Microanalytical determination of the activities of phospholipases A, C, and D and of their mixtures

ANALYTICAL

BIOCHEMISTRY

58, 301-309

(1974)

Microanalytical Determination of the Activities of Phospholipases A, C, and D and SHLOMO

of their

GROSSMAN,2 J. G. COBLEY;

Department of Biochemistry San Francisco, Cnlifornia Veterans AdmirGstration Received

duly

Mixtures1

G. OESTREICHER; P. K. HOGUE, AND THOMAS P. SINGER and Biophysics, University of California, 94149, and Division of Molecular Biology, Hospital, San Francisco, California 94121

13, 1973 ; accepted

September

7, 1973

In the course of studies on the catabolite repression of succinate dehydrogenase in yeast (1) , the need arose to monitor simultaneously phospholipases A and D activities in broken cell preparations. Although a variety of assay methods are described in the literature for both of these enzymes, none of them appeared to provide a simple and unambiguous measure of both activities simultaneously at the very low enzyme concentrations and with the crude enzyme preparations used. Therefore, a method was developed, based on the use of uniformly labeled 14C-lecithin, which provides simple and relatively easy assays for phospholipases A, C, and D, as well as for mixtures of the three enzymes, and permits initial-rate measurements. Details are described in this paper. Comparison of the methods described with other assays for phospholipases is presented in the Discussion. EXPERIMENTAL

Materials Unlabeled egg yolk L-a-lecithin was obtained from General Biochemicals, Inc., uniformly labeled L-W W-lecithin (1.85 Ci/mmole) from New England Nuclear. Phospholipase A was partially purified from Naja naja venom through the Sephadex G-75 step of the procedure of Cremona and Kearney (2). The preparation was diluted, just before use, with lo/O ‘Supported by a Grant GB 30078-01 from the National ’ On leave of absence from Bar Ilan University, Ramat-Gan, ’ Predoctoral Fellow of Organization of American States. ’ Fellow of the European Molecular Biology Organization. Copyright All rights

301 @ 1974 by Academic Press, Inc. of reproduction in any form reserved.

Science Foundation. Israel.

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ET

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(w/v) neutralized bovine serum albumin to a protein concentration of 60 pg/ml. Phospholipase C from Clostridium Welchii was purchased from the Sigma Chemical Co. (specific activity = 2.0 U/mg) and was dissolved in water to a protein concentration of 1.84 mg,/ml. Phospholipase D from cabbage (specific activity = 0.47 U/mg) was a lyophilized preparation from Calbiochem and was dissolved in 0.1 M Tris-acetate buffer, pH 5.7, to a protein concentration of 3.32 mg/ml. Assay

of Phospholipase

A

The method given below is a modification of the procedure of Doi et al. (3). A mixture of unlabeled lecithin (20 pmoles in 0.4 ml of ethanol) and ‘%-lecithin (0.27 ,uCi in 0.2 ml of ethanol) was dried under a stream of N, and suspended, with the aid of a Vortex mixer, in a solution composed of 1.2 ml 0.9 M Tris-Cl, pH 7.9, 0.5 ml 0.1 M CaCI,, 0.5 ml 0.12 M NaCI, and 1 ml 5% (v/v) Triton X-100 (purified to remove volatile impurities). Aliquots of 0.32 ml were placed in test tubes, and water was added to give a final volume of 1 ml during assay. The tubes were brought to 30°C and the reaction started by the addition of phospholipase A. The reaction was terminated by adding 3.75 ml of methanolchloroform (2: 1, v/v), mixed with a Vortex mixer, and chilled in ice to 0°C. After returning to room temperature, 1.25 ml of chloroform was added and the samples mixed with a Vortex mixer for 30 sec. Immediately, 1.25 ml of water was added to each tube, and the suspension was once again mixed with a Vortex for 30 sec. After brief centrifugation at room temperature to separate the layers, the upper layer was removed and the lower layer evaporated to dryness under N,. The resulting residue from each tube was dissolved in 0.1 ml of chloroform-methanol-water (65:25:4, v/v), and 20-~1 aliquots were applied to a Mallinckrodt Chrom AR sheet 1000, and tic was conducted in the same chloroformmethanol-water solvent mixture. Lecithin, lysolecithin, and fatty acids were visualized with I, vapor; the corresponding spots were cut out and suspended in 2 ml of water; 8 ml of scintillation gel (Aquasol, New England Nuclear) was added, and radioactivity was determined by scintillation counting. Assay of Phospholipase

C

Carrier lecithin (50 pmoles in 1 ml of ethanol) and 0.2 pLCi of 14clecithin in 0.15 ml of ethanol were dried under a stream of N2, and SUSpended, as in the phospholipase A method, in a mixture of 10 ml of 0.1 M Tris-Cl buffer, pH 7.5, 0.8 ml 5% (v/v) Triton X-100, and 0.1 ml 0.1 M CaCl,. Aliquots of 1.09 ml and water to give a final volume of 2.5 ml

