title: Location of N-cyclohexyl-N'-(4-dimethyl-amino-alpha-naphthyl)carbodiimide- binding site in sarcoplasmic reticulum Ca2+-transporting ATPase.
abstract: The Ca2+-transporting ATPase has been labeled with N-cyclohexyl-N'-(4-dimethyl-amino-alpha-naphthyl)carbodiimide (NCD-4), a fluorescent carbodiimide which reacts with carboxyl groups of acidic residues. It has been reported that NCD-4 labels a transmembrane portion of the protein at the high-affinity calcium-binding sites. We have determined the depth of the calcium-sensitive probe by quenching the fluorescence by nitroxide-substituted fatty acids with its spin probe located at different carbons of the fatty acid chain (5, 7, 10, 12 and 16-nitroxide derivatives). We have found that all the calcium-sensitive fluorescence is quenched and that the efficiency of quenching decreases as the n-(4,4-dimethyl-3-oxazolinyloxy) (Doxyl) group is deeper in the membrane. We conclude that the NCD-4 label which is involved in the high-affinity calcium-binding site is located near the water/lipid interface. The fluorescence of the NCD-4 bound to that site can be quenched by acrylamide and Cu2+ but not by iodide, probably due to its anionic nature which will be repulsed by the abundance of negative charges of Glu and Asp residues of NCD-4 located at this site. The hydrophobic location of NCD-4 was confirmed by the fact that its fluorescence could be quenched by the spin label 2,2,6,6-tetramethyl-1-piperidine-N-oxyl but not by 4-hydroxy-2,2,6,6-tetramethyl-1-piperidine-N-oxyl which is much less hydrophobic.
authors.affiliation: Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, Spain.
authors.forename: I
authors.initials: I
authors.lastname: Velasco-Guillén
authors.forename: S
authors.initials: S
authors.lastname: Corbalán-García
authors.forename: J C
authors.initials: JC
authors.lastname: Gómez-Fernández
authors.forename: J A
authors.initials: JA
authors.lastname: Teruel
mesh_terms: D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D002234:Carbodiimides; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008051:Lipid Bilayers; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D014867:Water
chemical_list: D002234:Carbodiimides; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D013113:Spin Labels; D014867:Water; C038088:N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide; D000252:Calcium-Transporting ATPases; C003959:TEMPO