title: Substrate specificity of lysophosphatidic acid acyltransferase beta -- evidence from membrane and whole cell assays.
abstract: 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-beta activity using two different assays. In a membrane-based assay, LPAAT-beta 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-beta activity in cells: PC-3 and DU145 cells converted exogenously added lysoPM and (14)C-labeled 18:1 into (14)C-labeled phosphatidylmethanol (PM). The rate of PM formation was higher in cells that overexpressed LPAAT-beta and was inhibited by the LPAAT-beta inhibitor CT-32501. In contrast, if lysoPM and (14)C-labeled 20:4 were added to PC-3 or DU145 cells, (14)C-labeled PM was also formed, but the rate was neither higher in cells that overexpressed LPAAT-beta nor inhibited by CT-32501. We propose that LPAAT-beta catalyzes the intracellular transfer of 18:1, 18:0, and 16:0 acyl groups but not 20:4 groups to lysoPA.
authors.affiliation: Department of Biochemistry, Cell Therapeutics, Inc., Seattle, WA 98119, USA. dhollenback@earthlink.net
authors.forename: David
authors.initials: D
authors.lastname: Hollenback
authors.forename: Lynn
authors.initials: L
authors.lastname: Bonham
authors.forename: Laura
authors.initials: L
authors.lastname: Law
authors.forename: Eddie
authors.initials: E
authors.lastname: Rossnagle
authors.forename: Lisa
authors.initials: L
authors.lastname: Romero
authors.forename: Heather
authors.initials: H
authors.lastname: Carew
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Tompkins
authors.forename: David W
authors.initials: DW
authors.lastname: Leung
authors.forename: Jack W
authors.initials: JW
authors.lastname: Singer
authors.forename: Thayer
authors.initials: T
authors.lastname: White
mesh_terms: D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D016367:Baculoviridae / Q000378:metabolism; D001681:Biological Assay; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008566:Membranes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D000217:Acyltransferases; D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; C018764:2-acylglycerophosphate acyltransferase