Purification and characterization of a lysophosphatidic acid-specific phosphatase.
Lysophosphatidic acid (LPA)-specific phosphatase was purified 3300-fold from bovine brain cytosol. The purification was achieved by (NH4)2SO4 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 1-phosphate or sphingosine 1-phosphate, although phosphatidic acid was hydrolysed slightly. The purified enzyme hydrolysed 1-oleoyl LPA at a rate of 1. 1 micromol/min per mg of protein when assayed with LPA as Triton X-100 mixed micelles. The Km value for LPA was 38 microM. 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.
Hiroyama
M
M
Department of Biochemistry, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan.
Takenawa
T
T
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008246:Lysophospholipids / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D010712:Phosphatidic Acids; C076020:lysophosphatidic acid phosphatase; D010744:Phosphoric Monoester Hydrolases