Occluded calcium sites in soluble sarcoplasmic reticulum Ca2+-ATPase

Area of Science:

• Biochemistry
• Biophysics
• Enzyme kinetics

Background:

• Rabbit muscle sarcoplasmic reticulum Ca2+-ATPase binds gadolinium ions (Gd3+) at two high-affinity Ca2+ sites.
• Bound Gd3+ exhibits long electron spin relaxation times, suggesting site occlusion and reduced solvent contact.

Purpose of the Study:

• To investigate the nature of Gd3+ binding sites in solubilized Ca2+-ATPase.
• To determine the accessibility of these sites to solvent water molecules.

Main Methods:

• Proton relaxation assays to determine dipolar correlation times (tau c) of 1H-Gd3+ interactions.
• Analytical ultracentrifugation and size-exclusion chromatography to assess enzyme quaternary structure.

Main Results:

• Solubilized Ca2+-ATPase yielded distinct tau c values for Gd3+ at site 1 (1.04 x 10(-9) s) and site 2 (1.98 x 10(-9) s).
• Frequency dependence of tau c indicated dominance by Gd3+ electron spin relaxation.
• The enzyme was found to be monomeric in solution.
• Long tau c values suggest poor accessibility of Gd3+ sites to water, especially site 2.

Conclusions:

• Occlusion of Ca2+ sites in Ca2+-ATPase is primarily determined by the tertiary structure of the monomeric enzyme.
• Site occlusion is independent of multimeric membrane structures.