Properties of deoxycholate solubilized sarcoplasmic reticulum Ca2+-ATPase

Area of Science:

• Biochemistry
• Membrane Protein Biochemistry
• Enzymology

Background:

• The Ca2+-dependent ATPase of sarcoplasmic reticulum is a crucial membrane protein involved in calcium transport.
• Understanding its structure and oligomeric state is essential for elucidating its enzymatic mechanism and membrane interactions.

Purpose of the Study:

• To characterize the molecular forms and properties of solubilized Ca2+-dependent ATPase after lipid removal.
• To investigate the relationship between the oligomeric state, lipid content, and enzymatic activity of the ATPase.

Main Methods:

• Solubilization of sarcoplasmic reticulum ATPase using deoxycholate.
• Lipid removal via gel chromatography.
• Analytical ultracentrifugation for molecular weight and shape determination.
• Circular dichroism for conformational analysis.
• Sedimentation velocity studies to assess enzymatic activity of different forms.

Main Results:

• Solubilized ATPase exists as a mixture of monomer, dimer, and higher aggregates, with monomer molecular weight estimated at 115,000 Da and an asymmetric shape.
• Enzymatic activity is largely lost upon delipidation but can be temporarily retained in the presence of phospholipid and sucrose, primarily from the monomeric form.
• Conformational changes and delipidation accompany inactivation, with no significant differences found between monomer and dimer fractions via isoelectric focusing or amino acid analysis.

Conclusions:

• The Ca2+-dependent ATPase monomer, possessing an asymmetric shape, is likely the primary species responsible for enzymatic activity.
• Lipid removal and conformational changes lead to ATPase inactivation.
• The findings support models of an elongated ATPase with limited hydrophobic membrane interaction.