ANALYTICAL BIOCHEMISTRY 231, 277–281 (1995)

A Chemiluminescence Method to AnalyzePhosphatidylcholine–Phospholipase Activity in PlasmaMembrane Preparations and in Intact Cells

Miguel Lucas,1 Vıctor Sanchez-Margalet, Consolacion Pedrera, and Ma Luz BellidoDepartment of Medical Biochemistry and Molecular Biology, Virgen Macarena Hospital,Medical School of Sevilla, Avenida Sanchez Pizjuan 4, Sevilla 41009, Spain

Received June 1, 1995

such as the labeling of the cell preparation or targetWe present a new method to analyze the hydrolysis substrate with a radioactive tracer followed by extrac-

of phosphatidylcholine by either phospholipase C tion with organic solvents and chromatography. There-(PLC) or phospholipase D. The method is nonradioac- fore, a method to assay PLC and PLD in a single steptive, rapid, and very sensitive and is based on chemilu- could be quite useful in the study of signals transduc-minescence. It relies on the peroxidase-catalyzed che- tion in cells such as neutrophils (2,3) or cellular mem-miluminescent oxidation of luminol by the H2O2 brane preparations.derived from choline oxidation. The enzyme activity Different chemiluminescent reactions have beencan be quantified by calculation of produced choline used for the analysis of a variety of compounds (4). Wein a standard curve. Data are accurate and reproduc- took advantage of the high sensitivity of the chemilumi-ible in a large range of choline concentration. In fact, nescent oxidation of luminol to analyze in a single pro-the response of PLC to GTP in plasma membranes was cedure the hydrolysis of phosphatydylcholine by eithersimilar to the activity measured with radioactive

PLC or PLD. The method relies on the measurementmethods of diacylglycerol or phosphatidylinositol tri-of choline following its oxidation to H2O2 by cholinephosphate determination. The method can be appliedoxidase (5) which is detected by the peroxidation ofto study the hydrolysis of phosphatidylcholine inluminol in the presence of horseradish peroxidase. Weplasma membrane preparations and in intact cells.propose a single procedure to analyze phosphatidylcho-q 1995 Academic Press, Inc.line hydrolysis by either PLC or PLD. The method isvery sensitive and relies on the peroxidase-catalyzedchemiluminescent oxidation of luminol by the H2O2 de-rived from choline oxidation. The sequence of enzy-Phosphatidylcholine (PC)2 turnover cycles can gener-matic and chemiluminescent reactions is the following:ate second messengers (including diacylglycerol (DAG))

and arachidonic acid. The diacylglycerol can be gener-ated directly via phospholipase C (PLC) or by the action For PLC (1)of phospholipase D (PLD) to yield phosphatidic acid

PC / H2O r DAG / P-choline(PA) which is hydrolyzed to DAG by phosphatidic acid–phosphohydrolase (1). The procedures to assay PLD

P-choline / H2O r Pi / cholineand PLC activity are time consuming, requiring stepsFor PLD (2)

1 To whom correspondence should be addressed at DepartmentoPC / H2O r PA / cholinede Bioquımica Medica y Biologıa Molecular, Facultad de Medicina,

Avenida Sanchez Pizjuan 4, Sevilla 41009, Spain. Fax: 34-5-4557481.Chemiluminescent reaction (3)2 Abbreviations used: PC, phosphatidylcholine; DAG, diacylglyc-

erol; PLC, phospholipase C; PLD, phospholipase D; PA, phosphatidicCholine / 2O2 / H2O r betaine / 2H2O2acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline;

fMLP, N-formyl-Met-Leu-Phe; PMA, 4b-phorbol-12b-myristate-13a-acetate. Luminol / 2H2O2 r aminophthalate/ N2 / light.

2770003-2697/95 $12.00Copyright q 1995 by Academic Press, Inc.All rights of reproduction in any form reserved.

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LUCAS ET AL.278

It is a suitable and very sensitive method that canbe applied to the study of PLC and PLD in plasmamembrane preparations and in intact cells.

