Purification and Characterization of Phosphatidylcholine ...

VOl. 269, No. 49, Issue of December 9, pp. 3120741213, 1994

THEJOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in U.S.A.

Purification and Characterizationof Phosphatidylcholine Phospholipase D from Pig Lung* (Received for publication, August 16, 1994)

Shin-ichi Okamura and Satoshi YamashitaS From the Department of Biochemistry, Gunma University School of Medicine, Maebashi 371, Japan

Dawson, 1964). DAG formation from PC causes a delayed, proPhospholipase D, whichmediatesphosphatidylcholine hydrolysisin response to agonist stimulation, is an longed increase in DAG as compared with an early but tranimportant component of signal transduction. We now sient increase inDAG produced by hydrolysis of inositol phosreport the purification of this enzyme to homogeneity pholipids (Wright et al., 1988; Motozaki and Williams, 1989; from pig lung microsomes. The enzyme was solubilized Nakashima et al., 1991; Leach et al., 1991; van Blitterswijk et withheptylthioglucosideandpurified2,200-fold by al., 1991). Thus the agonist-induced stimulation of PLD elicits on sulfate-Cellulofine, successive chromatography ether-Toyopearl, chelate-Toyopearl, Q-Sepharose, hepa-prolonged activation of specific isoforms of protein kinaseC and thereby plays a role in long term cellular responses, such as rin-Toyopearl,andhydroxyapatite.Thefinalenzyme M,= 190,000 on proliferation and differentiation (Asaoka et al., 1992; Nishipreparation gave a single protein of band zuka, 1992). Furthermore, recent studies demonstrated that SDS-polyacrylamidegelelectrophoresis.Theenzyme hydrolyzed phosphatidylcholine but not lysophosphati-PA, the direct product of PLD, acts as an activatorfor protein dylcholine, phosphatidylethanolamine, and phosphati- kinase. Bocckino et al. (1991) demonstrated PA-dependent prodylinositol. Optimum pH was 6.6. Half-maximal activity tein phosphorylation with rat tissue extracts and suggested the was obtained at0.8 m~ dipalmitoylglycerophosphocho- existence of a PA-dependent protein kinase(s). A DAG-indeline. The products were identified as phosphatidic acid pendent isoform of protein kinaseC, 6, was purified and shown of ethanol, phosphati- to be activated by PA (Nakanishi and Exton, 1992). More reand choline, but in the presence dylethanol was produced at the expense of phosphatidic cently, Khan et al. (1994) partially purified and characterizeda acid. Ethanolamine and serine were not utilizedas the new protein kinase from human platelets, which was also acphosphatidylacceptor.Althoughnotobligatory,Ca2+ and Mg2' were stimulatory at high concentrations. The tivated by PA. The kinase was different from any of the curenzyme was markedly stimulated by unsaturated fatty rently identified protein kinase C isozymes since it did not of Mg2' but not inits absence or by cross-react with antibodies raised against them. The identifiacids in the presence cation of PA-dependent protein kinases has suggested a new saturated fatty acids. N-Ethylmaleimide and detergents were inhibitory. Sucrose monolaurate had an aberrant role for PLD in the protein kinasecascade of cell signaling. Only limited information is available about the molecular effect on enzyme activity. properties of PLD because the enzyme has