Purification and characterization of a lysophospholipase from a macrophage-like cell line P388D1.
Two lysophospholipase activities (designated I and II) were identified in the macrophage-like cell line P388D1. Lysophospholipase I was purified (8,500-fold) to homogeneity by DEAE-Sephacel, Sephadex G-75, Blue-Sepharose, and chromatofocusing chromatography. Lysophospholipase II was separated from the lysophospholipase I in the Blue-Sepharose step. The apparent molecular mass of lysophospholipase I and II are 27,000 and 28,000 daltons, respectively, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their pI values were 4.4 and 6.1 respectively, as determined by isoelectric focusing. Lysophospholipase I exhibited a broad pH optimum between 7.5-9.0. The double-reciprocal plot of the substrate dependence curve of the purified lysophospholipase I showed a break around the critical micelle concentration of the substrate (1-palmitoyl-sn-glycerol-3-phosphorylcholine). The apparent Km, determined from substrate concentrations above 10 microM was 22 microM, and the apparent Vmax was 1.3 mumol min-1mg-1. The purified enzyme did not have phospholipase A1, phospholipase A2, acyltransferase, or lysophospholipase-transacylase activity. No activity was detected toward triacylglycerol, diacylglycerol, p-nitrophenol acetate, p-nitrophenol palmitate, or cholesterol ester. The enzyme did, however, hydrolyze monoacylglycerol although at a rate 20-fold less than lysophospholipid, 0.06 mumol min-1mg-1. The lysophospholipase I was inhibited by fatty acids but not by glycerol-3-phosphorylcholine, glycerol-3-phosphorylethanolamine, or glyc-fjerol-3-phosphorylserine. A synthetic manoalide analogue 3(cis,cis,-7,10)hexadecadienyl-4-hydroxy-2-butenolide inhibited the enzyme with half-inhibition (IC50) at about 160 microM. Triton X-100 decreased the enzymatic activity, although this apparent inhibition can be explained by a "surface dilution" effect. The pure lysophospholipase I was stable for at least 5 months at -20 degrees C in the presence of glycerol and beta-mercaptoethanol. Lysophospholipid also demonstrated a protective effect during the later stage of purification.
Zhang
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Department of Chemistry, University of California, San Diego, La Jolla 92093.
Dennis
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EA
D000818:Animals; D002413:Cations, Divalent; D002460:Cell Line; D002845:Chromatography; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008245:Lysophospholipase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology*; D008823:Micelles; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification*; D012996:Solutions; D013379:Substrate Specificity
D002413:Cations, Divalent; D005227:Fatty Acids; D008823:Micelles; D012996:Solutions; D010740:Phospholipases; D008245:Lysophospholipase; D005990:Glycerol