Identification of a Spectroscopic Marker for the Ca²⁺-Binding Site of (Ca²⁺ + Mg²⁺)-ATPase of Sarcoplasmic Reticulum in the Occluded State

Abstract

The ⁷F₀ → ⁵D₀ excitation spectrum of Eu³⁺ bound to the high-affinity calcium sites of SR (Ca^{2 +} + Mg²⁺)-ATPase diminishes upon occlusion of the Eu³⁺ into the interior of the enzyme. This “quenching” was found to be caused by the enzyme itself because trypsin digestion could relieve it. The level of digestion needed to relieve the quenching is beyond the level needed to eliminate occlusion; thus, the two processes are not related. Ca²⁺ is required during digestion to preserve the quenching, indicating close proximity between the Ca²⁺ site(s) and the quenching segment. Synthetic peptides were found that could mimic the native enzyme's ability to quench the Eu³⁺ fluorescence, although no native sequence has yet been identified that could emulate the enzyme

Key Words:

• Calcium
• occlusion
• (Ca²⁺ + Mg²⁺)-ATPase
• sarcoplasmic reticulum
• fluorescent spectroscopy
• europium