Phospholipase C from two bacterial strains acts differently on pure phospholipids and membrane bound glycosylphosphatidylinositol (GPI) anchors.
Phospholipase C (PLC) was purified to homogeneity from the culture filtrate of Bacillus cereus (65-fold, 540 U/mg protein) and B. thuringiensis (76-fold, 306 U/mg protein) by conventional techniques of enzyme purification. The purified enzymes have the molecular mass of 34 kDa and 38 kDa respectively, as determined by SDS-PAGE. Both the PLCs exhibited identical sensitivity to pH, temperature, cations, anions and inhibitors like glutathione and p-chloromercuribenzoate. PLC-Bc showed a preference for phosphatidylinositol, while PLC-Bt favoured phosphatidylcholine as the substrate. Although both the enzymes were able to hydrolyze pure phosphatidylinositol, distinct differences were observed in their activity on phosphatidylinositol-anchored membrane proteins. PLC-Bc cleaved and released alkaline phosphatase, a GPI-anchored marker enzyme from microsomal membranes to a greater extent, than PLC-Bt. Experiments with sperm membranes, followed by SDS-PAGE revealed that the pattern of proteins released from their GPI-anchors by PLC-Bc and PLC-Bt were dissimilar. Although some proteins were cleaved in common by both PLCs, some others including a prominent 57 kDa protein were resistant to PLC-Bt, but sensitive to cleavage by PLC-Bc. The type of modification in the GPI anchor, special environment on membranes, and relative charge of host plasma membrane to the charge of PLC may be the factors that are responsible for the differential action of two enzymes.
Reproductive Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttranchal 247 667, India.
Arshi
A
Rastogi
Tarun E
TE
Hutchinson
Ben M J
BM
Pereira
D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology*; D001413:Bacillus thuringiensis / Q000201:enzymology*; D002462:Cell Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006041:Goats; D007668:Kidney / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D013737:Testis / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
D017261:Glycosylphosphatidylinositols; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010738:Type C Phospholipases