LOCATION OF -DIMETHYL-AMINO-ALPHA-NAPHTHYL)CARBODIIMIDEBINDING SITE IN SARCOPLASMIC-RETICULUM CA2+ TRANSPORTING ATPASE

The Ca2+-transporting ATPase has been labeled with -N'-(4-dimethyl-ami no-alpha-naphthyl)-carbodiimide (NCD-4), a fluorescent carbodiimide wh ich reacts with carboxyl groups of acidic residues. It has been report ed that NCD-4 labels a transmembrane portion of the protein at the hig h-affinity calcium-binding sites. We have determined the depth of the calcium-sensitive probe by quenching the fluorescence by nitroxide-sub stituted fatty acids with its spin probe located at different carbons of the fatty acid chain (5, 7, 10, 12 and 16-nitroxide derivatives). W e have found that all the calcium-sensitive fluorescence is quenched a nd that the efficiency of quenching decreases as the n-(4,4-dimethyl-3 -oxazolinyloxy) (Doxyl) group is deeper in the membrane. We conclude t hat the NCD-4 label which is involved in the high-affinity calcium-bin ding 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 r epulsed by the abundance of negative charges of Glu and Asp residues o f NCD-4 located at this site. The hydrophobic location of NCD-4 was co nfirmed by the fact that its fluorescence could be quenched by the spi n label 2,2,6,6-tetramethyl-1-piperidine-N-oxyl not by 4-hydroxy-2,2,6 ,6-tetramethyl-1-pipelidine-N-oxyl which is much less hydrophobic.