Measuring H(+) Pumping and Membrane Potential Formation in Sealed Membrane Vesicle Systems

Area of Science:

• Biochemistry
• Membrane Biology
• Biophysics

Background:

• Enzyme activity in active transport across lipid bilayers is often indirectly measured via energy consumption (e.g., ATP hydrolysis).
• Directly assaying transport activity is challenging because substrates are not converted to products and movement is on the nanometer scale.

Purpose of the Study:

• To describe two novel methods for measuring active proton pumping across lipid bilayers.
• To demonstrate the concomitant formation of a membrane potential during proton transport.
• To exemplify these methods using the Arabidopsis thaliana plasma membrane H(+)-ATPase.

Main Methods:

• Utilized fluorescent dyes 9-amino-6-chloro-2-methoxyacridine (ACMA) and oxonol VI to monitor proton transport and membrane potential.
• Reconstituted purified Arabidopsis thaliana plasma membrane H(+)-ATPase (proton pump) into proteoliposomes.
• Applied the developed methods to assay the activity of the reconstituted proton pump.

Main Results:

• Successfully measured active proton pumping across lipid bilayers using ACMA and oxonol VI.
• Demonstrated the formation of a membrane potential correlated with proton transport.
• Validated the efficacy of the new methods with a well-characterized proton pump.

Conclusions:

• The described methods provide a direct and convenient way to assay active proton pumping by membrane proteins.
• These techniques are valuable for studying the function of transporters and ion pumps.
• The methods are applicable to various systems, including heterologously expressed and reconstituted proteins.