A phospholipase C with a high specificity for platelet-activating factor in rabbit liver light mitochondria.
The light mitochondrial fraction from rabbit liver was found to catalyze the hydrolysis of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by the phospholipase C reaction to form 1-O-alkyl-2-acetyl-glycerol and phosphocholine. The highest specific phospholipase C activity occurred in the liver and kidney. A subcellular survey showed that the enzyme was of lysosomal origin. The enzyme was solubilized with 2% Triton X-100 from rabbit liver light mitochondria and purified ca. 600- to 700-fold with a 17% yield using procedures that included hydroxyapatite, Sepharose 4B and isoelectric focusing column chromatography followed by fast protein liquid chromatography. The enzyme consists of two forms having a pl of 4.7 and 5.8. Each form was purified to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The enzyme migrated to positions corresponding to apparent molecular weights of 33,000 and 75,000, respectively. The purified enzymes of pl 4.7 and 5.8 had pH optima of 8.2 and 8.5 and apparent Km values of 55.6 and 45.5 microM for PAF, respectively. Furthermore, their phospholipase C activity was significantly inhibited by the addition of 1 mM EDTA. EDTA-inactivated enzyme, however, recovered completely upon addition of Ca2+ to the original level. p-Chloromercuribenzoate markedly inhibited enzyme activity, suggesting that phospholipase C is a -SH enzyme. The physiological role of the enzyme should be evaluated, considering its specificity for a highly potent, biologically active ether-phospholipid.
D000818:Animals; D002413:Cations, Divalent; D002458:Cell Fractionation / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D010972:Platelet Activating Factor / Q000378:metabolism*; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism
D002413:Cations, Divalent; D010972:Platelet Activating Factor; D010738:Type C Phospholipases