RUTHENIUM RED AFFECTS THE INTRINSIC FLUORESCENCE OF THE CALCIUM-ATPASE OF SKELETAL SARCOPLASMIC-RETICULUM

We have studied the effect of Ruthenium red on the sarcoplasmic reticulum Ca2+-ATPase. Ruthenium red does not modify the Ca2+ pumping activity of the enzyme, despite its interaction with cationic binding sites on sarcoplasmic reticulum vesicles. Two pools of binding sites were distinguished. One pool (10 nmol/mg) is dependent upon the presence of micromolar Ca2+ and may therefore represent the high-affinity Ca2+ transport sites of the Ca2+-ATPase. However, Ruthenium red only slightly competes with Ca2+ on these sites. The other pool (15-17 nmol/mg) is characterized as low-affinity cation binding sites of sarcoplasmic reticulum, distinct from the Mg2+ Site involved in the ATP binding to the Ca2+-ATPase. The interaction of Ruthenium red with these low-affinity cation binding sites, which may be located either on the Ca2+-ATPase or on surrounding lipids, decreases tryptophan fluorescence level of the protein. As much as 25% of the tryptophan fluorescence of the Ca2+-ATPase is quenched by Ruthenium red (with a dissociation constant of 100 nM), tryptophan residues located near the bilayer being preferentially affected.