never been highly purified. Taki and Kanfer (1979) purified PLD 240-fold from rat brain after solubilization with Miranol H2M freeze-dried Phospholipase D (PLD)' catalyzes the hydrolysis of phosand cholate. The specific activity of their preparation wasvery phatidylcholine (PC) in response to a variety of hormones, neurotransmitters, and growth factors and is believed to play a low, 2 nmol min-' mg protein", partly because the activator of t Their preparation utilized not fundamental role in signal transduction of cells (for reviews, PLD was notknown a t t h atime. see Pelech and Vance (1989), Billah and Anthes (1990), and only PC but also phosphatidylethanolamine (PE) with pH opExton (1990, 1994)). The primarylipid product of PLD is phos- timum at 6.0. Later, Chalifour and Kanfer (1982) identified phatidic acid (PA), and a major metabolic fate ofPA is PA unsaturated fatty acid as an activator of PLD in rat brain phosphatase-mediated dephosphorylation t o form diacylglyc- microsomes. Although the enzyme was located in the memrat tissues(Chalifourand Kanfer, 1982; erol (DAG) (Smith et al., 1957; Billah et al., 1989; Gruchalla et branefractionin al., 1990; Qian and Drewes, 1990; Siddiqui and Exton, 1992; Kobayashi and Kanfer, 1987) and Madin-Darby canine kidney Kanoh et al., 19921, representing an alternative pathway for cells (Huang et al., 19921, Wang et al. (1991) demonstrated that DAG formation to the hydrolysis of phosphatidylinositol 4,5- in bovine tissues a majority of enzyme activity was cytosolic. bisphosphate (PIP,) by phospholipase C (Thompson and The cytosolic enzyme waspurified 20-fold and shownto hydrolyze various phospholipids in the order of PE > PC > PI. The enzyme had a high K,,, whereas the membrane-boundenzyme * This work was supported in part by grants-in-aid for scientific re- was specific t o PC with a low K,,,.The cytosolic and membranesearch and cancer research from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were de- bound PLDs are believed to be different isoforms. The present study was undertaken to obtain a highly purifrayed inpart by the payment of page charges. This article musttherefore be hereby marked "advertisement"in accordance with 18 U.S.C. fied preparation of membrane-bound PLD. We used pig lung as Section 1734 solely to indicate this fact. the enzyme source because lung contains the highestPLD ac$ To whom correspondence should beaddressed. Tel: 81-272-20-7940; tivity(Chalifourand Kanfer,1982;Kobayashi and Kanfer, Fax: 81-272-20-7948. The abbreviations used are: PLD, phospholipase D; PC, phosphati- 1987; Wang et al., 1991). "he purified PLD was a 190-kDa dylcholine; PE,phosphatidylethanolamine; PI, phosphatidylinositol; protein. It catalyzed not only hydrolysis but also transphosGPC, sn-glycero-3-phosphocholine;PA,phosphatidic acid; DAG, 1,2- phatidylationand selectivelyutilized PC as substrate. Un4,5-bisphosphate; PAGE, diacylglycerol; PIP,, phosphatidylinositol saturated fatty acids were a potent activator of the enzyme. CHAPS, 3-[(3-cholamidopropolyacrylamide gel electrophoresis; Unlike base-exchange enzyme (Kanfer, 1972), PLD catalyzed pyl)dimethylammonio]-1-propanesulfonic acid; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l-propanesulfonicacid. transphosphatidylation in the absenceof Ca".

