Development of a High-Throughput Assay for Two Inositol-Specific Phospholipase Cs Using a Scintillation Proximity Format

Abstract

Inositol-specific PLCs comprise a family of enzymes that utilize phosphoinositide substrates, e.g., PIP(2), to generate intracellular second messengers for the regulation of cellular responses. In the past, monitoring this reaction has been difficult due to the need for radiolabeled substrates, separation of the reaction products by organic-phase extraction, and finally radiometric measurements of the segregated products. In this report, we have studied the enzymatic characteristics of two novel PLCs that were derived from functional genomic analyses using a phospholipid-modified solid scintillating support. This method allows for the hydrophobic capture of the [(3)H]phosphoinositide substrate on a well defined scintillation surface and the homogenous measurement of the enzymatic hydrolysis of the substrate by proximity effects. Our results show that the assay format is robust and well suited for this class of lipid-metabolizing enzymes.

The Phosphoinositide-Specific Phospholipase C Inhibitor U73122 (1-(6-((17β-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) Spontaneously Forms Conjugates with Common Components of Cell Culture Medium

“…Inhibition of PLC␥ by U73122 in the presence or absence of dithiothreitol (DTT; 1 mM) was assessed as described previously (Bembenek et al, 2003;Katan et al, 2003). In brief, after addition of enzyme, the rate of tritiated PIP 2 hydrolysis was monitored in the presence or absence of U73122 (0.5-100 M) with or without DTT (1 mM).…”

“…Ca 2؉ Release Assay in A431 Cells. U73122 was incubated with A431 (American Type Culture Collection via LGC Promochem, Middlesex, UK), an epidermoid carcinoma cell line overexpressing epidermal GFR (Bembenek et al, 2003;Katan et al, 2003). A431 cells cultured in DMEM containing 2 mM glutamine were harvested using TrypLE Express (Invitrogen Corporation, Paisley, UK) and seeded at a density of 5 ϫ 10 4 cells per well in 100 l in a sterile flat, clear-bottomed, black-walled 96-well microtiter plate (Corning B.V. Life Sciences, Schiphol-Rijk, The Netherlands).…”

Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen*

“…Consequently, we also assessed the three inhibitors in two cellbased assays. First, we investigated whether cellular PLCs were inhibited by ATA, 3013, or 3017 using previously published protocols to measure phospholipase activity (38,39). Briefly, 1321N1 human astrocytoma cells stably expressing the UDPactivated P2Y 6 receptor were metabolically prelabeled with myo- [2-3 H]inositol, and UDP-stimulated [ 3 H]inositol phosphate accumulation was quantified in the presence of various concentrations of ATA, 3013, or 3017 (Fig.…”

Phosphorothiolate Analogues of Phosphatidylinositols as Assay Substrates for Phospholipase C

“…[9] A number of continuous-assay techniques have been developed that employ substrates that release chromogenic, [10][11][12] fluorescent, [13][14][15] and chemiluminescent [16] products upon cleavage of the assay substrate. The common feature of these substrates is a severe modification of their leaving groups compared to DAG (the natural substrate).…”