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.
Faber
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Department of Biochemistry, Tel Aviv University, Israel.
Aviram
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D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000096:biosynthesis; D010738:Type C Phospholipases / Q000097:blood* / Q000302:isolation & purification
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010738:Type C Phospholipases