ASSAY

OF

PHOSPHOLIPASES

A, C, AND

G

303

during assay were brought to 37°C. At zero time, enzyme was added, rapidly mixed with a Vortex, and the tubes incubated for 10 min at 37°C. The reaction was terminated by adding 1 ml of 6% (w/v) HClO,. The solution was extracted three times with ether with centrifugations (10 min at 1,200g) in between. The radioactivity remaining in the aqueous phase was taken as a measure of phosphorylcholine liberated. (The ahsence of other radioactive components was ascertained by tic). Radioactivity was counted in 1 ml aqueous phase plus 15 ml of scintillation solution (4) in a liquid scintillation counter. When it was desirable to determine the diglyceride liberated, the latter was separated from unreacted lecithin as follows. The first ether extract was t’aken to dryness under N, at 5O”C, dissolved in 0.1 ml chloroform-methanol-water (70:26:4, v/v), and 20-~1 aliquots chromatographed on silica gel sheets (Mallinckrodt, Chrom AR sheet 1000) as in the method for phospholipase A. After drying the sheet, lipids were visualized with I2 vapor, the diglyceride spot (R.B = 0.95) cut out and suspendedin 2 ml of H,O, dispersed with 8 ml of Aquasol gel, and counted. Assay of Phospholipase D The method is based on the determination of the radioactivity of perchloric acid-soluble products liberated from ‘“C-lecithin. The conditions of incubation are similar to those recommended by Kates and Sastry (5) and Yang (6). Fifty micromoles of carrier lecithin in 1 ml ethanol plus 0.2 PC1 14C-lecitl~in in 0.15 ml ethanol were dried under a stream of N,, resuspended with the aid of a Vortex mixer in 2 ml 0.4 M Tris-acetate, pH 5.7, and 1 ml 1 M CaCl,. Aliquots of 0.3 ml were placed in stoppered test tubes; 0.6 ml ether and sufficient water to give a final volume of 2.5 ml during assay were added, and the suspensionwas mixed with a Vortex. After bringing the suspensionto 3O”C, enzyme was added, the suspension again mixed with a Vortex, and incubated for 10 min at 30°C. The reaction was terminated with 1 ml of 6% (w,/v) HClO,. The solution was extracted three times with 5-ml portions of ether as in the phospholipase C procedure. The radioactivity remaining in the aqueous phase was determined as in the phospholipase C assay and was taken as the choline liberated. Phosphatidic acid was separated from the first ether extract and its radioactivity determined as in the case of diglycerides liberated by phospholipase C. (The first ether extract contains >95% of the phosphatidic acid.) Simultaneous Determination of Phospholipases il, C, and D Carrier lecithin (100 pmoles in 2 ml ethanol) and 0.6 ,&% of W-lecithin in 0.3 ml ethanol were dried under a stream of N, and resuspended in a

304

ClROSShlAN

ET

AL.

mixture of 10 ml 0.1 M Tris-Cl buffer, pH 7.5, 0.25 ml 1 M CaCl,, and 1.0 ml 5% (v/v) Triton X-100 with the aid of the Vortex mixer. Aliquots of 1.125 ml were diluted with water to give a final volume of 2.5 ml during assay and brought to 30°C. The reaction was started by adding a solution of the three phospholipases. After incubation for 10 min at 3O”C, the reaction was terminated by adding 1 ml 6% (w/v) HClO, to each tube, followed immediately by 5 ml of ether. The suspension was extracted three times with ether as in the phospholipase C assay. The aliquots of the aqueous phase were counted directly (see Phospholipase C method) for the determination of the sum of choline and phosphorylcholine liberated. The first ether extract was evaporated to dryness under N, at 5O”C, the residue dissolved in 0.1 ml petroleum ether(bp = 6O110°C) :diethylether: acetic acid (90: 10: 1, v/v), and lecithin, lysolecithin, diglyceride, phosphatidic acid, and fatty acids separated by two-dimensional tic. This was accomplished by applying 10 ,LL~ of the redissolved ether extract on Chrom AR sheets and developing the chromatogram in the solvent petroleum ether: diethyl et’her: acetic acid (90: 10: 1, v/v). The chromatographic sheets were dried and the lipids visualized by ultraviolet light (phospholipids, Rf = 0; diglycerides, Rf = 0.063; phosphatidic acid, Rf = 0; fatty acids, Rf = 0.24). The tic sheet was then run again at right angles to the original direction of migration in the solvent chloroform :methanol :water (70 :26 : 4, v/v). The lipids were visualized with I, vapor. (The Rf values were lecithin, 0.75; lysolecithin, 0.5; phosphatidic acid, 0.95; diglycerides and fatty acids, 0.95.) After cutting out the individual spots, they were suspended and counted as in the methods for phospholipase C. Control experiments with one enzyme present. at a time gave the same Rf values and yields as in mixtures. RESULTS