31207

Purification of Phospholipase D

31208

I

I

I

0

20

I

I

In

I

I

I

I

I

IO

D

1 I)

I

2

3

4

20

I:ractInn Ilun1hcr

FIG.1. Chromatography of pig lung phospholipaseD. A, sulfate-Cellulofine; B , chelate-Toyopearl; C, Gigapite. PLD activity was assayed a s described under "Experimental Procedures." The thick lines above the activity peaks indicate the fractions that were pooled. D, SDS-PAGE. Fractions from Gigapite column chromatography were subjected to SDS-PAGE on a 10% gel under the reducing conditions, followed by silver staining. Lanes1 3 ,neighboring fractionsfrom Gigapite with activity peak a t lane 2. Lane 4, molecular mass markers (from top to hottom):myosin (205 kDa), 6-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), and carbonic anhydrase (29 kDa). a t 600 x g for 5 min. The upper phase containing the released choline was transferred to a vial, and radioactivity was counted in a toluene/ Materials-Sulfate-Cellulofine and Gigapite (hydroxyapatite) were Triton X-100 scintillant with a Beckman LS7000 liquid scintillation purchased from Seikagaku Kogyo (Tokyo,Japan). Ether-Toyopearl, chespectrometer. late-Toyopearl, and heparin-Toyopearl were obtained from Toso (Tokyo, Purification of PLD fromPig Lung-All procedures were carried out Japan), andQ-Sepharose was obtained from Pharmacia (Uppsala,Swea t 4 "C. Pig lung wasminced and homogenized in 3 volumes of 0.25 M den). Sucrose monolaurate, n-heptyl-P-n-thioglucoside, n-octyl-p-n-glucoside, and other detergents except for Triton X-100 were from Dojindo sucrose containing 5 mM Hepes-HC1, pH 7.2, 1 mM dithiothreitol, and 0.2 mu phenylmethylsulfonylfluoride. The homogenate was centrifuged Laboratories (Kumamoto, Japan). Triton X-100 was purchased from a Wako (Osaka,Japan). 1,2-Dipalmitoyl-sn-glycero-3-phospho[methyl- t 1,000 x g for 10 min to remove cell debris and thena t 12,000 x g for a t 100,000 x g for 60 "Hlcholine (1,2-dipalmitoyl-lmethyl-"HIGPC, 40-80 Ci/mmol), l-palmi- 20 min. The supernatant was further centrifuged toyl-2-ll-'.'Clpalmitoyl-GPC (50-60 mCi/mmol), 1-1l-l"Clpalmitoyl-GPC min. The resultant pellet (microsomes) was suspended in a minimal (50-60 mCi/mmol), I-acyl-2-1 l-"'Clarachidonoyl-sn-glycero-3-phospho- volume of the homogenizing buffer and kept a t -80 "C until use. mM ethanolamine (50-60 mCi/mmol), and 1,2-diacyl-sn-glycero-3-phos- Thawed microsomes were adjusted to10 mg of proteidml with 10 Hepes-HC1, pH 7.2, containing 1 mM dithiothreitol, 0.2 mM phenylpho[2-"Hlinositol (10-15 Ci/mmol) were obtained from Amersham International (Amersham, U. K.). Other lipids were obtainedfrom Sigma methylsulfonyl fluoride, and 0.02% NaN,. Following the addition of heptylthioglucoside to a final concentration of2%, the mixture was and Wako. a t 100.000 x g for 60 min. PLD Assay-1,2-Dipalmitoyl-lmethyl-~H1GPC was mixed with 1,2- gently stirredfor 30 min and then centrifuged fluffy layer bedipalmitoyl-GPC, suspended in 0.05% Triton X-100 to 10 mM by soni- The supernatant was carefully withdrawn, leaving the column (2.5 x 5 cm) cation, and used as the substrate. The standard assay mixture con- hind, and then applied to a sulfate-Cellulofine tained 50 mM sodium dimethylglutarate, pH 6.6, 1 mM 1,2-dipalmitoyl- equilibrated with buffer A(40 mhl potassium phosphate, pH 7.2, 0.06% sucrose monolaurate, and 0.02% NaN,) containing 0.5 M NaCI. After ImethyZ-:'H1GPC (3,700 dpm/nmol), 2 mu sodium oleate, 2 mM MgCl,, washing with the same buffer, enzyme was eluted witha 300-ml linear and enzyme in a total volume of 0.1 ml. Reaction was started by the addition of 1,2-dipalmotoyl-GPC, allowed to proceed for 20 or 40 min at NaCl gradient (0.5-3.0 M) in buffer A. Active fractions were collected, 30 "C, and terminated by the addition of 3 ml of chlorofordmethanol brought to 1.6M with solid ammonium sulfate, and then applied toan (2:l) and0.6 ml of saline. The mixture was shaken and centrifuged a t ether-Toyopearl column (1x 13 cm) equilibrated with buffer A contain600 x g for 5 min. The upper layer was quantitatively transferred to a ing 1.6 M ammonium sulfate. The column was eluted with a reverse tube, shaken with 2ml of butyronitrile containing30 mg/ml tetraphen- gradient of ammonium sulfate from 1.6 to 0 M in 120ml of buffer Aand ylboron and 0.5 ml of 70 mM sodium phosphate, pH7.2, and centrifuged subsequently with 30 ml of buffer A. Eluate containing activity was EXPERIMENTAL PROCEDURES

Purification of Phospholipase D TABLE I Purification of phospholipase D from pig lung Total Protein activity

Specific activity

Purification

Yield

umollrnin

pmol m i r r - 1 mg"

-fold

% ,

3.12 Microsomes 1893 2.81 Heptylthioglucoside 1626 0.992 Sulfate-Cellulofine 9.2 0.797 Ether-Toyopearl 3.1 Chelate-Toyopearl 0.62 0.123 0.246 Q-Sepharose Heparin-Toyopearl 0.033 790 0.045 Hydroxyapatite 0.003 0.011

