Identification of a human cDNA clone for lysosomal type Ca2+-independent phospholipase A2 and properties of the expressed protein

J Biol Chem. 1997 Jan 24;272(4):2542-50. doi: 10.1074/jbc.272.4.2542.


A Ca2+-independent phospholipase A2 (PLA2) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA2 with dipalmitoylphosphatidylcholine as substrate were Km = 0.25 mM and Vmax = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A1, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [35S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca2+-independent phospholipase A2 group of enzymes.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • DNA, Complementary / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Hydrogen-Ion Concentration
  • Lung / enzymology
  • Lysosomes / enzymology*
  • Molecular Sequence Data
  • Oocytes / enzymology
  • Peptide Mapping
  • Phospholipases A / genetics*
  • Phospholipases A1
  • Phospholipases A2
  • Protein Biosynthesis
  • Rats
  • Xenopus laevis


  • DNA, Complementary
  • Phospholipases A
  • Phospholipases A1
  • Phospholipases A2

Associated data

  • GENBANK/D14662