Detergent-solubilized sarcoplasmic reticulum ATPase. Hydrodynamic and catalytic properties

Area of Science:

• Biochemistry
• Membrane Proteins
• Enzyme Kinetics

Background:

• Sarcoplasmic reticulum ATPase (SERCA) is crucial for muscle calcium regulation.
• Understanding SERCA's structure-function relationship requires its isolation and characterization.
• Detergent-based solubilization is a common method for studying membrane proteins.

Purpose of the Study:

• To investigate the effect of nonionic detergent dodecyl nonaoxyethylene alcohol (C12E9) on sarcoplasmic reticulum Ca2+ ATPase activity.
• To characterize the biophysical properties of the detergent-solubilized monomeric ATPase.
• To explore the calcium binding and activation kinetics of the solubilized enzyme.

Main Methods:

• Solubilization of sarcoplasmic reticulum vesicles using C12E9.
• Assay of Ca2+ ATPase activity.
• Calcium ionophore-mediated permeability measurements.
• Sepharose 6B chromatography for complex analysis.
• Time-resolved fluorescence anisotropy decay for rotational dynamics.
• Determination of Stokes radius and axial ratio.
• Calcium binding affinity measurements.
• Analysis of ATP hydrolysis activation by calcium (Hill coefficient).

Main Results:

• C12E9 solubilization increased Ca2+ ATPase activity approximately 5-fold, likely due to enhanced calcium permeability.
• The detergent-solubilized monomeric ATPase maintained complete activity and high calcium affinity (9 nmol Ca2+/mg protein).
• The C12E9-ATPase complex exhibited significant asymmetry (axial ratio 5-6) with a Stokes radius of ~55-59 A.
• Calcium-dependent ATP hydrolysis activation was cooperative (Hill coefficient 1.8), similar to the vesicular form.

Conclusions:

• Detergent-mediated solubilization with C12E9 effectively activates and preserves the functional integrity of sarcoplasmic reticulum Ca2+ ATPase.
• The detergent-solubilized monomeric ATPase retains essential properties, including high calcium affinity and cooperative calcium activation.
• The observed cooperativity suggests intramolecular interactions between calcium binding sites within the ATPase enzyme.