Presence of a phospholipase D (PLD) distinct from PLD1 or PLD2 in human neutrophils: immunobiochemical characterization and initial purification

• Published in: Biochimica et biophysica acta Vol. 1530; no. 1; pp. 97 - 110
• Main Authors: Horn, Jeffrey M, Lehman, Jason A, Alter, Gerald, Horwitz, Joel, Gomez-Cambronero, Julian
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.01.2001
• Subjects: Adenosine Triphosphate; Cations, Divalent; Chromatography, Ion Exchange; Cloning, Molecular; Detergents; Dithiothreitol; Enzyme characterization; Enzyme Inhibitors - pharmacology; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Neutrophil; Neutrophils - enzymology; Oleic Acid - pharmacology; Phospholipase D; Phospholipase D - analysis; Phospholipase D - genetics; Phospholipase D - isolation & purification; Phospholipase D - metabolism; Phosphotyrosine; Signal transduction; PC, phosphatidylcholine; PLC, phospholipase C; PC8, 1,2-dioctanoyl- sn-glycero-3-phosphocholine; PKC, protein kinase C; Phosphotyrosine; DAG, diacylglycerol; PIP 2, L-α-phosphatidyl- D- myo-inositol-4,5- bis-phosphate; Phospholipase D; Enzyme characterization; PLA 2, phospholipase A 2; Signal transduction; PY, phosphotyrosine; PLD, phospholipase D; Neutrophil; PA, phosphatidic acid; ARF, ADP-ribosylating factor
• DOI: 10.1016/S1388-1981(00)00172-4
• ISSN: 1388-1981; 0006-3002

Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8–8.0 was determined. PIP 2, EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-β-glucopyranoside, divalent cations, GTPγS and H 2O 2. The apparent K m for the butanol substrate was 0.1 mM and the V max was 6.0 nmol mg −1 h −1. Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of ∼90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme’s regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granulocyte PLD isoform.