0.0017 0.0017 0.108 0.257 0.197

Fraction

mg

0.16

Front-

1

3 I209

A

100

1.51

1 64 151 120 890

1.35 3.67

90 32 26 3.9 7.9 1.4

2200

0.4

PA-

adjusted topH 8.0 with 1 M &PO, and applied to a zinc-treated chelateToyopearl column (1 x 5 cm) equilibrated with buffer A that contained 10 my Tris-HC1, pH 8.0, instead of potassium phosphate buffer, and 0.5 M NaCI. Enzyme was eluted with 60-ml a linear histidine gradient (0-25 Frontmbl) constructed in the samebuffer, diluted with 2volumes of buffer B (20 mhl Tris-HCI, pH 8.0,0.06% sucrose monolaurate, and 0.029 NaN,), and then adsorbed to a Q-Sepharose column (0.7 x 5 cm) equilibrated with buffer B containing 0.15 M NaCI. The column was eluted with a PEt30-ml linear NaCl gradient(0.15-1 RI) in bufferB. Active fractions were combined, diluted with 1 volume of buffer A, applied to a heparin0.2 Toyopearl column (0.7x 7 cm) equilibrated with buffer A containing M NaCI, and then eluted with a40-ml linear NaCl gradient(0.2-1.6 M) PAin buffer A. Eluted enzyme was adsorbed to a 0.8-ml Gigapite column equilibrated with bufferA. The loaded column was sequentially eluted with a16-ml linear gradient ofpotassium phosphate (0.04-1 M ) in buffer A and then with5 ml of 1 M potassium phosphate in buffer A to obtain the purified enzyme. Identification of the ReactionProducts-Enzyme was incubated with 1,2-dipalmitoyl-[methyl-:'H1GPC or l-palmitoyl-2-lr4Clpalmitoyl-GPC 4 6 8 10 12 14 16 18 Control under the standard assay conditions. The reaction mixture was mixed FIG.2. Reactionproducts. A, phosphatidicacid; B , phosphatiwith 3 ml ofchloroform/methanol(2:1)and shaken with0.6 ml of saline. dylethanol. Gigapite column fractions (20 pl each) shown in Fig. IC When 1,2-dipalmit0yl-[methyl-~~H]GPC was used as substrate, the upwere incubated with l-palmitoyl-2-[ '"C Ipalmitoyl-GPC in the absence per phase was saved for the analysis of the water-soluble product, (A) orpresence ( B1of ethanol under the standard assay conditions, and concentrated, and separatedby thin-layer chromatography (TLC) on a the chlorofordmethanol-soluble products were separated by TLC and Silica Gel 60 plate (Merck, Darmstadt, Germany) with methanol, 0.6% autoradiographed as described under "Experimental Procedures." ArNaCI, 30% ammonium hydroxide (20:20:1). The entire chromatogram rows indicate the locations of the authentic standards: PA, phosphatidic was divided into equal segments, and each segment was counted. When acid; PEt, phosphatidylethanol. The numbersbelow the lanes indicate l-palmit0yl-2-['~C1palrnitoyl-GPC was used as the substrate, thelower the fraction numbers from the Gigapite column. Control indicates the phase was isolated for the analysisof the lipid product and concentrated mock reaction without enzyme. in aVapor Mix evaporator (Toyo Rika, Tokyo, Japan). Radioactive lipids were separated by TLC with chlorofodmethanovacetic acid (13:3:1) Solubilized enzyme was first separated on a sulfate-Celluand autoradiographed using a Fuji RX film. lofine column (Fig. LA 1. Over 99% of protein passedthrough the ~ansphosphatidylationAssay-Enzyme was incubated with l-palmit0yl-2-['~C1palmitoyl-GPC in the presence of 400 mkl ethanol under column while about 40% of the PLD activity was adsorbed to the standard assay conditions, and the phosphatidylethanol formed was the column. PLD was eluted witha NaCl gradient, resulting in separated by TLC with chloroform/methanol/acetic acid (13:3:1) and 64-fold purification with 32% yield. The eluted enzyme was autoradiographed. applied to a hydrophobic column, ether-Toyopearl. The use of Other Analytical Methods-SDS-polyacrylamide gel electrophoresis hydrophobic adsorbents with larger aliphatic or aromatic hy(SDS-PAGE) was carried outby the method of Laemmli (1970). Protein low yields. The ammoniumsulfate concentration was determined by the method of Bradford (1976) using drocarbon chains resulted in eluate was then loaded onto a chelate column that had been the Bio-Rad protein assay kit. RESULTS

