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. Author manuscript; available in PMC: 2021 May 4.
Published in final edited form as: Methods Mol Biol. 2021;2251:225–236. doi: 10.1007/978-1-0716-1142-5_17

Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes

Adam J Carr 1, Edhriz Siraliev-Perez 2, Weigang Huang 1, John Sondek 2,3,4, Qisheng Zhang 5,6,7
PMCID: PMC8094607  NIHMSID: NIHMS1693341  PMID: 33481244

Abstract

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer’s disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.

Keywords: Phospholipase C; phosphatidylinositol 4,5-bisphosphate; Lipid vesicles; Enzymatic assay; Fluorogenic reporter; High-throughput screen

1. Introduction

The 13 mammalian PLC isozymes are divided into 6 subgroups (−β, −γ, −δ, −ε, −ζ, and −η) based on sequence similarity and share a conserved catalytic core [1]. Due to autoinhibition, the basal activities of PLCs are minimal in cells [2, 3]. When activated, PLCs hydrolyze the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane to generate the second messengers 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). These second messengers activate protein kinase C (PKC) isozymes and promote the release of intracellular Ca2+ stores, respectively [4]. The consumption of PIP2 also contributes to changes in the functions of membrane proteins such as ion channels [5]. Consequently, PLCs are important signaling proteins that regulate diverse cellular processes, including proliferation, migration, and nerve conductance. Conversely, aberrant regulation of PLCs contributes to various diseases such as cancer [6–8], Alzheimer’s disease [9, 10], and rheumatoid arthritis [11, 12]. PLCs, particularly the PLC-γ isozymes, have emerged as promising therapeutic targets.

Despite extensive studies on PLCs, it remains challenging to continuously and directly monitor PLC activity at membranes with high sensitivity and throughput. Traditionally, the phospholipase activities of PLCs have been quantified using radiolabeled substrates [13–16]. However, these formats have substantial limitations. Primarily, they do not allow for continuous monitoring of PLCs. In addition, the use of radioactive materials requires special training, handling, storage, equipment, and facilities that are less common than in previous decades. Indeed, radioactive PIP2 has recently been discontinued by all chemical vendors, and it is expensive to obtain such reagents through custom synthesis. Several chromogenic or fluorogenic PIP2 analogs have been developed to continuously monitor PLC activity [17–21]. However, most of these compounds are either inefficient substrates of PLCs or have other issues such as requiring a secondary enzymatic reaction not related to PLCs. Importantly, none of these compounds reliably report PLC activity at the membranes.

We created XY-69 as a membrane-associated PIP2 analog that works as a selective fluorogenic reporter of PLC activity [22]. XY-69 contains fluorescein as a fluorophore and 4-(dimethylaminoazo)benzene-4-carboxylic (DABCYL) acid as a dark quencher that absorbs emission energy from fluorescein and dissipates it as heat. PLC isozymes hydrolyze XY-69 to separate DABCYL from fluorescein, leading to a large increase in fluorescence intensity that is readily monitored in real time. The assay works both in detergent micelles and in lipid vesicles that more closely resemble biological membranes and avoids the quench and workup steps otherwise needed in radioactivity-based assays. Furthermore, XY-69 functions in a microplate format, which enables high-throughput discovery of molecules that modulate PLC activity. When presented in lipid vesicles, XY-69 captures the membrane-dependent activation of PLC activity by Gαq [22], the subunit of G-protein Gq that is known to activate PLC-β isozymes [14, 23, 24], or by conformational changes due to mutations [25]. Consequently, XY-69 is a compelling replacement for radioactive PIP2 to monitor PLC activity with the added advantages of continuous reaction monitoring and high-throughput. Assays based on XY-69 are readily applicable to both purified PLC isozymes and lysates from cell lines or animal tissues.

