Phospholipase C from Bacillus cereus has sphingomyelinase activity.

Area of Science:

• Biochemistry
• Enzymology

Background:

• Phospholipase C (PLC) is an enzyme involved in lipid metabolism.
• Understanding the catalytic properties and metal ion dependencies of PLC is crucial for elucidating its biological roles.

Purpose of the Study:

• To characterize the different forms of purified phospholipase C (PLC) obtained by isoelectric focusing.
• To investigate the role of zinc ions in the activity of PLC and its associated sphingomyelinase activity.

Main Methods:

• Purification of phospholipase C (PLC) to electrophoretic homogeneity.
• Isoelectric focusing in sucrose gradients to separate enzyme variants.
• Enzyme activity assays for lecithinase and sphingomyelinase.
• Metal ion substitution experiments (Zn2+, Co2+, Ca2+, Mg2+).

Main Results:

• PLC preparations separated into two peaks by isoelectric focusing, with different isoelectric pH values and zinc content.
• The main peak (pI 6.6-6.8) contained two Zn2+ per molecule, while the acidic peak (pI ~6.2) contained one Zn2+ and showed reduced specific activity.
• Adding Zn2+ restored the activity of the acidic peak.
• Sphingomyelinase activity was intrinsically associated with PLC, as evidenced by its co-elution and enhancement with Co2+ substitution for Zn2+.

Conclusions:

• Phospholipase C (PLC) exists in at least two forms with differing zinc content and specific activities.
• The enzyme's sphingomyelinase activity is an endogenous function, modulated by metal ions, particularly zinc and cobalt.