Depolymerization of solubilized gastric (H+ + K+)-ATPase by n-octylglucoside or cholate

Area of Science:

• Biochemistry
• Membrane protein biochemistry

Background:

• Active (H+ + K+)-ATPase was previously extracted from gastric apical membranes using n-octylglucoside.
• This extract contained a large holomeric enzyme (390-420 kDa) retaining significant K+-stimulated ATPase activity.

Purpose of the Study:

• To investigate the depolymerization and inactivation of (H+ + K+)-ATPase during solubilization.
• To characterize the effects of different detergents on enzyme structure and activity.

Main Methods:

• Solubilization of gastric apical membranes using n-octylglucoside.
• Enzyme depolymerization studies using increasing concentrations of n-octylglucoside and cholate.
• Analysis of enzyme structure and activity using molecular sieve chromatography and glycerol gradient centrifugation.

Main Results:

• Inactivation of (H+ + K+)-ATPase correlated with the appearance of smaller polymorphic structures (330-360 kDa and 240-250 kDa).
• Cholate effectively depolymerized the enzyme into different sized structures without significant loss of ATPase or phosphatase activity.
• Higher cholate concentrations (2.5%) inactivated the ATPase and led to inactive protein aggregates.

Conclusions:

• Solubilization of (H+ + K+)-ATPase involves initial polymer extraction followed by depolymerization.
• Cholate is a suitable detergent for depolymerizing the enzyme while preserving activity, suggesting different oligomeric states (monomers, dimers, trimers, tetramers) exist.
• Monomeric forms of the enzyme appear inactive under the tested conditions.