Phospholipase D-catalyzed hydrolysis of phosphatidylcholine occurs with PO bond cleavage

Biochimica et Biophysica Acta, 108~11991) 155-158

155

© It:91 ElsevierScience PublishersB.V. 0005-2760/9!/503.51) ADONIS 0005276091001974 BBALIP 53676

Phospholipase D-catalyzed hydrolysis of phosphatidylcholine occurs with P - O bond cleavage P.G. Holbrook, L.K. Pannell and J.W. Daly Laboratory of Bioorganic Chemistry, National Institute of Diabetes Digestive and Kidney Diseases. National Institutes of Health, Bethesda. MD ( U.S.A. )

(Received 7 January 1991)

Key words: PhospholipaseD; Phosphodiesterbond; Phosphatidylcholine:Phosphatidicacid: Signal-transduction; [ ISO]Oxygenlabeling Mammalian phospholipase D has been implicated in signal-transduction mechanisms, most recently in association with stimuli that enhance phosphatidylcholine (PC) turnover. It was previously unknown whether hydrolysis of PC by phospholipase D proceeds via P - O or C - O bond cleavage. Commercially available pbospholipase D isolated from Streptomyces chromofuscus was used to hydrolyse distearoyl phosphatidylcholine (PC) in a detergent-containing buffer consisting of 90% tSO-water. The product of hydrolysis, phosphatidic acid (PA), was purified by thin-layer chromatography and analyzed using californium-252 plasma desorption mass spectrometry. An increase of two mass units was observed, compared to a distearoyl PA control, consistent with a reaction mechanism involving cleavage of the P - O bond.

Introduction Phospholipase D catalyzes cleavage of the phosphodiester bond of a phospholipid molecule to produce phosphatidic acid (PA) and an alcoht~l; such as choline, ethanolamine, serine or inositol [1:. The mammalian enzyme has been purified from human eosinophils [2] and rat brain [3] and recently has received attention as an activity that attends PC-breakdown, stimulated either by receptor-activation or the tumor-promoting photbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) (reviewed in Refs. 4-6). A role for phospholipase D in receptor-activated phospholipid turnover was first suggested by the demonstration that the water-soluble product of phosphatidylinositol (Pl)-breakdown, elicited by acetylcholine in mouse pancreas, was the alcohol inositol rather than the expected product of phospholipase C-activation, inositol phosphate [7]. Nearly a decade later, it was shown that during stimulation of PI breakdown by formylmethionyl-leucyl-phenylalanine (fMLP) in neutrophiis prelabeled with 32Pi, 32p-PA was formed at significantly lower specific activity than the ATP

* Correspondence: P.G. Holbrook; Building 8 Room IA-15; National Institutes of Health; Bethesda, MD 20892, U.S.A.

pool [8]. This result is consistent with formation of PA directly by phospholipase D and not with formation exclusively by phosphorylation of diacyiglycerol (DAG) produced subsequent to phospholipase C-activation [9]. Recent observations suggest that phospholipase Dactivation may underlie enhanced PC-turnover elicited by TPA or by activation of a variety of 'calcium-mobilizing' receptors. An involvement of phospholipase D was suggested by studies demonstrating that TPA stimulates release of [3H]phosphocholine a n d / o r [3H]choline into the cellular medium of cells prelabeled with [3H]choline [10-13]. In perfused chicken heart and rat cortex, choline efflux is stimulated by muscarinic agonists [14] and a mechanism involving phospholipase D has been proposed [15]. In hepatocytes stimulated with vasopressin, PA is elevated prior to DAG and the fatty acid composition of the PA indicates that it is derived in part from PC [16]. Both products of phospholipase D-catalyzed hydrolysis of PC, namely choline and PA, can be derived front other metabolic pathways, so their formation is not conclusive evidence for involvement of phospholipase D. Perhaps more compelling are studies that demonstrate increased incorporation of low molecular weight alcohols, such as ethanol, into their corresponding phospholipids. The ability of phospholipase D to catalyze the incorporation of lipid and nonlipid-derived

