title: Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C.
abstract: We have purified to homogeneity the 33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) from the culture fluid of Listeria monocytogenes, a facultative intracellular pathogen. The protein was overexpressed, and secretion of PI-PLC was further enhanced by the addition of divalent cations to the culture medium. The basic protein (pI, approximately 9.4) was complexed with anionic proteins in the crude culture fluid. It bound to DEAE-Sepharose and was eluted from Sephacryl S-200 near the void volume in low-ionic-strength buffer, suggesting aggregates of greater than or equal to 150 kDa. Gel filtration chromatography on Sephacryl S-200 in the presence of 1 M ammonium sulfate resulted in disaggregation and complete separation of PI-PLC, which interacted with the column matrix. Amino-terminal sequencing of the pure protein gave results consistent with the previously deduced sequence and showed that the signal cleavage site was between alanine 29 and tyrosine 30. The enzyme was specific for PI and showed no activity with phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It did not cleave PI-4-phosphate or PI-4,5-bisphosphate, but it was active on the membrane form of the variable surface glycoprotein from Trypanosoma brucei, a PI-glycan-anchored protein. When assayed with deoxycholate-mixed micelles of PI, activity was highly dependent on added salt. Activation by salt was also observed with Triton X-100-mixed micelles. The optimal concentration of CaCl2 or MgCl2 was lower than that of KCl or (NH4)2SO4, but activity was not specifically dependent on divalent cations and was not inhibited by addition of EDTA. With deoxycholate, the optimum pH was 7.0. A broader pH optimum ranging from 5.5 to 6.5 was observed with Triton X-100-mixed micelles. These results are consistent with a postulated role for secreted PI-PLC in the acidified primary phagocytic vesicle of infected cells.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
authors.forename: H
authors.initials: H
authors.lastname: Goldfine
authors.forename: C
authors.initials: C
authors.lastname: Knob
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000254:growth & development; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D010738:Type C Phospholipases / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D008274:Magnesium; D002118:Calcium