MATERIALS AND METHODS

Preparation of Cells

Human lymphocytes were prepared from freshly ve-nesected blood of healthy donors according to (6).Briefly, blood was diluted 1/2 with 0.9% (w/v) NaCl.Aliquots (6 ml) were layered over 3 ml of lymphocytesisolation solution (Hypaque–Ficoll) and centrifuged(47C, 20 min, 600g) to obtain mononuclear leukocytes. FIG. 1. Lineal regression fitting chemiluminescence rate versusThe cells were washed by low-speed centrifugation choline concentration. The indicated concentrations of choline–Cl

were added to test tubes containing the assay mixture (see Materials(47C, 5 min, 200g) in 0.9% NaCl. Afterward, to elimi-and Methods). The reaction was triggered by injecting 1 ml of cholinenate monocytes of this preparation of mononuclear leu-oxidase and the chemiluminescence rate values were recorded fromkocytes, 5 ml of the suspension, 3 1 106/ml, was poured the digital output of the chemiluminometer as the integral of counts

on a petri dish and incubated at 377C for 60 min. Non- in 30 s. The values are the mean{ SEM of four to six determinations.adhered cells were collected and washed by low-speedcentrifugation in 0.9% NaCl. Finally, cells were sus-pended in PBS buffer (137 mM NaCl, 2.6 mM KCl, 8.1 recorded and afterward the oxidation of choline wasmM Na2HPO4, 2 mM KH2PO4, pH 7.4) supplemented triggered by addition of 1 ml choline oxidase. Determi-with 1.2 mM CaCl2, 1.2 mM MgCl2. Cells were stored nation protocols include the preincubation of extractson ice and assayed within 2 h. The cells were almost with or without alkaline phosphatase to discriminate100% lymphocytes, as they were identified by light mi- between PLC and PLD. Further details are describedcroscopy after Giemsa staining. Human neutrophils in the legends to figures.were prepared by dextran sedimentation, Ficoll–Hy-paque separation and hypotonic lysis of remaining

DAG Assayerythrocytes (7). Cells were resuspended in Krebs–Ringer–Hepes buffer. Cell viability, checked with ei- The assay of DAG was carried out by the DAG kinasether 0.25% Trypan blue or 50 mmol/liter ethidium bro- method (10) as previously described (11). Briefly, mem-mide, was over 95%. branes were incubated with different concentrations of

GTP for 15 min at 307C. Lipid fraction was extractedby chloroform/methanol (2/1, v/v), dried under N2, andPreparation of Liver Membranesstored at0207C until the assay for DAG analysis. Sam-Rat liver membranes were prepared as described by ples were analyzed by the DAG kinase assay reagentNeville (8) up to step 11. The protein concentrations system from Amersham International (Amersham,were measured with a protein assay kit (Bio-Rad) Bucks, UK).based on the method of Bradford (9), with BSA as

standard.IP3 Assay

IP3 was determined as previously described (12).Chemiluminescence Reaction to Measure CholineBriefly, membranes were incubated with different con-

Aliquots of the cell and membrane preparations were centrations of GTP for 15 min at 307C. After proteinextracted by heating at 957C for 5 min followed by cen- precipitation, IP3 is determined in the supernatant bytrifugation at 12,000g. The samples were analyzed a radioimmunoassay from Amersham International us-within 1 h following the extraction. Luminol-dependent ing D-myo-inositol [3H] 1,4,5-triphosphate as a tracer.chemiluminescence was measured by means of aBerthold LB 9500 C luminometer, attached to a chart

Chemicalsrecorder, modified to enable the injection of reagentsvia a microsyringe through a light-tight septum. Sam- Lymphocyte isolation solution was purchased from

Lagitre SRL (Milano, Italy). Luminol (5-amino-2,3-ples for chemiluminescence determination were pre-pared by adding aliquots of the cell or membrane ex- dihydro-1,4-phthalazinedione), fMLP (N-formyl-Met-

Leu-Phe), and PMA (4b-phorbol-12b-myristate-13a-ac-tract to PBS buffer supplemented with 10 mM luminoland 2 units/ml horseradish peroxidase. After equilibra- etate) were from Sigma Chemical Co. (St. Louis, MO).

Alkaline phosphatase and choline oxidase were fromtion at 377C, the basal chemiluminescence rate was

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CHEMILUMINESCENT DETECTION OF PHOSPHOLIPASE ACTIVITY 279

ranging variable degree of biological effects. In fact,a high marge of chemiluminescence intensity can bemeasured when analyzing the response to an effectorin comparison to the maximal response induced by ex-ogenously added phospholipase C. Figure 2 showstraces of the chemiluminescence reaction from an ex-periment performed with peripheral blood lymphocytesstimulated with the indicated effectors (note that thetrace corresponding to exogenously added PLC wasscaled down 10 times). The calcium ionophore A23187and the potassium ionophore valinomycin elicited aclear activation of PLC. PMA stimulated slightly PLCactivity and we found a clear stimulation of PLD whichcould be detected without preincubating with alkalinephosphatase.