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740 7so 760 MASS Fig I. Mass spectra of distearoyI-PA. DistearoyI-PCor distearoyI-PA were incubated with phospholipase D in the presence or absence of ~O-wlaer as described in Materials and Methods. DistearoyI-PAwas extracled, purified by TLC and analyzed using californium-252 plasma dcsorption massspectrometryas described in methods:(A) DistearoyI-PAincubatedwithenzymein Zf'O-water.tB) Distearoyl-PCincubi:ted with enzyme in t~O-w,aer.(C~DistearoyI-PAincubatedwith enzymein 180-water. alcohols into phospholipid was described in early studies of the purified plant enzyme and referred to as transphosphatidylation [17,18]. 'l-ransphosphatidylation is stimulated by TPA in several different cell lines [19-21] and by vasopressin in hepatocytes [22], thrombin in platelets [23], and ATP via a P2y-purinergic receptor in endothelial cells [24]. When the PC pool was prelabelcd with exogenous lyso-PC, either TPA [25,26] or receptor-activation [26-28] stimulated formation of labelled PA and, with ethanol present, phosphatidylethanol (PEt). Transphosphatidylatlon assays using exogenous alcohols provide a direct demonstration of phospholipase D-like activity in cells, but may not be adequate to

provide a quantitative measure of the PA pool formed by phospholipase D under physiological conditions. The extent to which transphosphatidylation competes with hydrolase activity has not been determined and thus the true extent of activation of phospholipase D in intact cells is not known. Furthermore, it is not clear whether transphosphatidylation assays distinguish true phospholipase D activity from calcium-dependent, phospholipase D-like, base-exchange enzymes that eatalyze incorporation of lipid-derived alcohols, choline, ethanolamine or serine, into phospholipid, by exchange with the alcohol moiety of a phospholipid molecule (reviewed in Refs. 29 and 30). The present study demonstrates that phospholipase D-catalyzed hydroly-

157 sis of PC occurs with P - O bond cleavage and incorporation of 180 into PA. It is suggested that 'SO-labeling of PA may prov!de a means to monitor the PA pool formed by phospholipase D in stimulated cells. Materials and Methods

Materials Phospholipase D from Streptomyces chromofuscus was from Sigma. Phospholipids, L-a-distearoylphosphatidylcholine and L-u-distearoyl-phosphatidic acid were from Avanti Polar Lipids. Tris(hydro.'cymethyl)aminomethane was from Bethesda Research Laboratories. Triton X-100 was from New England Nuclear. 18d-water (97-98 t80) was from Cambridge Isotope Laboratories. Thin-layer chromatography (TLC) plates were from Whatman. Methods Assay. Phospholipase D was assayed by a modification of the method described by lmamura and Horiuti [31]. The substrate, distearoyl-PC (1 /zmol), or product, distearoyI-PA (1 /.tmol), was suspended by sonication in 90/xl of a buffer consisting of 50 mM Tris-HCL, 10 mM CaCI z, 1% triton tpH 8.0), in either '~O-water or normal ~60-water. The reaction was started by addition of 10 p,I (20 units) of enzyme and was carried out for 30 min at 37°C. The reaction was stopped by addition of 3 ml of the extraction solvent, chloroform/ methanol (2: l, v/v). Samples were extracted by the method of Folch et al. [32], using 2 ml of 0.75% KCI in 50% methanol as the upper phase. The upper aqueous phase was aspirated and the lower organic phase was dried under vacuum. The resulting extracts were reconstituted in 100 b¢l of chloroform/methanol (1 : 1, v/v), applied to silica gel TLC plates (Whatman LK6-D), and developed with a mobile phase consisting of chlor o f o r m / ethanol/water/triethylamine (30 : 34 : 8 : 35,

