title: Cloning of PLD2 from baculovirus for studies in inflammatory responses.
abstract: The enzyme PLD hydrolyzes phosphodiester bonds of lipids in cell membranes. Phosphatidic acid, a chief product of PLD enzymatic activity, is a pleiotropic second messenger with key roles in membrane trafficking, cell invasion, cell growth, and anti-apoptosis. We describe in the present study molecular, cellular, and physiological methods to understand the mechanism of how the PLD2 isozyme regulates the process of inflammation. We describe here (1) a method that details phospholipase D2 (PLD2) cloning in the pBac expression vector, (2) the large-scale infection of Sf21 insect cells for protein production, (3) protein purification by TALON cobalt metal affinity matrix and subsequent assessment of PLD2 protein and lipase activity, (4) application of purified PLD2 protein for the study of Rac2 GTPase biology involving GTP binding by a pull-down assay and GTP/GDP exchange activity, (5) a method of PLD2 expression that involves mammalian cells, (6) a physiological application as relates to adhesion, chemotaxis, and phagocytosis, and (7) a model that integrates the results of a PLD-GTPase interaction from the molecular to the physiological contexts.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH, USA. julian.cambronero@wright.edu
authors.forename: Julian
authors.initials: J
authors.lastname: Gomez-Cambronero
authors.forename: Karen M
authors.initials: KM
authors.lastname: Henkels
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D019556:COS Cells; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D002633:Chemotaxis; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology; D010587:Phagocytosis; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D018411:Spodoptera; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D010712:Phosphatidic Acids; D010739:Phospholipase D; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C105606:phospholipase D2