Interactions between sarcoplasmic reticulum calcium adenosintriphosphatase and nonionic detergents.

Area of Science:

• Biochemistry
• Membrane Protein Studies
• Enzyme Kinetics

Background:

• The Ca2+ ATPase enzyme from sarcoplasmic reticulum plays a crucial role in muscle contraction.
• Understanding detergent interactions is vital for studying membrane proteins in their native-like states.
• Nonionic detergents like Triton X-100 are commonly used to solubilize and stabilize membrane proteins.

Purpose of the Study:

• To investigate the binding of Triton X-100 and other nonionic detergents to a delipidated Ca2+ ATPase.
• To elucidate the relationship between detergent binding, micelle formation, and ATPase activity reactivation.
• To infer the nature of phospholipid interactions with the Ca2+ ATPase in the native membrane.

Main Methods:

• Radiolabeled Triton X-100 binding assays using column chromatography.
• Measurement of ATPase activity at varying detergent concentrations.
• Analysis of detergent micelle structure using cis-parinaric acid fluorescence and differential scanning calorimetry.
• Determination of temperature dependence of ATPase activity via Arrhenius plots.

Main Results:

• Two classes of Triton X-100 binding sites were identified on the delipidated ATPase.
• Monomeric Triton X-100 binding below the critical micelle concentration (cmc) activated ATPase activity.
• Full ATPase activity recovery occurred near the cmc, with polar detergent portions being key for reactivation.
• Detergent micelle structure and ATPase polypeptide behavior influence temperature-dependent activity.

Conclusions:

• The Ca2+ ATPase possesses discrete binding sites for phospholipids, with polar head groups showing stronger interaction than hydrophobic chains.
• Nonionic detergent binding and micelle properties are critical for restoring and modulating Ca2+ ATPase activity.
• Both the detergent's physical state and the intrinsic properties of the ATPase polypeptide govern its temperature-dependent function.