To further optimize the assays based on XY-69 to measure PLC activity, we have investigated the impact of varying lipid composition, pH, free Ca2+ concentration, and vesicle size on assay performance. Here, we describe optimized assay conditions and a detailed protocol to measure the activity of purified, cancer-associated mutant PLC-γ1 (D1165H) [8] with XY-69 embedded in lipid vesicles. The described protocol also applies to other PLC isozymes and may be modified to include kinases, effectors, peptides, and small molecules in the assay.

2. Materials

2.1. Equipment and Supplies

  1. Basic pH meter.

  2. 0.45-μm polyethersulfone syringe filters.

  3. 12 × 75 mm borosilicate culture tubes.

  4. Dry nitrogen (N2) line equipped with a plastic hose and glass Pasteur pipette.

  5. Vacuum pump.

  6. Black 384-shallow well microplate.

  7. Ultrasonic dismembrator.

  8. 5/64” probe microtip for sonicator.

  9. Multimode plate reader.

  10. Ring stand.

  11. Dynamic light scattering instrument.

  12. 40-μL plastic cuvettes with stoppers.

  13. Water bath.

  14. Dewar flask.

  15. Septum.

  16. Pipette.

  17. Pipette tips.

  18. Syringe.

  19. Vortex mixer.

2.2. Lipids and Other Chemicals

  1. 10 mg/mL (13.2 mM) l-α-phosphatidylethanolamine (PE; liver, bovine) stock solution in chloroform (CHCl3), stored at −20 °C under N2.

  2. 1 mg/mL (912 μM) l-α-phosphatidylinositol-4,5-bisphosphate (PIP2; brain, porcine; ammonium salt) stock solution in CHCl3:methanol (MeOH):H2O (20:9:1; v/v/v), stored at −20 °C under N2.

  3. 210 μM XY-69 stock solution in H2O, stored at −80 °C under N2, wrapped in aluminum foil.

  4. HPLC-grade MeOH.

  5. Acetone.

  6. Sodium hydroxide (NaOH).

  7. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES).

  8. Potassium chloride (KCl).

  9. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA).

  10. Calcium chloride (CaCl2).

  11. 1,4-Dithiothreitol (DTT).

2.3. Proteins

  1. PLC-γ1 (21-1215) referred to as PLC-γ1 (WT).

  2. PLC-γ1 (21-1215) D1165H referred to as PLC-γ1 (D1165H).

  3. Fatty acid-free bovine serum albumin (FAF BSA).

3. Methods

3.1. Overview

The final assay is carried out in a 384-well plate at a volume of 12 μL. The assay mixture contains HEPES (33.9 mM, pH 7.4), KCl (70 mM), EGTA (3 mM), CaCl2 (2.35 mM), DTT (2 mM), FAF BSA (0.17 mg/mL), PE (192 μM), PIP2 (48 μM), XY-69 (0.5 μM), and the desired concentration of PLC isozyme (see Note 1). Under these conditions, the free Ca2+ concentration is approximately 390 nM (see Notes 2 and 3). The assay takes approximately 3 h from reagent setup to completion of data acquisition.

3.2. Preparation of Buffers

  1. Prepare stock solution A (see Note 4) containing 120 mM HEPES (pH 7.4) (see Note 5), 420 mM KCl, 18 mM EGTA, and 14.1 mM CaCl2.

  2. Make 1 mL of lipid vesicle assay buffer (LVAB) fresh for the assay by adding 12 μL of 1 M DTT (stored at −20 °C) to 988 μL of the stock solution A.

  3. Make 1.2 mL of PLC dilution buffer (PLCDB) by mixing 200 μL of LVAB, 120 μL of 10 mg/mL FAF BSA, and 880 μL of H2O (see Note 6).

  4. Make lipid sonication buffer containing 20 mM HEPES (pH 7.4).

3.3. Generation of Lipid Films and Lipid Vesicles

  1. Briefly vortex and spin-down a thawed aliquot of XY-69 stock solution (210 μM in H2O), and then transfer 1.5 μL to the bottom of a borosilicate culture tube.