Purification of PLD from Pig Lung-After testing a number of detergents, we found heptylthioglucoside (Shimamoto et al., 1985) to be most suitable for the solubilization of PLD from pig lung microsomes. When examined a t a fixed microsomal protein concentration of 10 mg/ml, this nonionic detergent was most effective a t 2% with a recovery of 70-100% PLD activity in the 100,000 x g supernatant. Octylglucoside, octylthioglucoside, and Mega-9 (n-nonaoyl-N-methylglucamide) gave less satisfactory results. Triton X-100, CHAPS, CHAPSO, Mega-8 (n-octanoyl-N-methylglucamide), and Mega-10 (n-decanoy1-Nmethylglucamide)yielded poor results. Aftersolubilization with heptylthioglucoside, PLD was subjected to sequential column chromatographicprocedures. To keep enzyme solubleand minimize nonspecific adsorption, we included a low concentration of sucrose monolaurate (0.06%) in all solutions used for column chromatography.

pretreated with ZnC1, (panel B ) . PLD could be eluted by a gradient increase in histidine concentration. Probably because of inhibition by contaminated zinc (see below), total activity was decreased to 15% but was restored considerably after QSepharose chromatography. As the final step, we used hydroxyapatite (panel C). Overall, PLD was purified 2,200-fold from pig lung microsomes with a yield of 0.4%. Table I summarizes the typical purification. SDS-PAGE, followed by silver staining, revealed a single protein band of M,= 190,000 (panel D). The band correlated well with enzyme activity. The purification procedure was repeated a t least five times with reproducible results. Purified PLD was stable a t 4 "C for a t least 3 weeks without a significant loss of activity. Reaction Products-To confirm the identity of the purified enzyme, the reaction products were identified. Active fractions from the hydroxyapatite column (see Fig. 1C) were incubated with l-palmit0yl-2-['~CC]palmitoyl-GPC in the presence and absence of 400 mM ethanol under the standard assayconditions,


31210

120

200

90

x

+A

9

100

0

0 4

5

I

6

8

PH

FIG.3. pH profile. Activity was assayed using heparin-Toyopearl eluate (56 ng) as enzyme as described under “ExperimentalProcedures” except that the buffer was changed to 100 mM sodium dimethylglutarate buffer (0)or sodium phosphate buffer (0) of the indicated pH values.

and theradioactive lipids were separatedby TLC. As shown in of ethanol Fig. 2 A , the radioactive lipid producedin the absence comigrated with authenticPA. In thepresence of ethanol, however, the same fractions catalyzed the formation of phosphatidylethanol at the expense of PA. I t should be noted that phosphatidylethanol formationoccurred in the absenceof Ca2+ (see “Experimental Procedures”). These results provide conclusive evidence for the widely accepted view that mammalian PLD catalyzes not only hydrolysis but transphosphatidylation as well (Kobayashi and Kanfer,1987; Gustavssonand Alling, 1987; Bocckino et al., 198713). Unlike transphosphatidylation catalyzed by base-exchange enzyme (Kanfer, 1972), ethanolamine and serine were not utilized as the phosphatidyl acceptor (data notshown). To identify the water-soluble product, purified PLD was inthe upper layer cubated with 1,2-dipalmitoyl-[rnethyl-3HlGPC, of chlorofodmethanol extraction was separatedby TLC, and the chromatogram was analyzed for radioactive product as described under “Experimental Procedures.” Radioactivity comigrated with authentic choline, and no radioactivity was associated with phosphocholine and GPC (data not shown). Thus, choline was thesole water-soluble product. In the routine PLD assay, released choline was extracted from the upper layer of chlorofodmethanol extraction with butyronitrile in the presence of tetraphenyl-boron (Murray et al., 1990) and counted (see “ExperimentalProcedures”). Taken together, these results demonstrate that the purified enzyme catalyzedthe hydrolysis of PC to PA and choline and, in the presence of ethanol, the formation of phosphatidylethanol. Properties of Purified PLD-The molecular mass of purified PLD was estimated to be 190 kDa by SDS-PAGE under the reducing conditions (Fig. v)). SDS-PAGE performed under the nonreducing conditions gavethe same results (data shown). not PLD showed an anomalous behavior in gel filtration matrices, such as Sephacryl S-200, S-300, and S-400 (Pharmacia), Toyopearl HW65 (Toso), and Protein Pak G-300 (Waters, Milford, M A ) . Its elution was markedly retarded in these gels probably because of interaction with the gels. Thus it was difficult to determinethenative molecular mass by gel filtration. As shown in Fig. 3, PLD showed a rather sharp pH profile with maximum activity at pH 6.6 in sodium dimethylglutarate

