Purification and characterization of a lysophasphatidic acid-specific phosphatase

Lysophosphatidic acid (LPA)-specific phosphatase was purified 3300-fold from bovine brain cytosol. The purification was achieved by (NH4)(2)SO4 fractionation and several chromatography steps, such as Q-Sepharose, DEAE-5PW, Superdex 200 and heparin-Sepharose. The final enzyme preparation showed a single band of molecular mass 44 kDa on SDS/PAGE under reducing conditions. The enzyme activity was completely dependent on the presence of detergents such as Triton X-100, CHAPS, cholate and octyl-beta-glucoside. The activity was independent of Mg2+; other cations were inhibitory. The enzyme hydrolysed LPA specifically but not cardiolipin, tetraoleoyl-bisphosphatidic acid, ceramide I-phosphate or sphingosine 1-phosphate, although phosphatidic acid was hydrolysed slightly. The purified enzyme hydrolysed I-oleoyl LPA at a rate of 1.1 mu mol/min per mg of protein when assayed with LPA as Triton X-100 mixed micelles. The K-m value for LPA was 38 mu M. NaF and N-ethylmaleimide markedly inhibited the activity, but propranolol had a less potent inhibitory effect. The LPA-specific phosphatase might have an important role in LPA elimination.