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v/v). The substrate PC was clearly separated from the product PA. The PA band was scraped, the silica gel placed into a Pasteur pipette and the PA eluted with 3 ml chloroform/methanol (1:1, v/v). The sample was then dried, reextracted as described above, using an upper phase containing 1 M NaCI and dried under vacuum. Californium-252 plasma desorption mass spectrometry'. The sample, in chloroform/methanol (1:1), was deposited by electrospray onto an aluminized Mylar foil disk. The instrument and operation were as previously described by Pannell et al. [33], but addition of a reflectron provided enhanced resolution and rejection of metastable ions. Spectra were accumulated for approx. 10 h. Results and Discussion

Phospholipase D-catalyzed hydrolysis of PC might theoretically occur either by cleavage of the P - O or C - O bond with incorporation of IsOlt from H~O into either PA or choline, respectively. Studle~ differentiating between these two possibilities have not been previously reported [1], although most studies of the mechanism of enzymatic cleavage of phosphate esters support cleavage of the P - O ester bond rather than the relatively stable C - O alcohol bond [34]. in the study described here, distearoyI-PC was hydrolysed with phospholipase D from S. chromofitscus in a buffer consisting of 90% lZO-water. As a control, the reaction was also carried out in normal water. The lipid product of hydrolysis, PA, was isolated by TLC and analyzed by plasma desorption mass spectrometry (Fig. 1), An increase of two mass units was observed (Fig. IB) (MW = 751.9/disodium salt, compared to a PA standard (data not shown) or to the product, distearoyI-PA, isolated after a control reaction in normal water (Fig. IA) (MW = 749. 9/disodium salt). The result demon-

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158 strates that phospholipase D-catalyzed hydrolysis of PC involves clclavage of the p - O bond (Fig. 2). W h e n distearoyl-PA was incubated with e n z y m e in the presence of 180-water and then isolated, t h e r e was no change in mass (Fig. IC), indicating that the e n z y m e does not catalyze exchange of the hydroxyl groups of PA. O t h e r studies with ~80-water have d e m o n s t r a t e d that hydrolysis of phospholipid by phospholipase A a proceeds by O-acyl cleavage [35,36]. lsO-labeling also has been used to study the dynamics of cyclic nucleotide metabolism in various signal transduction p a r a d i g m s [37], including stimulus-secretion coupling [38]. In such studies the y-phosphoryl or A T P was shown to equilibrate rapidly with ~80-water [39]. It is proposed that 180-labeling might be exploited to monitor formation of PA in intact cells either by phospholipase D with incorporation of one a t o m of 180 or by phosphorylation of D A G , s u b s e q u e n t to phospholipase C activation, with incorporation of m o r e than one a t o m of ~ O from A'FP. Acknowledgement We wish to t h a n k Martin G a r r a f f o for his expert advice. P.G.H. was s u p p o r t e d by an associateship from the National R e s e a r c h Council. References 1 Dennis, E.A. (19831 The Enzymes, Vol. XVI, pp. 307-353, Academic Press. New York. 2 Kater. L.A. Goctzl, E.J. and Auslen, K.F. (19761 J. Clin. Invest. 57, 1173-1180. 3 Taki, T. and Kanfer, J.N. (19791 J. Biol. Chem. 254, 9761-9765. 4 Pelech, S.L. and Vance, D.E. (19891 Trends Biochem. Sei., 14, 28-30. 5 Loffelholz, K. (19891 Biochem. Pharmacol. 38, 1543-1549. 6 Extort, J.H. (19901 J. Biol. Chem. 265, I-4. 7 Hokin-Neaverson, M., Sadeghian, K., Majumder, A.L. and Eisenberg, F. (19751 Biochem. Biophys. Res. Commun. 67, 1537-1544. 8 Cockcroft, S. (19841 Biochim. Biophys. Acta 795, 37-46. 9 Michell, R.H. (19751 Biochim. Biophy. Acta 415, 81-147. 10 Guy, ^.R. and Murray, A.W. (19821 Cancer Res. 42, 1980-1985. II Liscovitch, M., Blusztajn, J.K., Freese, A. and Wurtman, R.J. (19871 Biochem. J. 241, 81-86.

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