[17]

ANALYSIS

OF CHOLINE

AND PHOSPHOCHOLINE

191

[17] Q u a n t i t a t i v e Analysis of W a t e r - S o l u b l e P r o d u c t s of Cell-Associated P h o s p h o l i p a s e C- a n d P h o s p h o l i p a s e D - C a t a l y z e d H y d r o l y s i s of P h o s p h a t i d y l c h o l i n e

By DONALD A. KENNERLY Introduction Increasing evidence points to activation of cellular phospholipase C (PLC) and/or phospholipase D (PLD) on endogenous phosphatidylcholine (PC) in receptor-stimulated cells (reviewed by Extonl). Whereas PLC catalyzes the direct formation of 1,2-diacylglycerol, PLD may importantly contribute to the formation of this second messenger by an "Indirect Pathway" involving phosphatidic acid phosphohydrolase-catalyzed conversion of phosphatidic acid to diacylglycerol. Although studies have utilized [3H]choline to prelabel cellular PC in order to examine the liberation of labeled choline and/or phosphorylcholine as a result of cell activation, this approach involves a number of problems inherent to the use of radioisotopes, particularly in cells which cannot be labeled to isotopic equilibrium. To overcome this limitation, we have significantly extended the mass-based methods initially developed by others 2'3 for quantitating choline.4 Although other methods can provide similar sensitivity, they require high-performance liquid chromatography (HPLC) with postcolumn enzyme-catalyzeddetection,3 a technique frequently not in the repertoire of cell biologists and biochemists. Further, we have improved methods of differential extraction of cell-associated choline and phosphorylcholine and developed a new assay of the latter. An overview of the experimental approach is illustrated in Fig. 1. Extraction of Cellular Choline

Principle. Choline present in the methanol-water upper phase of a Bligh and Dyer~ extract forms a lipid-soluble ion pair with heptanonesoluble sodium tetraphenylboron. 1 j. H. Exton, J. Biol. Chem. 265, 1 (1990). 2 W. D. Reid, D. R. Haubrich, and G. Krishna, Anal. Biochem. 42, 390 (1971). 3 N. M. Barnes, B. Costal, A. F. Fell, and R. J. Naylor, J. Pharm. Pharmacol. 39, 727 (1987). 4 j. j. Murray, T. T. Dinh, and D. A. Kennedy, Biochem. J. 270, 63 (1990). 5 E. Bligh and W. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).

METHODS IN ENZYMOLOGY, VOL. 197

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

PHOSPHOLIPASE ASSAYS,KINETICS,SUBSTRATES

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SAMPLE

~ extraction BIIgh&Dyer ORGANIC PHASE

I

CHOLINE ASSAY

AQUEOUS PHASE (choline + P.chollne) Lipid Analysis ion pair extraction

\

ORGANIC PHASE (choline)

Choline Klnase

[32p]P-choline

[32p] ATP

AQUEOUS PHASE (P-choline)

l alkaline pho.phct.. AQUEOUS PHASE (choline + PI)

PHOSPHORYLCHOLINE ASSAY

Ion pair act on

F

ORGANIC PHASE (choline)

Choline Kinase

~ ~'32p]P-choline

[32p] ATP

AQUEOUS PHASE

FIG. 1. Extractionand quantitation of cellularcholineand phosphorylcholine(P-choline).

Reagents Sodium tetraphenylboron, 5 mg/ml in 4-heptanone Sodium phosphate buffer, 12 mM (pH 7.0) HC1, 0. I N [3H]Choline chloride, 4.5 /zCi/ml in ethanol (specific activity 87 Ci/mmol) Procedure. Unless otherwise indicated, all procedures were performed at room temperature. Lipids and water-soluble cellular components are extracted from a pellet containing 105-107 cells by adding 0.5 ml of methanol, 0.25 ml of chloroform, and 0.2 ml of water. After removing insoluble materials at 200 g for 10 min, the supernatant is transferred to a tube containing 45 nCi of [3H]choline, and phase separation is accomplished by adding 0.25 ml of chloroform and 0.25 ml of water. A 0.85-ml sample of the 0.925-ml choline-containing upper methanol-water phase is removed for further analysis of choline, and the lipid-containing lower phase can be dried and for analysis of cellular lipids. After adding 0.65 ml of 12 mM

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ANALYSIS OF CHOLINE AND PHOSPHOCI-IOLINE

193

sodium phosphate (pH 7.0) and mixing briefly, cellular choline is extracted into the upper heptanone phase generated by adding 1.0 ml of sodium tetraphenylboron in heptanone. After vigorous mixing and phase separation by centrifugation, 0.9 ml of the heptanone phase is removed and added to 0.3 ml of 0.1 N aqueous HCI (to disrupt the ion pair and extract free choline into the acidic aqueous lower phase). Following brief centrifugation in a Microfuge, the heptanone layer is removed by vacuum aspiration, 20/~l of the choline-containing aqueous phase is placed in a scintillation vial (to assess [3H]choline recovery) and 0.25 ml of the aqueous phase is dried either by using a Speed Vac or by heating in an oven at 65° overnight. Remarks. Although choline and phosphorylcholine are freely soluble in water, many investigators may find it useful to be able to examine both lipid- and water-soluble products of endogenous PLC and/or PLD activation. As a result, methods were developed to permit efficient differential extraction of choline and phosphorylcholine from the methanol/ water upper phase of a Bligh and Dyer cell extract (neither choline nor phosphorylcholine is found to any significant degree in the lower chloroform-containing phase). We exploit the ability of the tetraphenylborate ion to selectively complex with choline6 (but not with phosphorylcholine4) to render the choline-tetraphenylboron complex soluble in heptanone in preference to water/methanol. Aqueous sodium phosphate is added to the Bligh and Dyer upper phase to maintain a pH of approximately 7.0 and to reduce the concentration of methanol in order to increase the efficiency of extraction of the choline-tetraphenylborate complex into heptanone (80 - 3 compared to 67 - 5% when no additional water is added4). Purposefully incomplete sampling (69% of the theoretical maximum) is employed to improve the reproducibility of the multiple extractions required in the assay (average standard error of the mean equals 3.5%). Although adding NaC1 in a Bligh and Dyer extraction promotes phase separation and disrupts protein-lipid interactions, increasing the ionic strength interrupts the choline-tetraphenylboron ion pair and reduces the effectiveness of choline extraction into tetraphenylboron-containing heptanone. Because 25 mM NaCI causes a 10% reduction and 100 mM a 25% reduction in choline recovery,4 the addition of unnecessary salt is discouraged. Extraction of Cellular Phosphorylcholine To obtain cellular phosphorylcholine, the 1.5-ml choline-depleted methanol/water phase described above is subjected to a second identical extraction with sodium tetraphenylboron in heptanone to further reduce 6 F. Fonnum, Biochem. J. 113, 291 (1969).

194

PHOSPHOLIPASE ASSAYS, KINETICS, SUBSTRATES

[17]

residual choline (to

Quantitative analysis of water-soluble products of cell-associated phospholipase C- and phospholipase D-catalyzed hydrolysis of phosphatidylcholine.

[17] ANALYSIS OF CHOLINE AND PHOSPHOCHOLINE 191 [17] Q u a n t i t a t i v e Analysis of W a t e r - S o l u b l e P r o d u c t s of Cell-Associ...
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