Human neutrophil cytosolic phospholipase C: partial characterization

The activity of neutrophil cytosolic phospholipase C on PIP₂ and PI was compared employing [³H]inositol-labeled heat-inactivated membranes of differentiated HL-60 cells, into which tracer [³²P]PIP₂ was incorporated. Hydrolysis of PIP₂ did not require Ca²⁺ and was stimulated when the content of PIP₂ in the membrane was increased by incorporation of unlabeled inositol lipid. At equal concentrations of PI and PIP₂ in the membrane, hydrolysis of PIP₂ was faster and no evidence of competition between the two substrates was obtained. Incorporation of PI into PE-[³²P]PIP₂ vesicles, accelerated PIP₂ hydrolysis also at conditions that favor hydrolysis of PI. Partial purification of neutrophil cytosolic PLC on Q Sepharose, phenyl Sepharose and heparin-Agarose columns is described. From heparin-Agarose column, two PLC activity peaks exhibiting different substrate specificities were eluted. The elution profile of the main PLC species from Superose 12 gel filtration column was compatible with an approx. 150 kDa protein.