Characterization of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum by laser-excited europium luminescence

Abstract

The molecular environment of Ca²⁺ translocating sites of skeletal muscle sarcoplasmic reticulum (SR) (Ca²⁺+ Mg²⁺)‐ATPase has been studied by pulsed‐laser excited luminescence of Eu³⁺ used as a Ca²⁺ analogue. Interaction of Eu³⁺ with SR was characterized by investigating its effect on partial reactions of the Ca²⁺ transport cycle. In native SR vesicles, Eu³⁺ was found to inhibit Ca²⁺ binding, phosphoenzyme formation, ATP hydrolysis activity and Ca²⁺ uptake in parallel fashion. The non‐specific binding of Eu³⁺ to acidic phospholipids associated with the enzyme was prevented by purifying (Ca²⁺+ Mg²⁺)‐ATPase and exchanging the endogenous lipids with a neutral phospholipid, dioleoylglycerophosphocholine. The results demonstrate that the observed inhibition of Ca²⁺ transport by Eu³⁺ is due to its binding to Ca²⁺ translocating sites. The ⁷F₀→⁵D₀ transition of Eu³⁺ bound to these sites was monitored. The non‐Lorentzian nature of the excitation profile and a double‐exponential fluorescence decay revealed the heterogeneity of the two sites. Measurement of fluorescence decay rates in H₂O/D₂O mixture buffers further distinguished the sites. The number of water molecules in the first co‐ordination sphere of Eu³⁺ bound at transport sites were found to be 4 and 1.5. Addition of ATP reduced these numbers to zero and 0.6. These data show that the calcium ions in translocating sites are well enclosed by protein ligands and are further occluded down to zero or one water molecule of solvation during the transport process.