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

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. PIP2, EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100 , n-octyl-beta -glucopyranoside, divalent cations, GTP gammaS and H2O2. The apparent K-m for the butanol substrate was 0.1 mM and the V-max was 6.0 nm ol mg(-1) h(-1). Immunochemical analysis by anti-pan PLD antibodies reveale d a neutrophil PLD of similar to 90 kDa and other bands recognized minimall y by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phos phorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protei n partial purification using column liquid chromatography was performed aft er cell subfractionation. Based on the enzyme's regulatory and inhibitory f actors, and its molecular weight, these data indicate an enzyme isoform tha t might be different from die mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granul ocyte PLD isoform. (C) 2001 Elsevier Science B.V. All rights reserved.