  2. Dry with a gentle stream of N2 for 5 min.

  3. Allow PIP2 and PE stock solutions to equilibrate to room temperature, and then vortex briefly.

  4. Transfer 34.1 μL of PIP2 stock solution (912 μM) to the bottom of the tube containing XY-69. Tap the tube gently to re-dissolve the XY-69 flake.

  5. Add 9.4 μL of PE stock solution (13.2 mM) into the PIP2/XY-69 solution, followed by 20 μL of HPLC-grade MeOH (see Note 7). Dry this mixture down to an opaque film on the bottom of the tube using a gentle stream of N2 (see Note 8).

  6. Leave the lipid film (see Note 9) under a gentle stream of N2 for at least another 60 min to remove the bulk of organic solvent, and then remove remaining traces of solvent by applying high vacuum (0.5 mTorr) for at least 60 min (see Note 10).

  7. Add 450 μL of lipid sonication buffer to the dry lipid film, and then clamp the tube securely to a ring stand (see Note 11).

  8. Submerge the probe tip of a sonicator halfway down into the solution. The probe tip should be positioned vertically and centered in the tube (see Note 12).

  9. Place the tube in a Dewar flask containing iced water (ice bath) and cool the solution for 45 s.

  10. Pulse the sonicator at 20% output for three cycles of 5 s ON, 15 s OFF (see Note 13).

  11. Remove the tube from the ice bath and cover the opening with a septum. Confirm that the solution is free of particulate matter and that no lipid film remains on the bottom of the tube. Allow the solution to equilibrate to room temperature for at least 15 min.

  12. Subject the lipid vesicles to freeze-thaw (see Note 14) by immersing the tube in a Dewar flask containing acetone-dry ice (dry ice bath, −78 °C) for 40 s with gentle shaking, followed by immersing in a water bath (35 °C) for 70 s with gentle shaking. Repeat this process for a total of six cycles.

  13. Add 90 μL of LVAB directly to the solution at room temperature, and then vortex on a medium intensity setting for 20 s.

  14. Protect the resulting lipid vesicle solution (LVS) from light and store at room temperature (~20 °C) until needed for the assay. This solution is calculated to give 1.2 times the desired concentration of lipids in the final assay since 10 μL of LVS will be mixed with 2 μL of PLC solution to make a 12-μL reaction mixture.

3.4. Measurement of PLC Activity

  1. Dilute PLC proteins using ice-cold PLCDB to six times the final concentration in the assay since PLC solution composes 2 μL; out of the final 12 μL in the reaction (see Note 15).

  2. Keep PLC solutions on ice until needed for the assay.

  3. Warm up the plate reader and allow it to equilibrate to assay temperature (see Note 16).

  4. Set the excitation wavelength at 485 nm and emission detection wavelength at 520 nm by using a FITC 485 excitation filter and 520 emission filter (see Note 17).

  5. To perform the assay in quadruplicate, distribute 2 μL aliquots of PLC solution of a given concentration into four sequential wells of a 384-well microplate (see Note 18). Repeat for each desired concentration of enzyme. Blank PLCDB can be used as a PLC-free or “BSA-only” control.

  6. Add 10 μL of LVS into each well containing PLC solution to initiate the assay and immediately insert the assay plate into the plate reader for measurement (see Note 19).

  7. Record fluorescence at required time points at 60-s intervals for 20–60 min (see Note 20).

  8. Use the remaining LVS for dynamic light scattering (DLS) analysis.

  9. Add 8 μL of blank PLCDB to a cuvette, followed by 40 μL of LVS and mix gently with a pipette. The resulting mixture contains the same buffer and lipid component concentrations as the microplate PLC assay. Cap the cuvette and insert the sample into the DLS instrument for measurement at a temperature matching the relevant PLC assay conditions (see Note 21).

Fig. 1.

