Solubilization and reconstitution of ca pump from corn leaf plasma membrane

Area of Science:

• Plant Physiology
• Membrane Biology
• Biochemistry

Background:

• The calcium (Ca(2+)) transport system in corn (Zea mays) leaf plasma membranes involves a Ca(2+) pump and a Ca(2+)/H(+) antiporter.
• Characterizing these transporters requires effective methods for solubilization, isolation, and reconstitution into liposomes.

Purpose of the Study:

• To establish procedures for solubilizing and reconstituting the Ca(2+) pump from corn leaf plasma membranes.
• To identify the most effective detergent for Ca(2+) pump extraction and functional reconstitution.

Main Methods:

• Seven detergents were screened for their effectiveness in extracting and reconstituting the Ca(2+) pump.
• Octaethyleneglycol monododecyl ether (C(12)E(8)) was identified as the most effective detergent.
• High-performance liquid chromatography (HPLC) with a DEAE anion exchange column was used for further separation.
• ATP-dependent (45)Ca(2+) uptake assays were performed on reconstituted liposomes.

Main Results:

• C(12)E(8) effectively solubilized and allowed functional reconstitution of the Ca(2+) pump.
• HPLC separated Ca(2+)-ATPase from Mg(2+)-ATPase.
• Reconstituted liposomes with Ca(2+)-ATPase exhibited ATP-dependent Ca(2+) uptake.
• The Ca(2+)/H(+) antiporter was not detected in the Ca(2+)-ATPase preparation.

Conclusions:

• The Ca(2+)-ATPase isolated from corn leaf plasma membranes functions as a Ca(2+) pump.
• The detergent C(12)E(8) is suitable for solubilizing and reconstituting this Ca(2+) pump.
• The study provides a foundation for further characterization of plant plasma membrane Ca(2+) transporters.