Luminescence studies of Tb3+ bound to the high affinity sites of the Ca2+-ATPase of sarcoplasmic reticulum.

Direct excitation of lanthanide luminescence with a pulsed dye laser has been used to probe the molecular environment of the high affinity sites of the sarcoplasmic reticulum Ca2+-ATPase. The direct excitation spectrum of Tb3+ bound to these sites has been determined and a luminescence lifetime of approximately 1 ms measured. Measurements of the difference in lifetime of the Tb X ATPase complex in H2O and D2O indicate that there are approximately 2 H2O molecules in the first coordination sphere of Tb3+ bound at the high affinity sites of the ATPase. The results are compared with the properties of Tb3+ binding to high affinity sites of other Ca2+ binding proteins. The binding constant of Tb3+ to the ATPase is in the range of 0.3-5.0 X 10(8) M-1 as inferred from the KI for inhibition of ATP hydrolysis, in agreement with a previous report (Highsmith, S. R., and Head, M. R. (1983) J. Biol. Chem. 258, 6858-6862). The values of the Ca2+ binding constant (approximately 2 X 10(6) M-1) and the cooperative nature (n = 1.9) of Ca2+ protection of Tb3+ inhibition indicate that Tb3+ and Ca2+ compete for the high affinity sites of the ATPase. The results demonstrate that directly-excited Tb3+ luminescence provides unique information on the environment of the Ca2+ binding-transport sites of the SR ATPase.