Assaying phospholipase A2 activity.

Mammalian cells contain many structurally and functionally diverse phospholipases A2 (PLA2) that catalyze the hydrolysis of sn-2 fatty acid from membrane phospholipid. Assays are described for measuring the activity of Group IVA cytosolic PLA2alpha(cPLAalpha) and for secreted PLA2s (sPLA2) that are suitable for purified enzymes and for measuring activity in crude cell lysates and culture medium. The assay for cPLA2alpha involves measuring the calcium-dependent release of radiolabeled sn-2 arachidonic acid from small unilamellar vesicles of phosphatidylcholine. Methods are described for distinguishing cPLA2alpha activity in cell lysates from other PLA2s. sPLA2 activity is monitored using a fluorimetric assay that measures the continuous calcium-dependent formation of albumin-bound pyrene fatty acid from the sn-2 position of phosphatidylglycerol.

[1]  C. Leslie,et al.  Anionic phospholipids stimulate an arachidonoyl-hydrolyzing phospholipase A2 from macrophages and reduce the calcium requirement for activity. , 1990, Biochimica et biophysica acta.

[2]  M. Gelb,et al.  Trifluoromethyl ketones and methyl fluorophosphonates as inhibitors of group IV and VI phospholipases A(2): structure-function studies with vesicle, micelle, and membrane assays. , 1999, Biochimica et biophysica acta.

[3]  C. Leslie,et al.  The 85-kDa, arachidonic acid-specific phospholipase A2 is expressed as an activated phosphoprotein in Sf9 cells. , 1993, Archives of biochemistry and biophysics.

[4]  R. Kramer,et al.  Stimulation of Ca2+-activated human platelet phospholipase A2 by diacylglycerol. , 1987, The Biochemical journal.

[5]  J. D. Clark,et al.  A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP , 1991, Cell.

[6]  R. Kriz,et al.  Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2+)-dependent lipid-binding domain and a Ca(2+)-independent catalytic domain. , 1994, The Journal of biological chemistry.

[7]  C. Bon,et al.  A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin. , 1989, Analytical biochemistry.

[8]  E. Chi,et al.  Translocation of the 85-kDa Phospholipase A2 from Cytosol to the Nuclear Envelope in Rat Basophilic Leukemia Cells Stimulated with Calcium Ionophore or IgE/Antigen (*) , 1995, The Journal of Biological Chemistry.

[9]  M. Gelb,et al.  Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2. , 1993, Biochemistry.

[10]  R. Kriz,et al.  Molecular Characterization of Cytosolic Phospholipase A2-β* , 1999, The Journal of Biological Chemistry.

[11]  C. Leslie.  Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity. , 1991, The Journal of biological chemistry.

[12]  R. Kriz,et al.  A Novel Cytosolic Calcium-independent Phospholipase A2 Contains Eight Ankyrin Motifs* , 1997, The Journal of Biological Chemistry.

[13]  C. Leslie.  Properties and Regulation of Cytosolic Phospholipase A2 * , 1997, The Journal of Biological Chemistry.

[14]  C. Leslie,et al.  Enzymatic Properties of Human Cytosolic Phospholipase A2γ* , 2002, The Journal of Biological Chemistry.

[15]  R. Kramer,et al.  Molecular Cloning of Two New Human Paralogs of 85-kDa Cytosolic Phospholipase A2 * , 1999, The Journal of Biological Chemistry.

[16]  C. Leslie.  Macrophage phospholipase A2 specific for sn-2-arachidonic acid. , 1990, Methods in enzymology.

[17]  J. Balsinde,et al.  Irreversible inhibition of Ca(2+)-independent phospholipase A2 by methyl arachidonyl fluorophosphonate. , 1996, Biochimica et biophysica acta.

[18]  M. Gelb,et al.  Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2 * , 2002, The Journal of Biological Chemistry.

[19]  G. Lambeau,et al.  Increasing molecular diversity of secreted phospholipases A(2) and their receptors and binding proteins. , 2000, Biochimica et biophysica acta.

[20]  R. Kriz,et al.  A Novel Calcium-independent Phospholipase A2, cPLA2-γ, That Is Prenylated and Contains Homology to cPLA2 * , 1998, The Journal of Biological Chemistry.

[21]  M. Gelb,et al.  Kinetic analysis of a high molecular weight phospholipase A2 from rat kidney: divalent metal-dependent trapping of enzyme on product-containing vesicles. , 1992, Biochemistry.

[22]  Makoto Murakami,et al.  Phospholipase A2 enzymes. , 2002, Prostaglandins & other lipid mediators.

[23]  M. Peters-Golden,et al.  Redistribution of 5-lipoxygenase and cytosolic phospholipase A2 to the nuclear fraction upon macrophage activation. , 1993, Biochemical and biophysical research communications.

[24]  Christina C. Leslie,et al.  Regulation of Lysophospholipase Activity of the 85-kDa Phospholipase A2 and Activation in Mouse Peritoneal Macrophages (*) , 1995, The Journal of Biological Chemistry.

[25]  C. Leslie,et al.  A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7. , 1990, The Journal of biological chemistry.

[26]  J. Bonventre,et al.  Nuclear Translocation of Cytosolic Phospholipase A2Is Induced by ATP Depletion* , 2001, The Journal of Biological Chemistry.

[27]  Xianlin Han,et al.  Identification of calcium-independent phospholipase A2 (iPLA2) beta, and not iPLA2gamma, as the mediator of arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells. Enantioselective mechanism-based discrimination of mammalian iPLA2s. , 2002, The Journal of biological chemistry.

[28]  E. Dennis,et al.  Inhibition of Macrophage Ca-independent Phospholipase A by Bromoenol Lactone and Trifluoromethyl Ketones (*) , 1995, The Journal of Biological Chemistry.

[29]  C. Leslie,et al.  Intracellular Calcium Signals Regulating Cytosolic Phospholipase A2 Translocation to Internal Membranes* 210 , 2001, The Journal of Biological Chemistry.

[30]  E. Dennis,et al.  The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. , 2000, Biochimica et biophysica acta.

[31]  M. Gelb,et al.  Calcium-dependent and -independent interfacial binding and catalysis of cytosolic group IV phospholipase A2. , 1998, Biochemistry.

[32]  J. Balsinde,et al.  Calcium-independent phospholipase A(2): structure and function. , 2000, Biochimica et biophysica acta.