Purification and characterization of lysophospholipase D from rat brain.
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 Km and Vmax values for 1-palmitoyl-glycerophosphorylcholine were 176 microM and 0.3 micromol/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.
Sugimoto
Sayaka
S
Department of Molecular Biochemistry, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan.
Sugimoto
Hiroyuki
H
Aoyama
Chieko
C
Aso
Chizu
C
Mori
Masatomo
M
Izumi
Takashi
T
D000818:Animals; D001921:Brain / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D000099189:Lysophospholipase D
D008244:Lysophosphatidylcholines; D010727:Phosphoric Diester Hydrolases; D010972:Platelet Activating Factor; D011755:Pyrophosphatases; D000099189:Lysophospholipase D; C029271:O-deacetyl platelet activating factor