Isolation and characterization of three lysophospholipases from the murine macrophage cell line WEHI 265.1.
Anion exchange chromatography of WEHI 265.1 cell homogenates resolved the lysophospholipase activity into three peaks, when assayed using lysophosphatidylcholine as a substrate. Peaks 1 and 2 were purified by sequential hydrophobic interaction and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks 1 and 2 indicated homogeneous proteins with apparent masses of 28 and 27 kDa, respectively. Peak 3 lysophospholipases was partially purified by hydrophobic, hydroxyapatite and gel filtration chromatography. Peak 3 lysophospholipase also had calcium-dependent phospholipase A2 activity, which further co-purified with the lysophospholipase activity. The three lysophospholipases were characterized with respect to substrate specificity, additional enzymatic activities and the effects of lipids, metal ions and other compounds on enzymatic activity. Peaks 1, 2 and 3 hydrolyzed lysophosphatidylcholine most readily, but lysophosphatidylethanolamine also served as substrate for each enzyme. Furthermore, all three enzymes hydrolyzed platelet activating factor and acetylated lysophosphatidylcholine. Each lysophospholipase was inhibited by free fatty acids and by palmitoyl carnitine, although the relative sensitivities to these agents differed among the enzymes. The lysophospholipase activities of peaks 1 and 2, but not peak 3, were inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate and N-ethylmaleimide. Although they had similar masses, the amino acid compositions of peaks 1 and 2 differed, indicating that these are distinct proteins rather than posttranslational modifications of the same gene product.
Garsetti
D E
DE
Schering-Plough Research Institute, Bloomfield, NJ 07003.
Ozgür
L E
LE
Steiner
M R
MR
Egan
R W
RW
Clark
M A
MA
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008245:Lysophospholipase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008264:Macrophages / Q000201:enzymology*; D008670:Metals / Q000378:metabolism; D051379:Mice; D010172:Palmitoylcarnitine / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D005227:Fatty Acids; D008244:Lysophosphatidylcholines; D008670:Metals; D010743:Phospholipids; D010172:Palmitoylcarnitine; D008245:Lysophospholipase