche sur les Biopolymères Artificiels, URA 1465 CNRS, Faculté de Pharmacie, F-34060 Montpellier Cedex 2, France |
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| Abstract: | Summary 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. |
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| Keywords: | Blood-brain barrier Drug targeting Hydrophobisation Reversed micelles |
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