Active detergent-solubilized H+,K+-ATPase is a monomer.

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Protein Biochemistry

Background:

• Gastric acid secretion relies on H(+),K(+)-ATPase, crucial for stomach pH regulation.
• The structural organization of H(+),K(+)-ATPase in relation to ion transport remains incompletely understood.
• This enzyme comprises catalytic alpha and glycosylated beta subunits.

Purpose of the Study:

• To purify and characterize the pig gastric H(+),K(+)-ATPase.
• To determine the oligomeric state and functional properties of the purified enzyme.
• To elucidate the relationship between enzyme structure and function in membrane environments.

Main Methods:

• Solubilization using non-ionic detergent C(12)E(8) followed by detergent exchange with Tween 20.
• Purification via Superose 6 gel filtration chromatography.
• Characterization using mass spectrometry, analytical ultracentrifugation, sedimentation velocity, and light scattering.

Main Results:

• Pure, functionally active pig gastric H(+),K(+)-ATPase was obtained with a Stokes radius of 6.3 nm.
• Mass spectrometry confirmed beta-subunit glycosylation (9 kDa excess mass).
• Analytical ultracentrifugation and sedimentation velocity data indicated a monomeric α,β-protomer (147.3 kDa) with bound lipids and detergent, rejecting an α(2),β(2) dimer.

Conclusions:

• The purified H(+),K(+)-ATPase is active in detergent solution.
• The enzyme likely functions as a monomer, consistent with other P-type ATPases like Ca(2+)-ATPase and Na(+),K(+)-ATPase.
• This provides insights into the molecular mechanisms of gastric acid production.