Fig. 1

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Effects of pH and free Ca2+ concentration on hydrolysis of XY-69 by PLC-γ1 (D1165H). (a) pH effect. Lipid vesicles consisting of PE (192 μM), PIP2 (48 μM), and XY-69 (0.5 μM) in assay buffers with varying pH were added to PLC-γ1 (D1165H) (1 nM) at 20 °C or 30 °C. The initial velocity of XY-69 hydrolysis was measured and plotted against pH. The free Ca2+ concentration was 390 nM. (b) Effect of free Ca2+ concentration. The initial velocity of XY-69 (0.5 μM) hydrolysis by PLC-γ1 (D1165H) (1 nM) was measured with varying concentrations of free Ca2+ in the assay buffer (pH 7.4) at 20 °C or 30 °C

Fig. 2.

Fig. 2

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Phospholipase C activity of PLC-γ1 (D1165H) in lipid vesicles. (a) XY-69 (0.5 μM) was embedded into lipid vesicles containing PE (200 μM) and PIP2 (20 μM) and added to PLC-γ1 (D1165H) with indicated concentration to initiate the hydrolysis. The reaction progression at 20 °C was monitored continuously for 60 min by fluorescence (λex/λem = 485/520 nm). The final assay buffer has a pH of 7.4 and free Ca2+ concentration at 390 nM. (b) The concentration of PE and PIP2 in lipid vesicles was 192 and 48 μM, respectively. The experiment was run similarly as described in (a)

Fig. 3.

Fig. 3

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Size of lipid vesicle affects the relative rate of XY-69 hydrolysis by PLC-γ1 (WT) and PLC-γ1 (D1165H). (a, b) The Z-average (Zav) particle size of lipid vesicles was measured by dynamic light scattering (DLS). The % intensity of total scattered light was plotted versus particle size of lipid vesicles, which were formed either by probe sonication only (a) or by first probe sonication and then freeze–thaw (b). (c, d) XY-69 (0.5 μM) hydrolysis by PLC-γ1 (WT) or PLC-γ1 (D1165H) at 20 °C was monitored by fluorescence (λex/λem = 485/520 nm). Lipid vesicles were formed either by probe sonication only (c) or by first probe sonication and then freeze–thaw (d). All lipid vesicles contain PE (192 μM) and PIP2 (48 μM). The final pH of the assay buffer was 7.4, and the free Ca2+ concentration was 390 nM

Acknowledgments

This work was supported by the National Institutes of Health (GM057391 to J. S. and CA177993 to Q. Z.) and the North Carolina Biotechnology Center (TEG-2018-1505 to Q. Z.). We thank Dr. Xiaoyang Wang for providing XY-69 and the Center for Integrative Chemical Biology and Drug Discovery (CICBDD) at the UNC Eshelman School of Pharmacy for the access to a micro-plate reader.

Footnotes

1.

The parameters presented here are optimal for the range of 1–100 nM PLC-γ1 (WT) and 0.1–50 nM PLC-γ1 (D1165H).

2.

The concentration of free calcium ion is calculated using the Maxchelator program, which accounts for temperature, pH, and ionic strength of the assay mixture, as well as the concentrations of EGTA and total calcium (CaCl2).

3.

All PLC isozymes are calcium dependent, and a calcium ion binds to the active site within the TIM barrel. Therefore, the concentration of free Ca2+ is an essential component for signal generation in the XY-69 assay. Figure 1b shows that with 1.0 nM PLC-γ1 (D1165H), 100 nM free Ca2+ (this is near basal cytosolic levels) [26] produces a modest rate of hydrolysis. However, rates of hydrolysis plateau at concentrations of free Ca2+ greater than 400 nM. These results are consistent with a binding affinity (kd) of ~250 nM for CaCl2 in PLC1-γ (WT). At 100 μM free Ca2+, the assay reagents precipitated, which terminated PLC activity.

4.

