Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells.
A novel Ca2+-independent phospholipase A2 (iPLA2) has recently been purified and characterized from P388D1 macrophages (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme appears to play a key role in regulating basal phospholipid remodeling reactions. Also an iPLA2 from Chinese hamster ovary (CHO) cells has been purified, molecularly cloned, and expressed (Tang, J., Kriz, R., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J. Biol. Chem. 272, 8567-8575). We report herein that the cloned CHO iPLA2 is equivalent to the mouse enzyme purified from P388D1 cells. Polymerase chain reaction amplification of cDNA fragments from P388D1 cells using primers based on the CHO iPLA2 sequence, revealed a high degree of homology between the mouse and hamster enzymes at both the nucleotide and amino acid levels (92 and 95%, respectively). Identity between the two proteins was further demonstrated by using immunochemical, pharmacological, and biochemical approaches. Thus, an antiserum generated against the CHO enzyme recognized the P388D1 cell enzyme and gave similar molecular masses (about 83 kDa) for the two enzymes under the same experimental conditions. Further, the CHO enzyme has exactly the same sensitivity to inhibition by a variety of compounds previously shown to inhibit the P388D1 enzyme, including bromoenol lactone, palmitoyl trifluoromethyl ketone, and methyl arachidonyl fluorophosphonate. Additionally, covalent modification of the CHO enzyme by [3H]bromoenol lactone is dependent on active enzyme as is the P388D1 iPLA2. Finally, both enzymes have the same specific activities under identical experimental conditions.
Balboa
M A
MA
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0601, USA.
Balsinde
J
J
Jones
S S
SS
Dennis
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005260:Female; D007941:Leukemia P388 / Q000201:enzymology; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000378:metabolism; D010053:Ovary / Q000201:enzymology; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D054467:Phospholipases A2; D011753:Pyrones / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000201:enzymology
D009281:Naphthalenes; D011753:Pyrones; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D004228:Dithionitrobenzoic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium