Cholesteryl de-esterifying enzyme from Staphylococcus aureus: separation from alpha toxin, purification, and some...

Area of Science:

• Microbiology
• Enzymology
• Biochemistry

Background:

• Staphylococcus aureus produces alpha toxin, a known virulence factor.
• Partially purified alpha toxin preparations contained a cholesteryl de-esterifying enzyme.
• The relationship between alpha toxin and this esterase was not fully understood.

Purpose of the Study:

• To separate and purify the cholesteryl esterase from alpha toxin.
• To characterize the physical and biochemical properties of the purified esterase.
• To investigate the potential role of other molecules in esterase stability.

Main Methods:

• Isoelectric focusing and gel filtration chromatography for enzyme separation.
• Analytical ultracentrifugation, gel diffusion, immunoelectrophoresis, and SDS-PAGE for purity assessment.
• Molecular weight determination using gel filtration and SDS-PAGE.

Main Results:

• Cholesteryl esterase was successfully separated and purified, showing high purity.
• The enzyme exists as an associating system with a protomer molecular weight of 25,500 and a higher aggregate weight of 175,000.
• A non-cholesterol neutral lipid, possibly a glycolipid, was found and may stabilize the enzyme.
• The esterase has an isoelectric point of 9.1, is labile, and loses activity upon reversible binding to agarose.

Conclusions:

• Cholesteryl esterase is a distinct enzyme from alpha toxin in Staphylococcus aureus.
• The enzyme's properties suggest it is an associating system potentially stabilized by a lipid.
• The production of cholesteryl esterase is linked to alpha toxin production in the Wood 46 strain.