1

2

3

4

1,2-Dipahtoyl-GPC (mM)

FIG.4. Dependence on phosphatidylcholineconcentration. Activity was assayed with 19 ng of purified enzyme as described under “Experimental Procedures” except thatthe concentration of 1,2dipalmitoyl-GPC was varied as indicated.

buffer. A similar pH profile was obtained with sodium phosphate buffer. Activities measured in these two buffers were not significantly different. Similar pH optima were reported for partially purified rat brain PLD (Taki and Kanfer, 1979) and detergent-solubilized bovine lung PLD (Wang et al., 1991). Choo 80 line formation was proportional to the incubation time tup min under the standard assay conditions. Fig. 4 shows the effect of varying concentrationsof dipalmitoyl-GPC onenzyme activity. PLD did not follow a normal saturation kinetics. The substrate dependence curve was sigmoidal as previously noted with rat brain synaptosomes (Kobayashi and Kanfer, 1987). Half-maximalactivity wasattained at 0.8 mM dipalmitoylGPC. This value was close to the K , value measured with partially purified rat brain enzyme, 0.75 mM (Taki andKanfer, 1979). Effects of Divalent Cations and Detergents-Ca2+ has been implicated in the activationof PLD (Augert et al. 1989; Huang et al., 1991; Kanaho et al., 1992). In HL-60 cells and neutrophils, Ca2+ was required for G protein-mediated activation of PLD (Anthes et al., 1991; Olson et al., 1991; Geny et al., 1993; Brown et al., 1993; Cockcroft et al., 1994). Ca2+ requirement was also demonstrated in the protein kinaseC-dependent activation, for example, of CCL39 cell membrane PLD (Conricode et al.,1994). We were interested in examining whether Ca2+ had a direct effect on PLD enzyme. As shown in Fig. 5, purified enzyme exhibited no absolute requirement for Ca2+and M e . EDTA had no effect. Low Ca2+ and Mg2’ had no significant effect, but higher Ca2+and Mg2‘ increased PLD activity to a maximum of 2.5- and 1.7-fold at 1and 2 mM, respectively. Zn2+ was inhibitory. Other divalent cations, such asCu2+,Co2+,and Ni2+,were also inhibitory (data not shown). These results are fairly well consistent with the observations obtained with partially purified rat brain PLD and synaptosomal membrane PLD (Taki and Kanfer, 1979; Chalifa et al., 1990). Several reports showed the stimulation of PLD activity by detergents (Chalifour and Kanfer, 1982; Kobayashi and Kanfer, 1987; Kanohet al., 1991; Huang et al., 1992). We examined the effects of some detergents on purified enzyme. Although purified enzyme already contained 0.06% sucrose monolaurate, its final concentration in the assay mixture was calculated to be 0.012%, a concentration considerably lower than its critical micellar concentration, 0.021%. Thus the effect of the endoge-

3 1211


L

500 , ” .

s

v

2. 400 ‘.* 5 *

P 9 ‘2 300

-d 2

0

4

8

6

200

cam” (mM)

FIG.5. Effects of divalent cations. Assay was carried out under the standard assay conditions except that Mg2‘ (O),Ca2+(01, or Zn2+(x) of the indicated concentrations was present in the assay mixture.

loo

100

li-”l

0

1

2 Oleicacid

4

3 (mM)

FIG.7. Effect of oleic acid. The assay was carried out with 19ng of enzyme as describedunder“ExperimentalProcedures”except that varying concentrationsof oleic acid were addedin the presence (0)and absence (0) of M e .

TABLEI1 Effects of fatty acids on phospholipase D activity Assays wereperformed as describedunder“ExperimentalProcedures” except that the indicated fatty acids were added at concentrations of 2 mM. Fatty acid

activityRelative %

46 0

2

4 Detergent (mM)

6

60

acid 8 acid

None Palmitic acid Stearic acid Oleic Linoleic Arachidonic

100 300 162 490

acid FIG.6. Effects of detergents. Purified enzyme(10ng) was assayed in the presence of varying concentrations of detergents as described with the under “Experimental Procedures.” Detergents used were sucrose mono- The optimal oleate concentration varied somewhat concentration of PC and amountof enzyme protein present in laurate (0)and Triton X-100 (0).

