Characterization of a P-type Ca(2+)-ATPase from Flavobacterium odoratum

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Intracellular calcium homeostasis in bacteria is typically maintained by secondary exchangers using ion gradients.
• Prokaryotic calcium ATPases are rare, with none previously identified in Gram-negative bacteria.

Purpose of the Study:

• To investigate the mechanism of ATP-dependent calcium uptake in the Gram-negative bacterium Flavobacterium odoratum.
• To characterize the enzyme responsible for calcium transport in F. odoratum.

Main Methods:

• Everted membrane vesicle preparation from F. odoratum.
• Enzyme activity assays measuring calcium uptake and ATP hydrolysis.
• Inhibition studies using specific ATPase inhibitors (orthovanadate) and ionophores.
• Enzyme solubilization, partial purification, and kinetic characterization (Km, IC50).
• Phosphorylation assays using [gamma-32P]ATP and SDS-PAGE analysis.

Main Results:

• Demonstrated ATP-dependent calcium uptake in F. odoratum membrane vesicles.
• Identified a calcium-dependent ATPase activity inhibited by vanadate but not other inhibitors.
• Partially purified enzyme showed kinetic properties similar to eukaryotic Ca(2+)-ATPases.
• Phosphorylation studies confirmed the presence of an acylphosphate intermediate characteristic of P-type ATPases.

Conclusions:

• The primary mechanism for calcium transport in F. odoratum is a P-type ATPase.
• This discovery expands the known diversity of calcium transport systems in prokaryotes.
• The characterized enzyme shares functional similarities with eukaryotic sarcoplasmic reticulum calcium ATPase.