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Journal of Lipid Research, Vol. 47, 593-604, March 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Substrate specificity of lysophosphatidic acid acyltransferase ß—evidence from membrane and whole cell assays

David Hollenback¹, Lynn Bonham, Laura Law, Eddie Rossnagle, Lisa Romero, Heather Carew, Christopher K. Tompkins, David W. Leung, Jack W. Singer and Thayer White

Department of Biochemistry, Cell Therapeutics, Inc., Seattle, WA 98119

Published, JLR Papers in Press, December 20, 2005.

¹ To whom correspondence should be addressed. e-mail: dhollenback{at}earthlink.net

Membranes of mammalian cells contain lysophosphatidic acid acyltransferase (LPAAT) activities that catalyze the acylation of sn-1-acyl lysophosphatidic acid (lysoPA) to form phosphatidic acid. As the biological roles and biochemical properties of the six known LPAAT isoforms have yet to be fully elucidated, we have characterized human LPAAT-ß activity using two different assays. In a membrane-based assay, LPAAT-ß used lysoPA and lysophosphatidylmethanol (lysoPM) but not other lysophosphoglycerides as an acyl acceptor, and it preferentially transferred 18:1, 18:0, and 16:0 acyl groups over 12:0, 14:0, 20:0, and 20:4 acyl groups. The fact that lysoPM could traverse cell membranes permitted additional characterization of LPAAT-ß activity in cells: PC-3 and DU145 cells converted exogenously added lysoPM and ¹⁴C-labeled 18:1 into ¹⁴C-labeled phosphatidylmethanol (PM). The rate of PM formation was higher in cells that overexpressed LPAAT-ß and was inhibited by the LPAAT-ß inhibitor CT-32501. In contrast, if lysoPM and ¹⁴C-labeled 20:4 were added to PC-3 or DU145 cells, ¹⁴C-labeled PM was also formed, but the rate was neither higher in cells that overexpressed LPAAT-ß nor inhibited by CT-32501. We propose that LPAAT-ß catalyzes the intracellular transfer of 18:1, 18:0, and 16:0 acyl groups but not 20:4 groups to lysoPA.

Supplementary key words phosphatidic acid • lysophosphatidylmethanol • phosphatidylmethanol • acyl-CoA

Abbreviations: GFP, green fluorescent protein; LPAAT, lysophosphatidic acid acyltransferase; lysoPA, lysophosphatidic acid; lysoPC, lysophosphatidylcholine; lysoPM, lysophosphatidylmethanol; PA, phosphatidic acid; PLD, phospholipase D; PM, phosphatidylmethanol

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