nous detergent was thought be to negligible. As shown in Fig. 6, sucrose monolaurate exerted an aberranteffect on PLD activity. PLD activity was inhibited by low concentrations of this detergent but restored considerably by higher detergent concentrations. Unlike sucrose monolaurate, Triton X-100 showed only inhibitory effect with little restorationof activity even at high concentrations. The exact mechanism for the paradoxical effects of sucrose monolaurate is not clear. Probably, this detergent had dualeffects: l)“surface dilution effect,” a decrease in the surface concentration of substrate at the water-lipid interphase by the addition of detergent (Deems et al., 1975; Hendrickson and Dennis, 1984) and 2) activation of PLD. The former would account for the enzyme inhibition at low detergent concentrations, and the latter would explain the partial restoration of enzyme activity at high detergent concentrations. N-Ethylmaleimide, a thioreactive reagent, inhibited PLD activity by 75% at 10 mM. Effect of Fatty Acid and Complex Lipids-The stimulation of PLD by fatty acid has been studied with membrane preparations(Chalifour and Kanfer,1982;Kobayashi and Kanfer, 1987; Chalifa et al., 1990; Siddiqui and Exton, 1992). But ambiguity remained as to whether or not fatty acid affected the enzyme itself. Fig. 7 shows the effect of oleic acid on purified PLD. Maximal activation occurred at a concentration of 2 mM.

the assay mixture. InTable I1 the effects of various fatty acids were compared at fixed concentrations of 2 mM. The datashow that all the unsaturated fatty acids examined were effective, whereas all the saturated fattyacids were ineffective. Arachidonic acid was themost effective fatty acid. Interestingly, Mg2‘ was needed for maximal activation of PLD by fatty acid (Fig. 7). The stimulatory effects of M$+ and Ca” (see above) required the presence of fatty acid (data not shown). We next examined the effects of various complex lipids on PLD activity by adding the lipids at a concentration of 0.1 mM to the standardreaction mixture insteadof oleic acid. Whereas lyso-PC, PE, PI, PA, and DAG were inhibitory to the enzyme, lyso-PA and PIP, were slightlystimulatory (about20% increase in activity). This weak stimulatory effect of PIP, was unexpected because Brown et al. (1993) reported that HL-60 PLD was strongly stimulated by this phospholipid. This discrepancy may be caused by different isoforms of PLD present in HL-60 cells and lung or simply by different assay conditions. Phosphatidylserine had no effect. Substrate Specificity-We examined the activity of purified PLD toward different phospholipids. Enzyme was incubated l-a~yl-2-[1-’~C]with 1mM l-palmitoyl-2-[l-’4C]palmitoyl-GPC, arachidonoyl-sn-glycero-3-phosphoethanolamine,l-[l-l4C1palmitoyl-GPC, and 1,2-diacyl-sn-glycero-3-phospho[2-3H]inositol underthestandardassay conditions, and we examined