Figures 1 and 2 illustrate the assay of phospholipase C by the method described. Activity was proportional to enzyme concentration until the substrate concentration became rate limiting (Fig. 1). The kinetics were zero order, and the rates of liberation of diglyceride and phosphoryl choline were in good agreement (Fig. 2). Figure 3 shows enzyme proportionality and Fig. 4 the time course of product liberation in the phospholipase D assay. It may be seen that phosphatidic acid and choline formation agreed rather closely. Data for phospholipase A are not included since Doi et al. (3) have already reported on the validity of a phospholipase A assay based on the use of 14C-phosphatide as substrate and chromatographic separation of the products. It may be mentioned, however, that in the presence of 0.3 pg of purified enzyme (cf. Methods), the rate was linear for about 8 min,

ASSAY

200

OF

PHOSPHOLIPASES

A, C, AND

D

305

1

-

; s. 1602 i

0.4 08 PHOSPHOLIPASE

I I I 2 1.6 C (mg:

I 20

FIG. 1. Relation of the concentration of phoepholipase C to the amount of phosphorylcholine liberated. The reaction mixture was as described in Experimentnl; temperature, 37°C. Abscissa, milligrams of phosphotime of incubation, 10 min; lipase C preparation in assay mixture (1 mg = 0.46 mg of protein). Ordinate, nanomoles of product liberated per minute.

after which substrate limitation became evident and that the disappearance of lecithin closely paralleled the formation of lysolecithin and of fatty acids. Table 1 presents data on the simultaneous assay of phospholipases A, C, and D. The assay conditions (see Esperi~~ental) were selected so that all these enzymes would be at least partially active. Thus, the pH was 7.5 in between the pH optima of phospholipases A [pH 7.8 (7) J, C ]pH 7.2 (s)], and D [pH 5.6 (5)]. The Ca’+ concentration was reduced from the level used in the assay of phospholipase D (40 mM) to 10 m&l, because

2000

MINUTES

FIG. 2. Time course of phospholipase C action, Conditions were as in Fig. cept that 0.37 mg (protein basis) of phospholipase C preparation was present. nate, nanomoles of product liberated; A, phosphorylcholine ; B, diglyceride.

1, exOrdi-

306

GROSSMAN

0.4 0.8 PHOSPHOLIPASE

ET

1.2 D (mg)

AL.

1.6

2.0

FIG. 3. Relation of the concentration of phospholipase D to the amount of choline liberated. The reaction mixture was as specified in Experimental; time of incubation, 10 min; temperature, 30°C. Abscissa, milligrams of commercial phospholipase D preparation in assay mixture (1 mg solid = 0.83 mg of protein) ; ordinate, nanomoles of choline liberated per minut.e.

excess Ca2+ inhibits phospholipase C (8). As a result of suboptimal Ca’+ concentration, unfavorable pH and, possibly, the omission of ether, the phospholipase D activity was relatively low. It may be seen in Table 1 that the activities of phospholipase C and D, measured alone and in the presence of the other two phospholipases, agreed satisfactorily. Phospholipase A activity, as measured by fatty-acid liberation, was also the same in the mixture as when phospholipase A alone was assayed. The yield of lysolecithin, the other product of the action of phospholipase A, was 227% lower in the mixture than when phospholipase A alone was acting. Since this was a consistent finding, it raises the possibility that some of the lysolecithin formed by phospholipase A is further hydrolyzed by one of the other two enzymes in the mixture. Cabbage-leaf phospholipase D has been, in fact, reported to act

Fro. 4. Time course of phospholipase D action. Conditions were as in Fig. 3, except that 0.66 mg (protein basis) of phospholipase D preparation was present. Ordinate, nanomoles of product liberated in 10 min; A, choline; B. phosphatidic acid.

ASSAY

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D

TABLE 1 Comparison of the Activities of Phospholipases A, C, and D Assayed Individually and in Mixtures” Product liberated

Enzyme present

Fatty acid

Phospholipase A Phospholipase C Phospholipase D Phospholipases A+C+D

1.87

Lysolecithin

(moles/l0

Products in aqueous phaseb

min)

Diglyceride

Phosphatidic acid

1.66

1.98

3.26 0.20 3.40

1.30

2.95 2.64

0.20 0.25

0 Conditions were as described under Experimental. The amounts of the various enzyme preparations assayed on a protein basis were as follows: phospholipase A, 0.3 rg; phospholipase C, 0.37 mg; phospholipase D, 1.66 mg. b Choline and/or phosphorylcholine.

