IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
artículos
Título:
“Interactions between plasma membrane calcium pump and phospholipids studied with a photoactivatable probe”
Autor/es:
VILLAMIL GIRALDO, A. M.; CASTELLO PR; GONZÁLEZ FLECHA FL; DELFINO JM; ROSSI JPFC
Revista:
Cell biochem biophys
Referencias:
Año: 2006 vol. 44 p. 431 - 437
Resumen:
Abstract The functions of membrane proteins are highly dependent on their phospholipid environment. In this article, we have used a hydrophobic photolabeling method to study the noncovalent interactions between plasma membrane calcium pump (PMCA) and surrounding phospholipids. With this approach, we determined (1) the number of lipid molecules in close contact with the transmembrane surface, i.e., the lipid–protein stoichiometry, and (2) the distribution of lipid molecules among different regions of the protein. PMCA was photolabeled in mixed micelles containing detergent, the phosphatidylcholine photoactivatable analog 1-palmitoyl-2-[9-[2′-[125 I]iodo-4′- (trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (DMPC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 } 1 molecules of DMPC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N-terminal (1-322), the central (323-660), and the C-terminal (661-1205) regions was 26, 36, and 38%, respectively.′-[125 I]iodo-4′- (trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (DMPC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 } 1 molecules of DMPC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N-terminal (1-322), the central (323-660), and the C-terminal (661-1205) regions was 26, 36, and 38%, respectively.sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (DMPC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 } 1 molecules of DMPC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N-terminal (1-322), the central (323-660), and the C-terminal (661-1205) regions was 26, 36, and 38%, respectively.} 1 molecules of DMPC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N-terminal (1-322), the central (323-660), and the C-terminal (661-1205) regions was 26, 36, and 38%, respectively. Index Entries: Membrane proteins; plasma membrane calcium pump; photoactivatable phospholipids; lipid–protein interactions.Membrane proteins; plasma membrane calcium pump; photoactivatable phospholipids; lipid–protein interactions.