Cloning, expression, and catalytic mechanism of murine lysophospholipase I.
A lysophospholipase (LysoPLA I) has been purified and characterized from the mouse macrophage-like P388D1 cell line (Zhang, Y. Y, and Dennis, E. A. (1988) J. Biol. Chem. 263, 9965-9972). This enzyme has now been sequenced, cloned, and expressed in Escherichia coli cells. The enzyme contains 230 amino acid residues with a calculated molecular mass of 24.7 kDa. It has a high helical content in its predicated secondary structure, which is also indicated in its CD spectrum. The cloned LysoPLA I was purified to homogeneity from the transformed E. coli cells by a gel filtration column and an ion exchange column. The specific activity of the purified protein is 1. 47 micromol/min.mg toward 1-palmitoyl-sn-glycero-3-phosphorylcholine at pH 8.0 and 40 degrees C, corresponding to the reported value of 1.3-1.7 micromol/min.mg for the protein purified from the P388D1 cells. In addition, the cloned protein cross-reacted with an antibody raised against LysoPLA I also purified from the P388D1 cells. The deduced LysoPLA I sequence contains a well conserved GXSXG motif found in the active site of many serine enzymes, and the activity of the LysoPLA I was irreversibly inhibited by the classical serine protease inhibitor diisopropyl fluorophosphate. Furthermore, site-directed mutagenesis was employed to change Ser-119 in the GXSXG motif to an Ala. The resulting mutant protein lost all of its lysophospholipase activity, even though it had the same overall protein conformation as that of the wild-type LysoPLA I. Therefore, LysoPLA I has been demonstrated to be a serine enzyme with Ser-119 at the active site.
Wang
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A
Department of Chemistry and Biochemistry, School of Medicine and Revelle College, University of California at San Diego, La Jolla, California 92093-0601, USA.
Deems
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RA
Dennis
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D007941:Leukemia P388 / Q000201:enzymology; D008245:Lysophospholipase / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D018076:DNA, Complementary; D008245:Lysophospholipase