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2010 | 57 | 3 | 369-372
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Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets

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Phospholipase D plays a key role in the biosynthesis of phosphatidic acid, a second messenger involved in essential cellular processes. Oleate-activated phospholipase D was the first mammalian phospholipase D isoform to be discovered but is the least known. The study was aimed to test a fluorometric method of assessment of oleate-activated phospholipase D activity in different biological materials. The brain cortex of male Wistar rats, cultured rat brain astrocytes, and human platelets were processed to yield plasmatic membranes for experiments. To assess phospholipase D activity the modified fluorometric method was used. Previously, the method was used only to determine H2O2. In this enzyme-coupled assay phospholipase D activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine. First, phospholipase D cleaves exogenous phosphatidylcholine to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H2O2. Finally, in the presence of horseradish peroxidase, H2O2 reacts with 10-acetyl-3,7-dihydroxyphenoxazine to generate the highly fluorescent product, resorufin. The concentration of resorufin was measured using excitation and emission at 560 nm and 590 nm, respectively. The proposed optimal parameters of the tested assay are 25 µg of rat brain cortex protein, 50 µg of rat brain astrocyte protein, and 50 µg of human platelet protein in a reaction volume of 200 µL, and 2 min enzymatic reaction at 37°C. The fluorometric method may be applied to assay phospholipase D in different biological materials.
Physical description
  • Department and Clinic of Psychiatry and Psychotherapy, Medical University of Silesia, Katowice, Poland
  • Department of Pharmacology, Medical University of Silesia, Sosnowiec, Poland
  • Department of Neurology, Medical University of Silesia, Katowice, Poland
  • Department of Pharmacology, Medical University of Silesia, Katowice, Poland
  • Chalifa V, Mohn H, Liscovitch M (1990) A neutral phospholipase D activity from rat brain synaptic plasma membranes. J Biol Chem 265: 17512-17519.
  • David JL, Herion F (1972) Assay of platelet ATP and ADP by the luciferase method: some theoretical and practical aspects. Adv Exp Med Biol 34: 341-354.
  • English D, Cui Y, Siddiqui RA (1996) Messenger functions of phosphatidic acid. Chem Phys Lipids 80: 117-132.
  • Girotti S, Ferri E, Cascione ML, Orlandini A, Farina L, Nucci S, Di Graci F, Budini R (1991) Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes. Anal Biochem 192: 350-357.
  • Hanahan DJ, Chaikoff IL (1947) A new phospholipid-spliting enzyme specific for the ester linkage between the nitrogenous base and the phosphoric acid grouping. J Biol Chem 169: 699-705.
  • Hertz L, Juurlink BHJ, Szuchet S (1985) Cell Cultures. In Handbook of Neurochemistry. Lajtha A ed, 8: 603-661. Plenum Press, New York, London.
  • Horwitz J, Davis LL (1993) The substrate specificity of brain microsomal phospholipase D. Biochem J 295: 793-798.
  • Houle MG, Bourgoin S (1999) Regulation of phospholipase D by phosphorylation dependent mechanism. Biochim Biophys Acta 1439: 135-150.
  • Jelsema CL (1987) Light activation of phospholipase A2 in rod outer segments of bovine retina and its modulation by GTP-binding proteins. J Biol Chem 262: 163-168.
  • Jin JK, Ahn BH, Na YJ, Kim JI, Kim YS, Choi EK, Ko YG, Chung KC, Kozlowski PB, Min do S (2007) Phospholipase D1 is associated with amyloid precursor protein in Alzheimer's disease. Neurobiol Aging 28: 1015-1027.
  • Klein J (2005) Functions and pathophysiological roles of phospholipase D in the brain. J Neurochem 94: 1473-1487.
  • Krzystanek M, Trzeciak HI, Krupka-Matuszczyk I, Krzystanek E (2002) Antidepressant-like influence of olanzapine on membrane phospholipase D activity in rat brain. Eur Neuropsychopharmacol 12 (Suppl 3): S297.
  • Krzystanek M, Krupka-Matuszczyk I, Trzeciak IH, Krzystanek E (2003) Amitriptiline mechanism of action may relate to oleate activated phospholipase D inhibition. Eur Neuropsychopharmacol 13 (Suppl 4): S235.
  • Krzystanek M, Krupka-Matuszczyk I, Trzeciak HI, Krzystanek E (2004) Classical neuroleptics do not change the membrane phospholipase D activity. Eur Neuropsychopharmacol 14 (Suppl 3): S239-S240.
  • Krzystanek M, Krupka-Matuszczyk I, Trzeciak HI, Krzystanek E (2006) Phospholipase D activation as a possible apoptotic signal in ischemic astrocytes. Eur Neuropsychopharmacol 16 (Suppl 4): S486-S487.
  • Krzystanek E, Krzystanek M, Opala G, Trzeciak HI, Siuda J, Małecki A (2007) Platelet phospholipase A2 activity in patients with Alzheimer's disease, vascular dementia and ischemic stroke. J Neural Transm 114: 1033-1039.
  • Liscovitch M, Czarny M, Fiucci G, Lavie Y, Tang X (1999) Localization and possible functions of phospholipase D isozymes. Biochim Biophys Acta 1439: 245-263.
  • Lotharius J, Brundin P (2002) Impaired dopamine storage resulting from alpha-synuclein mutations may contribute to the pathogenesis of Parkinson's disease. Hum Mol Genet 11: 2395-2407.
  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275.
  • Meier KE, Gibbs TC, Knoepp SM, Ella KM (1999) Expression of phospholipase D isoforms in mammalian cells. Biochim Biophys Acta 1439: 199-213.
  • Nishida A, Emoto K, Shimizu M, Uozumi T, Yamawaki S (1994) Brain ischemia decreases phosphatidylcholine-phospholipase D but not phosphatidylinositol-phospholipase C in rats. Stroke 25: 1247-1251.
  • Saito M, Kanfer J (1973) Solubilization and properties of a membrane-bound enzyme from rat brain catalyzing a base-exchange reaction. Biochem Biophys Res Commun 53: 391-398.
  • Strosznajder J, Strosznajder RP (1989) Guanine nucleotides and fluoride enhance carbachol-mediated arachidonic acid release from phosphatidylinositol. Evidence for involvement of GTP-binding protein on phospholipase A2 activation. J Lipid Mediat 1: 217-229.
  • Taki T, Kanfer JN (1979) Partial purification and properties of a rat brain phospholipase D. J Biol Chem 254: 9761-9765.
  • Varga A, Alling C (2002) Formation of phosphatidylethanol in vitro in red blood cells from healthy volunteers and chronic alcoholics. J Lab Clin Med 140: 79-83.
  • Venable ME, Obeid LM (1999) Phospholipase D in cellular senescence. Biochim Biophys Acta 1439: 291-298.
  • Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP (1997) A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253: 162-168.
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