Purification and characterization of lysophospholipase D from rat brain

Abstract A lysophospholipase D (lysoPLD) was purified to apparent homogeneity from rat brain nuclear fractions using 1-[ 14 C]palmitoyl-glycerophosphorylcholine as a substrate. The abundance of autotaxin (ATX), a secretory lysoPLD, was also estimated for each fraction. The nuclear fraction had relatively high levels of lysoPLD activity but weak immunoreactivity with an anti-ATX antibody. LysoPLD activity was further purified 5550-fold by sequential chromatography. The final preparation migrated as a single band with a molecular weight of 35,000. Anti-ATX antibodies did not cross-react with the purified enzyme. Moreover, enzyme activity was highest at pH 7.0–7.5 and requires Mg 2+ . The K m and V max values for 1-palmitoyl-glycerophosphorylcholine were 176 μM and 0.3 μmol/min/mg, respectively. The purified enzyme hydrolyzed saturated forms of LPC more robustly than unsaturated forms. The enzyme could hydrolyze platelet-activating factor (PAF) to the same extent as 16:0-LPC, and showed a higher activity toward lysoPAF (1- O -hexadecyl-2-lyso-glycerophosphorylcholine). These results suggested that the lysoPLD purified from rat brain nuclear fractions in this work is a novel enzyme that hydrolyzes lysoPAF, PAF, and LPC to liberate choline.