Membrane ATPase of Proteus L-forms. Solubilization and molecular properties.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• The Mg2+-dependent ATPase (EC 3.6.I.3) is a crucial enzyme found in cell membranes.
• Understanding its structure and function is vital for comprehending cellular energy processes.
• Proteus L-forms present a unique model for studying membrane-bound enzymes.

Purpose of the Study:

• To solubilize and purify the Mg2+-dependent ATPase from Proteus L-form membranes.
• To characterize the molecular weight and subunit composition of the purified enzyme.
• To propose a structural formula for the native ATPase.

Main Methods:

• Solubilization using various agents including Tris-HCl shock-wash, EDTA, and Triton X-100.
• Enzyme purification via ultracentrifugation (105,000 x g) and acrylamide/agarose gel electrophoresis.
• Molecular weight determination using gel filtration and sedimentation coefficient.
• Subunit analysis using polyacrylamide disc-gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE).

Main Results:

• The optimal solubilization method involved a 33mM Tris-HCl (pH 7.5) shock-wash without Mg2+.
• The purified ATPase has a molecular weight of approximately 360,000.
• SDS-PAGE revealed two subunits, alpha (64,000 Da) and beta (58,000 Da), in a 1:1 ratio.
• A proposed formula for the native enzyme is alpha-3beta-3.

Conclusions:

• A robust method for solubilizing and purifying Proteus L-form ATPase was established.
• The enzyme is a heterooligomer composed of two distinct subunits.
• The findings provide insights into the structure of Mg2+-dependent ATPases and their potential similarities across different organisms.