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The Ca 2ϩ -transporting ATPase has been labeled with N-cyclohexyl-N′-(4-dimethyl-amino-A-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 Cu 2ϩ 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-Noxyl but not by 4-hydroxy-2,2,6,6-tetramethyl-1-piperidine-N-oxyl which is much less hydrophobic.Keywords : Ca 2ϩ -transporting ATPase; sarcoplasmic reticulum; fluorescent carbodiimide; fluorescence quenching.Calcium transport through sarcoplasmic reticulum memIt has been also described that the derivatization of sarcoplasmic reticulum ATPase with NCD-4 resulted in inhibition of branes is initiated by cooperative binding of calcium to the calcium-dependent ATP utilization but not of calcium-indepencalcium pump. Two high-affinity calcium-binding sites in the dent partial reactions such as enzyme phosphorylation by inorcalcium-transporting ATPase have been demonstrated: a molar ganic phosphate [6]. stoichiometry of calcium bound/phosphorylation site of 2 :1 was Another approach to locate the high-specific-binding site of found [1]; and one molecule of ATP is hydrolyzed to transport calcium has been site-directed mutagenesis, which showed that two calcium ions [2, 3]. However, little is known concerning the the mutations of any of six amino acids within the transmemprecise localization of these calcium-binding sites in the molebrane region of the ATPase interfere with enzyme activation by cule of Ca 2ϩ -ATPase, and their relationship with the phosphorycalcium [7]. lation site, which is an essential question to understand the moTherefore both types of approaches agree on the membrane lecular mechanism of calcium transport. location of this site, although both of them, and specially the It has been shown that N,N′-dicyclohexylcarbodiimide, a hysite-directed mutagenesis one, are substantially based on the asdrophobic reagent for acidic amino acid residues, inhibits the sumptions made by predictions on domain structure after hydroca...