Molecular characterization of a phosphatidylcholine‐hydrolyzing phospholipase C

摘要: 1While searching for a phospholipase C (PLC) specific phosphatidylcholine in mammalian tissues, we came across such an activity originating from contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing the cold. The secreted phosphatidylcholine-hydrolyzing PLC purified by combination chromatographic procedures. As substrates, enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol. active is monomer ≈ 40 kDa. other bacterial PLCs, requires Ca2+ Zn2+ activity; dithiothreitol affected due its chelation Zn2+, but this inhibition could be compensated addition ZnCl2. compound D609, described selectively inhibit phosphatidylcholine-specific caused half-inhibition P. fluorescens at ≈ 420 µm, while 50-fold lower concentrations similarly PLCs Bacillus cereus Clostridium perfringens. Partial peptide sequences obtained pure after tryptic cleavage were used clone DNA fragment 3.5 kb gene library prepared our laboratory isolate. It contains ORF 1155 nucleotides encoding PLC. There no significant sequence homology suggesting that represents distinct subclass PLCs. protein lacks cysteine residues consequently disulfide bonds. Interestingly, reference strain DSMZ 50090 devoid here as well relevant coding sequence.