Purification of a putative K+-ATPase from Streptococcus faecalis.

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Membrane-bound ATPases play critical roles in cellular transport and energy transduction.
• Understanding the diversity and function of ATPases in bacteria like Streptococcus faecalis is essential for microbial physiology research.

Purpose of the Study:

• To isolate and characterize a novel membrane ATPase from Streptococcus faecalis.
• To determine the biochemical properties and potential function of the purified ATPase.

Main Methods:

• Purification involved Triton X-100 extraction followed by sequential chromatography (DEAE-cellulose, hydroxylapatite, Cm-Sepharose).
• Enzyme activity was assessed by ATP hydrolysis, and kinetic parameters (Km, optimal pH) were determined.
• The purified enzyme was reconstituted into phospholipid vesicles to study coupled transport.

Main Results:

• A novel membrane ATPase was purified, consisting of a single 78,000 Mr polypeptide.
• The enzyme exhibited specific activity dependent on phospholipids and was inhibited by vanadate.
• It formed an acylphosphate intermediate and demonstrated coupled transport activity when reconstituted, stimulated by valinomycin and potassium.

Conclusions:

• The purified enzyme is an ion-motive ATPase, strongly suggesting it is a potassium ATPase (K+-ATPase).
• This finding contributes to understanding energy-coupling mechanisms in bacterial membranes.