The results in Table 1 show the activation of PLC inneutrophils by PMA, fMLP, and A23187. The degreeof activation in 10-min incubation was almost fourtimes in the case of the calcium ionophore A23187 andFIG. 2. Traces of the chemiluminescence records of the reaction ofapproached values close to 30 nmol/106 cells in 10 min.choline oxidation. Lymphocytes were incubated in the presence of

the indicated effectors and the experiments were done as described In a set of experiments we measured PLC activityunder Materials and Methods. The concentrations of the effectors in plasma membrane isolated from rat liver and wewere 50 nM PMA, 2 mM A23187, and 0.4 nM valinomycin. Phospholi- obtained a clear dependency on GTP, suggesting thepase C refers to the incubation of cells with exogenously added 75

involvement of G protein. The curve obtained was com-mg/ml (approximately 60 U/ml) phospholipase C. For comparativeparable to the activation by GTP of phospholipasepurpose the traces of phospholipase C were scaled down 10 times.

The cell extracts were preincubated with nothing (dashed traces) or when those activities were analyzed by the release ofwith (solid traces) alkaline phosphatase (20 U/ml). Chemilumines- DAG and by the release of IP3 (see Fig. 3). The shapecence reaction was triggered by the addition of 1 ml (0.05 U) choline of the curve shows a quite similar dependency onoxidase to the assay mixture (see Materials and Methods) containing

GTP concentrations reaching maximal activation atthe extract. The curves, representative of two other experiments, are1006 M GTP.hand-drawn traces from the actual records obtained in the chart

recorder.

DISCUSSION

The activation of PLC or PLD mediates the effect ofBoehringer-Mannheim. Others reagents were of ana-several agonists such as fMLP and PMA in the caselytical grade.of neutrophils (2). The mechanism of PLC activationinvolves a G protein and transient increases in cytosolic

RESULTS free calcium (13). G proteins are also known to regulatephospholipid base exchange in rat liver plasma mem-We present a single procedure to measure choline

released from PC by the action of PLC an PLD. Intactcells or plasma membrane preparations deserved astarget in experimental designs aimed to measure the TABLE 1activity of the above indicated enzymes. The extract Phospholipase C Activity in Human Neutrophilswas analyzed by the coupled enzymatic reaction of ex-ogenously added alkaline phosphatase, choline oxi- P-Choline production

Additions (nmol/106 cells 1 min01)dase, and peroxidase that in a final step trigger thechemiluminescent reaction of the oxidation of luminol

None 0.96 { 0.1by the H2O2 derived from choline oxidation. PMA (50 nM) 3.38 { 0.4The rate of choline oxidation by choline oxidase was fMLP (1 mM) 3.9 { 0.4

linear at choline concentrations in the range 0.1–10 A23187 (4 mM) 4.2 { 0.4mM. This was shown by measuring the initial rate of

Note. Freshly isolated human neutrophils were incubated (51 104/choline oxidation as the integral of light emitted in theml) for 10 min at 377C with the above-indicated effectors. Reactionsfirst 30 s following the addition of the enzyme choline were stopped by centrifugation and the release of P-choline into the

oxidase (see Fig. 1). The high degree of linearity of the supernatant was determined as described under Materials andMethods. Results are the mean { SEM of five separate cell batches.reaction allowed experimental designs with effectors

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LUCAS ET AL.280

line as an indirect determination of P-choline. It is wor-thy to point out that, without alkaline phosphatase,the basal phosphatase activity in our system is verylow and therefore P-choline is not further hydrolyzedto choline.

The activity of both enzymes was measured in neu-trophils and lymphocytes in response to fMLP andPMA. The activity could be quantified as a percentageof maximal phosphocholine released by exogenouslyadded phospholipase C or as direct values of cholineproduced. The last values were obtained from the stan-dard curve of the rate of choline oxidation by cholineoxidase in the chemiluminescence assay. These arevery reproducible and accurate values, in spite of thehigh range of choline production, because of the goodcorrelation between chemiluminescence rate and cho-line in a large range of choline concentration.