All stock solutions are made with Milli-Q-grade water, adjusted to the desired pH using 8 M NaOH, filtered through a 0.45-μm filter, and stored at 4 °C.

5.

The pH of the XY-69 assay buffer controls the protonation state of key amino acid residues and influences the concentration of free Ca2+ ions, thereby regulating PLC activity. PLC-γ isozymes are typically purified using HEPES buffer at a pH of 7.4. We have examined PLC-γ1 activity in 34 mM HEPES across a pH range of 6.5 to 8.5 at 20 °C and 30 °C with the results shown in Fig. 1a. These data suggest an optimal assay buffer pH of 7.4 ± 0.2, and that deviating from this range causes precipitous loss of PLC-γ1 activity.

6.

The final composition of PLCDB includes 20 mM HEPES (pH 7.4), 70 mM KCl, 3 mM EGTA, 2.35 mM CaCl2, 2 mM DTT, and 1 mg/mL FAF BSA. We recommend making PLCDB fresh for the assay and cool the solution on ice prior to making PLC dilutions.

7.

The addition of MeOH leads to a more even, homogeneous film as the volume is reduced under the N2 stream. Warmth from the hands also helps to prevent early precipitation of lipids due to evaporative cooling.

8.

The goal is to maintain XY-69, PE, and PIP2 on the bottom of the tube where the resulting film will be most efficiently reached by energy from the sonication probe tip. We recommend drying the film gently to avoid blowing lipid solutions up the sides of the tube and rotate the tube slowly while drying in order to form an even layer.

9.

We have investigated the effects of the composition of the lipid film on the assay. The assay tolerates a wide range of vesicles with different lipid compositions. We obtained the best data quality using lipid vesicles with a final PE/PIP2 content of 220/20 or 192/48 μM.

10.

We recommend the use of high vacuum when drying lipid films, but if one is not available, then leave the lipid films under streaming N2 for at least 2 h.

11.

The outcome of lipid vesicle formation by probe sonication depends on several factors, including solution volume, temperature, vessel size and shape, lipid concentration, transducer percent output, pulse time, probe tip immersion depth, and probe tip diameter. Due to the sensitivity of this assay to the properties of lipid vesicles, deviation from the recommended parameters may affect assay performance and require re-optimization.

12.

If probe tip placement is too high, then foaming of the solution may occur. If probe placement is too low, inadequate circulation of contents may cause a problem. Review the manufacturer’s instructions for additional tips on optimizing this step.

13.

If the scale of LVS is changed, this step may require re-optimization of tube size, sonication power output, sonication time, and/or probe tip depth.

14.

The freeze–thaw step increases the size of lipid vesicle.

15.

When the solutions containing PLC isozymes are mixed, we recommend gentle pipette mixing or inversion only.

16.

This assay has been successfully performed in our laboratory at temperatures ranging from 20 °C to 37 °C. Running the assay at room temperature offers simplicity, and PLC activity generally increases with temperature under the listed conditions without detriment to data quality.

17.

Other default settings may need to be optimized depending on the type of microplate and plate reader used.

18.

The use of multichannel pipettes is recommended for minimizing error and reducing the amount of time it takes to assemble the assay. Otherwise, the experimenter should work quickly to avoid losing early data points on highly active samples. Keep any microplates and feeder solutions covered as much as practical to exclude dust, minimize evaporation, and protect fluorescent molecules from light.

19.

The addition of 10 μL of LVS into 2 μL of PLC solution should provide adequate mixing of assay components. A single gentle pipette mix of each well may be useful for homogenizing more complex samples. Additionally, gentle tapping on the side of the microplate may help even out all menisci prior to placing in the reader.

20.

The representative enzymatic reaction progression curves with different concentrations of PLC-γ1 (D1165H) in two lipid vesicle conditions are shown in Fig. 2.

21.