31212


assaying phosphatidylbutanol synthesis using t3HIbutanol as labeled substrate. PE andphosphatidylserine were reported to give relatively small activities. In contrast, the present results are consistentwith the finding of Honvitz and Davis (1993) and Wang et al. (1991) as well. The latter authors showed that octylglucoside-solubilized membrane PLD from bovine lung was specific to PC. The hydrolysis of phospholipid other thanphosphatidylinosiPAto1 in response to agonist has been intensively studied. Several investigators showed that PE wasalso hydrolyzed together with PC in certain cell-agonist systems, such as NaF-stimulated mesangial cell microsomes (Harris and Bursten, 19921, LPAhydrogen peroxide- or linoleic acid hydroperoxide-stimulated endothelial cells (Natarajan et al., 1993), and phorbol ester- or Originthrombin-stimulated amnion cells (Mizunuma et al., 1993). But the results of most studies point to PC as the principal phos1 2 3 4 5 6 7 8 9 pholipid susceptible t o agonist-induced hydrolytic reaction. PC FIG. 8. Substrate specificity. Purified enzyme (19 ng) was incuhas been identified as the major hydrolyzable phospholipid bated with the indicated substrate (1 mM), and the reaction products were identified by TLC and autoradiography a s described under "Ex- based on the analysis of the fatty acid composition ofDAG perimentalProcedures."Substratesusedwerephosphatidylcholine (Grove and Schimmel, 1982) or PA (Bocckino et al., 1987a), (lanes 1 3 ) , phosphatidylethanolamine (lanes 4-6), and lysophosphatiidentification of the selectively decreasing phospholipid (Daniel dylcholine (lanes 7-9). Control experiments were performed without enzyme (lanes 3 , 6 , and 9 ) .Arrows indicate the locationsof the authen- et al., 1986; Bocckino et al., 1987a; Osada et al., 19921, and analysis of the molecular species of DAG (Augert et al., 1989; tic standards: PA, phosphatidic acid; LPA, lysophosphatidic acid. Pessin et al., 1990; Leach et al., 1991) or phosphatidylethanol whether radioactive PA or lyso-PA could be formed from the (Holbrook et al., 1992). These observations stronglysupport the PLD. former three phospholipids by TLC and autoradiography. When view that thePLD purified here is the signal-transducing Apparently, the present PLD is not involved in the agonist1,2-diacyl-sn-glycero-3-phospho[2-JHlinositol was used as substrate,the incubation mixturewasshakenwith 3 ml of induced hydrolysis of PE seen in some tissues (see above), chlorofodmethanol(2:l) and 0.6 ml of saline, and the aqueous where a different isoform of PLD (for example, cytosolic PLD phase was used to examine whether or not radioactive inositol with broader substrate specificity) (Wang et al., 1991) or some was formed. But the releaseof radioactive inositol could not be other enzymatic mechanism (Guy and Murray, 1983)may play demonstrated (data not shown). As shown in Fig. 8, PA was a role. PLD activity on PIhas been detectedinneutrophils liberated from PC but not from PE. Lyso-PA formation from (Balsinde et al., 1989) and Madin-Darby canine kidney cells lyso-PC could not be demonstrated. Thus PE, lyso-PC, and PI (Huang et al., 1992). The enzyme is Ca2+-dependent and cytowere not utilized by the enzyme. These results clearly show solic, thus different from the present PLD. These findings, tothat theenzyme was specific t o PC. gether with the present results,suggest that there areseveral PLDs with different substrate specificities. The present enDISCUSSION zyme should be adequately called phosphatidylcholine phosThe present study has shown that PLD can be efficiently pholipase D. An additional finding of some interest is thelack solubilized from lung microsomes with a nonionic detergent, of activity toward lyso-PC in the purified enzyme. Recently, heptylthioglucoside. The solubilized enzyme was purified to lyso-PA has attracted much attention as an intercellular sighomogeneity by sequential chromatographic procedures in the naling molecule (van Corven et al., 1989; van de Bend et al., presence of sucrose monolaurate. The molecular mass of the 1992; van Corven et al., 1993). This phospholipid is thoughtt o purified enzyme was considerably large, 190 kDa. A similar be synthesized from PA bythe action of a specific phospholipase value, 200 kDa, was also reported for the molecular mass of rat 4 in platelets(Billah et al., 1981; Gerrard andRobinson, 1989) brain PLD determined by gel filtration (Taki and Kanfer, 1979). and secreted in response to stimuli (Eichholtz et al., 1993). This size is even larger than those of PI phospholipases C-pand Theoretically, sequential action of, first, phospholipase 4 and, "y known to be activated by G protein and tyrosine kinase, second, PLD on PC could be considered as an alternative route respectively (for reviews, see Rhee et al. (1989) and Rhee and for the synthesis of lyso-PA, but the present results seem to Choi (1992)). Several factorshave been implicated in thereguexclude this possibility. lation of PLD activity, such as G protein, protein kinase C, and Ca2+.Although very little isknown about theregulation of lung REFERENCES PLD, we expect that this 190-kDa sequence might contain a Anthes, J. C., Wang, P., Siegel, M. I., Egan, R. W., andBillah. M. M. (1991) domain responsible for the control of enzyme activity by these Biochem. Biophys. Res. Commun. 175,236-243 factors. The specific activity of the present enzyme is 3.7 pmol Asaoka. Y.. Nakamura. S., Yoshida, K., and Nishizuka, Y. (1992)Dends Biochem. min" mg", but this activity will be considerably increased Sci. 17,414417 G., Bocckino, S. B., Blackmore, P. F., and Exton,J. H.(1989)J. Biol. Chem. when the regulatory mechanism for lung PLD activity is elu- Augert, 264,21689-21698 cidated and reconstitution of the regulation is accomplished. Balsinde, J., Diez, E., Fernandez, B., and Mollinedo, F. (1989)Eur:J . Biochem. 186, 7 17-724 ." 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