The method was applied to plasma membrane prepa-rations and we could detect a qualitative response ofPLC to GTP similar to the curves obtained by assayingthe production of either DAG or IP3. However, it isdifficult to make a quantitative comparison since: (i)IP3 is the enzymatic product of PI-PLC; (ii) DAG is theFIG. 3. Comparison of three methods to measure PLC activity incommon, nonspecific, hydrolysis product of differentplasma membranes from rat liver. Plasma membranes (1 mg/ml)

were incubated at 377C for 10 min in the presence of the indicated glycerophospholipids by PI-PLC and PC-PLC and; (iii)concentrations of GTP. Reactions were stopped and the hydrolysis phosphorylcholine and choline are the products of PC-products phosphorylcholine, diacyl glycerol, and IP3 were extracted PLC and PC-PLD.and measured as indicated under Materials and Methods. Top, cho-

The measurement of instantaneously produced cho-line released from phosphorylcholine following the incubation of theline in a continuous assay is an alternative to the dis-cell extracts with phosphatase alkalyne.continuous assay described in the present article andcould be a promising method for the direct visualizationand recording of the immediate phospholipase activity.brane (14) and signaling through the breakdown of

phosphatidylcholine has been shown in many systems In fact, this was achieved in some instances with liverplasma membranes at a higher concentration.(15), suggesting a physiological role in cellular control

mechanisms that require long-term activation of pro-tein kinase C. ACKNOWLEDGMENT

Although there is no evidence of a signaling role for This work was supported in part by Fondo de Investigacion Sani-choline or phosphocholine, phosphatidic acid has been taria (FIS 92/0399)suggested to have a potential role as a second messen-ger in processes such as the stimulus–secretion cou- REFERENCESpling (16) and the respiratory burst of neutrophils (17).

1. Besterman, J. M., Duronio, V., and Cuatrecasas, P. (1986) Proc.On the other hand, a role for DAG as a second messen-Natl. Acad. Sci. USA 83, 6785–6789.ger is now well accepted in many biological systems

2. Smith, D. M., and Waite, M. (1992) J. Leukocyte Biol. 52, 670–(18). Therefore, a nonradioactive procedure measuring678.

the activities of PLD and PLC would be a quite helpful3. Garland, L. G. (1992) Immunology 105, 229–238.

tool in the analysis of membrane signaling mecha-4. Campbell, A. K. (1986) Trends Biochem. Sci. 11, 104–108.

nisms.5. Hasegawa, Y., Kunow, E., Shindou, J., and Yuki, H. (1991) Lip-

Determination of choline to assay hydrolysis of phos- ids 26, 1117–1121.phatidylcholine has previously been reported (19–21). 6. Bellido, L., Lopez-Gonzalez, M. A., Pedrera, C., and Lucas, M.In this paper we describe a nonradioactive method that (1994) Life Sci. 54, 1909–1916.is rapid and very sensitive to measure choline produc- 7. Lucas, M., and Solano, F. (1992) Anal. Biochem. 206, 273–277.tion in both cell and membrane preparations and, 8. Neville, D. M. (1968) Biochim. Biophys. Acta 154, 540–542.therefore, to assay the activities of PLC and PLD. We 9. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254.used an excess of alkaline phosphatase to hydrolyze all 10. Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Neidel, J. E.,

and Bell, R. M. (1986) J. Biol. Chem. 261, 8597–8600.the P-choline produced, and thus to measure the cho-

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CHEMILUMINESCENT DETECTION OF PHOSPHOLIPASE ACTIVITY 281

11. Sanchez-Margalet, V., Lucas, M., and Goberna, R. (1994) Bio- 16. Billah, M. M., and Anthes, J. C. (1990) Biochem. J. 269, 281–291.chem. J. 302, 51–54.

17. Mitsuyama, T., Takeshige, K., and Minakami, S. (1993) FEBS12. Sanchez-Margalet, V., and Goberna, R. (1994) J. Cell. Biochem. Lett. 328, 67–70.55, 173–181.

18. Berridge, M. J. (1987) Annu. Rev. Biochem. 56, 159–193.13. Mullmann, T. J., Cheewatrakoolpong, J. C., Siegel, M. I., Egan, 19. Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton,

R. W., and Billah, M. (1993) J. Leukocyte Biol. 53, 630–635. J. H. (1987) J. Biol. Chem. 262, 15309–15315.20. Irving, H. R., and Exton, J. H. (1987) J. Biol. Chem. 262, 3440–14. Siddiqui, R. A., and Exton, J. H. (1992) J. Biol. Chem. 267, 5755–

3443.5761.21. Kanaho, Y., Nakai, Y., Katoh, M., and Nozawa, Y. (1993) J. Biol.

Chem. 268, 12492–12497.15. Exton, J. H. (1990) J. Biol. Chem. 265, 1–4.

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