Figure 5. Acyl chain length specificity and headgroup specificity of PLB.

(A) Recombinant PLB was assayed using three different PC species: cellular PC extracted from [³H]choline-labelled HL60 cells and [³H]choline-labelled dipalmitoyl-PC (Dipalm-PC) and didecanoyl-PC (Didec-PC). Assays were performed for 60 min at 37 °C. (B) Partially purified PLB (purified on Superdex-200, Heparin, and Mono-Q) was assayed using the same substrates as in (A). Samples containing 7.5 μg of partially purified PLB were incubated for 20 min at 37 °C. (C) Partially purified PLB [as in (B)] was assayed for activity using appropriately-labelled lipids purified from HL60 cells (grown for 48 h with [³H]choline, [³H]ethanolamine or [³H]inositol). Samples containing 1.5 μg of partially purified PLB were incubated for 60 min at 37 °C. Water-soluble products were extracted, separated into phosphorylated and non-phosphorylated metabolites, and quantified. Assays were performed in duplicate and the results shown are representative of three independent experiments. Results are shown for percentage hydrolysis of substrate, of which >95% in each case was the deacylated lipid (glycerophosphorylcholine, glycerophosphorylinositol or glycerophosphorylethanolamine).