title: Modification of catalytically active phospholipase D1 with fatty acid in vivo.
abstract: Phospholipase D1 (PLD1) was covalently labeled with 3H when expressed transiently in COS cells and immunoprecipitated following labeling of the cells with [3H]palmitate. Labeling of PLD1 was abolished by treatment with hydroxylamine at neutral pH, indicating that the fatty acid is linked via thioester to the enzyme. In pulse-chase studies the label persisted over a 3-h chase, indicating a slow rate of turnover. A catalytically inactive point mutant of PLD1 that changes serine at position 911 to alanine (S911A) was partially but not entirely redistributed to the cytosol, and it contained no detectable palmitate label. Similarly, N- and C-terminal domain fragments of the protein, encompassing in combination the entire coding region and all expressed to levels comparable with the wild type protein, showed no label with palmitate. Treatment of immunoprecipitated PLD1 with hydroxylamine diminished catalytic activity to background levels in a dose response manner that paralleled the removal of label from [3H]palmitate-labeled protein. We suggest that modification of PLD1 with palmitate is related to its catalytic activity and may be an important requirement for the function of this enzyme.
authors.affiliation: Department of Signaling, Babraham Institute, Cambridge CB2 4AT, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Manifava
authors.forename: J
authors.initials: J
authors.lastname: Sugars
authors.forename: N T
authors.initials: NT
authors.lastname: Ktistakis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000737:chemistry*; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests
chemical_list: D019308:Palmitic Acid; D010739:Phospholipase D; C106801:phospholipase D1