Membrane curvature and vesicle size have been shown to influence vesicle binding [27], domain conformation [28], and catalytic activity [29, 30] of phospholipase enzymes. Consequently, we investigated the effects of vesicle size on the XY-69 based assay. Lipid vesicles generated from probe sonication alone have Z-average size (Zav) in the range of 150–160 nm (Fig. 3a). We noticed that repeated freezing and thawing (freeze–thaw) of sonicated lipids produces larger vesicles ranging from 180 to 250 nm (Fig. 3b). The autoinhibited, wild-type PLC-γ1 has significantly higher activity in smaller-sized lipid vesicles (Fig. 3c) than in larger-sized ones (Fig. 3d). The relative activity of constitutively active mutant PLC-γ1 (D1165H) to that of wild-type enzyme is approximately two-fold in vesicles prepared from probe sonication alone. This ratio increased to nine-fold when lipid vesicles were prepared by first probe sonication and then freeze–thaw. Therefore, membrane-dependent regulation of PLC activity due to external stimulations or mutations is better monitored with XY-69 embedded in larger lipid vesicles.

References

  • 1.Harden TK, Sondek J (2006) Regulation of phospholipase C isozymes by Ras superfamily GTPases. Annu Rev Pharmacol Toxicol 46:355–379. 10.1146/annurev.pharmtox.46.120604.141223 [DOI] [PubMed] [Google Scholar]
  • 2.Hicks SN, Jezyk MR, Gershburg S et al. (2008) General and versatile autoinhibition of PLC isozymes. Mol Cell 31:383–394. 10.1016/j.molcel.2008.06.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Hajicek N, Charpentier TH, Rush JR et al. (2013) Autoinhibition and phosphorylation-induced activation of phospholipase C-γ isozymes. Biochemistry 52:4810–4819. 10.1021/bi400433b [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Rhee SG (2001) Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312. 10.1146/annurev.biochem.70.1.281 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Balla T (2013) Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 93:1019–1137. 10.1152/physrev.00028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Koss H, Bunney TD, Behjati S et al. (2014) Dysfunction of phospholipase C-γ in immune disorders and cancer. Trends Biochem Sci 39:603–611. 10.1016/j.tibs.2014.09.004 [DOI] [PubMed] [Google Scholar]
  • 7.Martins M, McCarthy A, Baxendale R et al. (2014) Tumor suppressor role of phospholipase C-ε in Ras-triggered cancers. Proc Natl Acad Sci U S A 111:4239–4244. 10.1073/pnas.1311500111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kataoka K, Nagata Y, Kitanaka A et al. (2015) Integrated molecular analysis of adult T cell leukemia/lymphoma. Nat Genet 47:1304–1315. 10.1038/ng.3415 [DOI] [PubMed] [Google Scholar]
  • 9.Sims R, van der Lee SJ, Naj AC et al. (2017) Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer’s disease. Nat Genet 49:1373–1384. 10.1038/ng.3916 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Magno L, Lessard CB, Martins M et al. (2019) Alzheimer’s disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph. Alzheimers Res Ther 11:16. 10.1186/s13195-019-0469-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cremasco V, Graham DB, Novack DV et al. (2008) Vav/phospholipase C-γ2-mediated control of a neutrophil-dependent murine model of rheumatoid arthritis. Arthritis Rheum 58:2712–2722. 10.1002/art.23757 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Afroz S, Giddaluru J, Vishwakarma S et al. (2017) A comprehensive gene expression meta-analysis identifies novel immune signatures in rheumatoid arthritis patients. Front Immunol 8:74. 10.3389/fimmu.2017.00074 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Waldo GL, Ricks TK, Hicks SN et al. (2010) Kinetic scaffolding mediated by a phospholipase C-β and Gq signaling complex. Science 330:974–980. 10.1126/science.1193438 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Smrcka AV, Hepler JR, Brown KO et al. (1991) Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science 251:804–807. 10.1126/science.1846707 [DOI] [PubMed] [Google Scholar]