on lysolecithin (9). Since the lysophosphatidic acid which would result from cleavage of choline from lysolecithin was not detected and since the total activity of phospholipase D on both lecithin and lysolecithin was less than the amount of lysolecithin missing, as judged by the yields of phoephatidic acid and choline, it seemspossible that phospholipase C might also have acted on the lysolecithin to a small extent. Hydrolysis of lysolecithin by phospholipase C would give rise to a monoglyceride ; t.his would explain the slightly lower recovery of diglyceride in the mixture of the three enzymes, as compared with the assay of phospholipase C alone. To our knowledge, the action of phospholipase C on lysolecithin has not been reported in the literature, but such a possibility is consistent with the broad specificity of the enzyme (8). This uncertainty reflects our incomplete knowledge of the enzyme in question rather than a shortcoming of the analytical procedure. As concerns the method itself, it seemsclear that it provides a simple and unambiguous means for following the activities of individual phospholipases even in complex mixtures and at low enzyme concentrations. DISCUSSION

While the use of radioactive phospholipids for the assay of phospholipases is not conceptually original since there are reports on the use of “H-labeled choline lecithin for the assay of phospholipases C (10) and D (11) and also on the assay of phospholipase A with I%-lecithin (3)) the present study represents useful applications and extensions of this technique.

First,

while

in

previous

methods

commercially

available,

308

GROSSMAN

ET

AL.

labeled phospholipids have only been used for the assay of phospholipase A (3), the present study shows that it also provides satisfactory and direct assays for phospholipases C and D. Moreover, since 14C-lecithin may be readily and quantitatively converted to ‘“C-lysolecithin by purified phospholipase A from snake venoms, the method may be undoubtedly extended to include the determination of phospholipase B activity. Among these applications, the assay of phospholipase C deserves emphasis, since other available procedures (8) are either indirect biological assays or too insensitive for use with crude enzyme preparations (e.g., acid liberation) or unspecific (e.g., liberation of acid-soluble phosphorus). Moreover, none of them lends themself readily to initial-rate measurements, whereas chromatographic separation of ‘“C-phosphorylcholine in complex mixtures or separation of acid-soluble 14C-phosphorylcholine in preparations devoid of phospholipase D readily lend themselves to kinetic studies. Second, the use of 3H-labeled choline lecithin for the assay of phospholipases C (10) and D (11) is impractical, because the substrate is not commercially available and requires elaborate biological synthesis and isolation. It should be emphasized that the use of l”C-lecithin and determination of the radioactivity of the acid-soluble products provide a far more sensitive and faster method than either precipitation of the choline liberated with periodide (12) or reineckate (13,14) or chromatographic separation of the phosphatidic acid liberated and its quantitative determination by Phosphorus analysis (6). Third, as shown in the present study, the use of W-lecithin, in combination with separation of the products by tic, permits the simultaneous determination of the activity of phospholipases. This, in turn, permits analysis of the enzymatic machinery of phospholipid catabolism or of alterations in membrane-bound phospholipids in complex systems, as has been demonstrated in the case of the glucose repression of aerobic yeast cells (1). REFERENCES 1. GROSSMAN, S., COBLET, J., HOGUE, P. K., KEARNEY, E. B., AND SINGER, T. P. (1973) Arch. Biochem. Biophqs. 158, 744. 2. CREMONA, T., AND KEARNEY, E. B. (1964) J. Biol. Chem. 239, 2328. 3. DOI, O., OHKI, M., AND NOJIMA, S. (1972) Biochi~~. Biophys. Acta 260, 244. 4. HORGAN, D. J., SINGER, T. P., AND CASIDA, J. E. (1968) J. Biol. Chem. 243, 834. 5. KATES, M., AND SASTRY, P. S. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 14, p. 197, Academic Press, New York. 6. YANG, S. F. (1969) ill Methods in Enzymology (Lowenstein, J. M., cd.), Vol. 14, p. 208, Academic Press, New York. 7. SALACH, J. I., SENC, R., TISDALE, H., AND SINGER, T. P. (1971) J. Biol. Chem. 246, 340. 8. OTTOLENCHI, A. C. (1969) in Methods in Enzymology (Lowenstein, J. M.. ed.), Vol. 14. p. 188, ilcademic Press, New York.

ASSAY

9. 10. 11. 12.

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AND

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LONG, C., ODAVIC, R., AND SARGENT, E. J. (1967) &o&em. J. 102, 216. DINER, B. A. (1970) Biochim. Biophys. Acta 198, 514. TZUR, R., AND SHAPIRO. B. (1972) Biochim. Biophys. Acta 280, 290. APPLEMN, H. D.. LADU. B. N.. LEVY, R. B., STEALE, J. M., AND BRODIE, (1953) J. Biol. Chem. 205,803. 13. GLICK, D. (1944) J. Bid. Chem. 156, 643. 14. CRATES, M. (1955) crcn. J. Biochem. Physiol. 33, 575.

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