Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7

Abstract

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Cyclic phosphatidic acid is produced by GDE7 in the ER lumen as a lysophospholipid mediator

“…Activity of recombinant GDE7 enzymes was con rmed using a selective assay system with a uorescent substrate, FS-3 15 . The FS-3-degrading activities of hGDE7-and mGDE7-expressing cells (0.65 ± 0.03 and 1.00 ± 0.06 µmol h − 1 per mg protein, respectively) signi cantly increased compared with control cells (0.05 ± 0.02 µmol h − 1 per mg protein) (Fig.…”

“…The cPA-and LPAproducing activities were 5.34 ± 1.98 and 0.83 ± 0.21 µmol h − 1 per mg protein, respectively. In order to analyze endogenous GDE7, we also prepared the membrane fractions of wild-type human breast cancer MCF-7 cells (WT) endogenously expressing GDE7, as well as GDE7-knockout MCF-7 cells (KO) 15 . FS-3degrading activity for KO was reduced to 37% of that for WT, and ectopic expression of mGDE7 in KO restored the activity to 242% (Fig.…”

“…A knockout cell line for human GDE7 gene (GDPD3) was previously established 15 . These cell lines were cultured at 37°C in a humidi ed atmosphere of 5% CO 2 and 95% air.…”

“…Fluorescence-based enzyme assays were based on a previous method 15 . Brie y, membrane fractions (5 µg of protein) were incubated with 5 µM FS-3 for 3 h at 37°C in 60 µL of 50 mM Tris-HCl buffer (pH 7.4) in the presence of 2 mM CaCl 2 and 0.1% (w/v) Nonidet P-40.…”

“…Radioactivity-based enzyme assays were performed as previously described 15 with modi cations. The membrane fractions were incubated at 37°C for 30 min with 25 µM of [ 14 C]LPC (20,000 cpm, dissolved in 5 µL of ethanol) in 100 µL of 50 mM Tris-HCl (pH 7.4) containing 2 mM of CaCl 2 and 100 µM of phosphatase inhibitor sodium orthovanadate to protect the product from degradation 11 .…”