Location of N-cyclohexyl-N'-(4-dimethyl-amino-alpha-naphthyl)carbodiimide- binding site in sarcoplasmic reticulum Ca2+-transporting ATPase.
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.
Velasco-Guillén
I
I
Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, Spain.
Corbalán-García
S
S
Gómez-Fernández
J C
JC
Teruel
J A
JA
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
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