Heterogeneity of tryptophanyl residues in the sarcoplasmic reticulum ATPase probed by fluorescence energy transfer between the protein and fluorescent ionophore X537A.

Sarcoplasmic reticulum vesicles and purified ATPase were studied by fluorescence spectroscopy. The preparations exhibited similar excitation and emission spectra, characteristic of tryptophan fluorescence. Addition of calcium to the preparations suspended in ethylene glycol bis(P-aminoethyl ether)N, N, N’, N’-tetraacetic acid (EGTA) solutions promoted a 3 to 4% increase in fluorescence intensity, both in sarcoplasmic vesicles and purified ATPase. This indicates that non-ATPase proteins, which account for 20 to 30% of the vesicular preparations, are not influencing significantly the fluorescence measurements. Maximal increase in fluorescence upon Ca2+ addition was always centered at 335 nm, when excited at different wavelengths, T h i s suggests that the increase in fluorescence corresponded to a specific change in exposure of fluorescent residues of the ATPase to the polar environment. Caz+ titration of the increase in fluorescence in sarcoplasmic vesicles showed that half-maximal change occurred at 5 p~ Ca2+. A Hill plot of the data gives an n of 2.04 2 0.10, indicating that the fluorescence change is associated with a cooperative phenomenon. The extent of interaction between the ATPase and membrane lipids was assessed by fluorescence energy transfer between lipid-soluble fluorescent ionophore XS37A (acceptor) and the Ca2+-ATPase (donor). It was observed that energy transfer was obtained at a concentration of X537A as low as 2 p ~ . The ionophore is preferentially excited through the ATPase up to concentrations of 20-40 PM X537A. It is suggested that the ionophore might have a particular affinity for the protein-lipid interface of the ATPase lipoprotein microenvironment. Addition of the acceptor ionophore causes quenching of the donor tryptophan fluorescence. Halfmaximal quenching was obtained at 12 p~ X537A in sarcoplasmic vesicles and 6.3 PM in purified ATPase. Maximal quenching with excess ionophore was 70% in sarcoplasmic vesicles and 78% in purified ATPase. This indicates that most of the fluorescent residues are accessible to the lipid-soluble ionophore probe at the lipid-protein interface. The population of ATPase tryptophans which transfer their fluorescence to the ionophore was found to be insensitive to CaZr. The fraction which does not transfer its fluorescence to the probe remains sensitive to Ca“ addition.