title: Preparation of the Full Set of Recombinant Mouse- and Human-Secreted Phospholipases A2.
abstract: A family of 14-20kDa, disulfide-rich, calcium-dependent secreted phospholipases A2(sPLA2s) that release fatty acids from the sn-2 position of glycerophospholipids can be found in mammals. They have a diverse array of tissue distribution and biological functions. In this chapter we provide detailed protocols for production of nearly all of the mouse and human sPLA2s mainly by expression in bacteria and in vitro refolding or by expression in insect cells. High-resolution mass spectrometry and enzymatic assays were, respectively, used to show that all disulfides are formed and that the enzymes are active, strongly suggesting that each sPLA2was prepared in the structurally native form. The availability of these proteins has allowed kinetic studies to be carried out, to prepare highly selective antisera, to screen for selective inhibitors, to study receptor binding, and to study the action of each enzyme on mammalian cell membranes and their in vivo biological roles.
authors.affiliation: University of Washington, Seattle, WA, United States.
authors.forename: F
authors.initials: F
authors.lastname: Ghomashchi
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia Antipolis and Centre National de la Recherche Scientifique (CNRS), Valbonne, France.
authors.forename: V
authors.initials: V
authors.lastname: Brglez
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia Antipolis and Centre National de la Recherche Scientifique (CNRS), Valbonne, France.
authors.forename: C
authors.initials: C
authors.lastname: Payré
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia Antipolis and Centre National de la Recherche Scientifique (CNRS), Valbonne, France.
authors.forename: L
authors.initials: L
authors.lastname: Jeammet
authors.affiliation: University of Sfax, Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Sfax, Tunisia.
authors.forename: S
authors.initials: S
authors.lastname: Bezzine
authors.affiliation: University of Washington, Seattle, WA, United States. Electronic address: gelb@uw.edu.
authors.forename: M H
authors.initials: MH
authors.lastname: Gelb
authors.affiliation: Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia Antipolis and Centre National de la Recherche Scientifique (CNRS), Valbonne, France. Electronic address: lambeau@ipmc.cnrs.fr.
authors.forename: G
authors.initials: G
authors.lastname: Lambeau
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000737:chemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D054497:Phospholipases A2, Secretory / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061987:Sf9 Cells; D018411:Spodoptera; D014018:Tissue Distribution
chemical_list: D004220:Disulfides; D007527:Isoenzymes; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D054497:Phospholipases A2, Secretory; D015951:Factor Xa
keywords: Interfacial enzymology; Lipid biochemistry; Lipolytic enzymes; Phospholipids