  • 15.Seifert JP, Snyder JT, Sondek J et al. (2006) Direct activation of purified phospholipase C-ε by RhoA studied in reconstituted phospholipid vesicles. Methods Enzymol 406:260–271. 10.1016/S0076-6879(06)06019-8 [DOI] [PubMed] [Google Scholar]
  • 16.Litosch I (2000) Regulation of phospholipase C-β1 activity by phosphatidic acid. Biochemistry 39:7736–7743. 10.1021/bi000022y [DOI] [PubMed] [Google Scholar]
  • 17.Liu Y, Mihai C, Kubiak R et al. (2007) Phosphorothiolate analogues of phosphatidylinositos as assay substrates for phospholipase C. Chembiochem 8:1430–1439. 10.1002/cbic.200700061 [DOI] [PubMed] [Google Scholar]
  • 18.Rukavishnikov AV, Zaikova TO, Birrell GB et al. (1999) Synthesis of a new fluorogenic substrate for the continuous assay of mammalian phosphoinositide-specific phospholipase C. Bioorg Med Chem Lett 9:1133–1136. 10.1016/s0960-894x(99)00166-3 [DOI] [PubMed] [Google Scholar]
  • 19.Zaikova TO, Rukavishnikov AV, Birrell GB et al. (2001) Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C. Bioconjug Chem 12:307–313. 10.1021/bc0001138 [DOI] [PubMed] [Google Scholar]
  • 20.Rose TM, Prestwich GD (2006) Synthesis and evaluation of fluorogenic substrates for phospholipase D and phospholipase C. Org Lett 8:2575–2578. 10.1021/ol060773d [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Huang W, Hicks SN, Sondek J et al. (2011) A fluorogenic, small molecule reporter for mammalian phospholipase C isozymes. ACS Chem Biol 6:223–228. 10.1021/cb100308n [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Huang WG, Wang XY, Endo-Streeter S et al. (2018) A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes. J Biol Chem 293:1728–1735. 10.1074/jbc.RA117.000926 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Waldo GL, Boyer JL, Morris AJ et al. (1991) Purification of an AlF4- and G-protein β-γ-subunit-regulated phospholipase C-activating protein. J Biol Chem 266:14217–14225 [PubMed] [Google Scholar]
  • 24.Harden TK, Morris AJ, Waldo GL et al. (1991) Avian G-protein-regulated phospholipase C. Biochem Soc Trans 19:342–346. 10.1042/bst0190342 [DOI] [PubMed] [Google Scholar]
  • 25.Hajicek N, Keith NC, Siraliev-Perez E et al. (2019) Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations. Elife 8:e51700. 10.7554/eLife.51700 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Clapham DE (2007) Calcium signaling. Cell 131:1047–1058. 10.1016/j.cell.2007.11.028 [DOI] [PubMed] [Google Scholar]
  • 27.Wehbi H, Feng J, Kolbeck J et al. (2003) Investigating the interfacial binding of bacterial phosphatidylinositol-specific phospholipase C. Biochemistry 42:9374–9382. 10.1021/bi034195+ [DOI] [PubMed] [Google Scholar]
  • 28.Uekama N, Aoki T, Maruoka T et al. (2009) Influence of membrane curvature on the structure of the membrane-associated pleckstrin homology domain of phospholipase C-δ1. Biochim Biophys Acta 1788:2575–2583. 10.1016/j.bbamem.2009.10.009 [DOI] [PubMed] [Google Scholar]
  • 29.Ahyayauch H, Villar AV, Alonso A et al. (2005) Modulation of PI-specific phospholipase C by membrane curvature and molecular order. Biochemistry 44:11592–11600. 10.1021/bi050715k [DOI] [PubMed] [Google Scholar]
  • 30.Burack WR, Biltonen RL (1994) Lipid bilayer heterogeneities and modulation of phospholipase A2 activity. Chem Phys Lipids 73:209–222. 10.1016/0009-3084(94)90182-1 [DOI] [PubMed] [Google Scholar]

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