title: Receptor-independent modulation of reconstituted Galpha(i) protein mediated by liposomes.
abstract: A cationic amphiphile, BC5 (N-pentadecylpiperidin-4-amine), was recently designed and tested for its ability to directly stimulate the activity of recombinant Galpha inhibitory subunits. However, amphiphilic drugs can self-associate and bind to plasma membranes, causing undesired side effects. In this contribution, we report on the incorporation of BC5 in 1,2-dipalmytoyl-sn-glycerophosphocoline (DPPC) liposomes and on the characterization of the mixed DPPC/BC5 systems at various lipid/drug mole ratios by means of dynamic light scattering, differential scanning calorimetry and fluorescence spectroscopy. The myristoylated Galpha(i) subunit (Galpha-mir) was reconstituted in 1,2-dimiristoyl-sn-glycerophosphocoline (DMPC) bilayers, as a mimic of the drug target. We compare several reconstitution procedures in liposomes and present for the first time a complete characterization of a Galpha subunit reconstitution in model membranes in terms of protein activity as a function of the reconstitution protocol. The incorporation of the drug in DPPC bilayers resulted in enhanced Gi-modulating efficiency (evaluated in terms of binding to GTPgammaS (guanosine-5'-(gamma-thio)-triphosphate)). A correlation of the physico-chemical features and binding activity of protein-containing membrane model is proposed.
authors.affiliation: Department of Chemistry and CSGI, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Luciani
authors.forename: Debora
authors.initials: D
authors.lastname: Berti
authors.forename: Martina
authors.initials: M
authors.lastname: Fortini
authors.forename: Piero
authors.initials: P
authors.lastname: Baglioni
authors.forename: Carla
authors.initials: C
authors.lastname: Ghelardini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pacini
authors.forename: Dina
authors.initials: D
authors.lastname: Manetti
authors.forename: Fulvio
authors.initials: F
authors.lastname: Gualtieri
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bartolini
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Di Cesare Mannelli
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D010880:Piperidines / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000737:chemistry / Q000378:metabolism
chemical_list: D008081:Liposomes; C542493:N-pentadecylpiperidin-4-amine; D010880:Piperidines; D013501:Surface-Active Agents; D015060:1,2-Dipalmitoylphosphatidylcholine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019206:GTP-Binding Protein alpha Subunits, Gi-Go