[57]

575

P H O S P H O L I P A S E D FROM RAT TISSUES

PLD does not hydrolyze phosphatidylinositol or phosphatidylcholine and produces [3H]phosphatidic acid as the only radiolabeled product when [3H]myristate-labeled VSG is used as the substrate. 6,7 The properties of the purified GPI-PLDs correspond closely to those established previously from studies with plasma, serum, or partially purified GPI-PLD from a number of mammalian sources. ~5

[57] S o l u b i l i z a t i o n a n d P u r i f i c a t i o n o f R a t T i s s u e Phospholipase D B y M U T S U H I R O KOBAYASHI

and JULIAN N.

KANFER

Introduction

A role for phospholipase D (EC 3.1.4.4) in signal transduction has emerged, but the mechanisms of its activation are still unknown. Two types of phospholipase D have been reported. There is a membrane-bound type which is rich in microsome and plasma membrane fractions and utilizes phosphatidylcholine and phosphatidylethanolamine as substrates. The other is phosphatidylinositol-glycan-specific and has been detected in serum. Mammalian phosphatidylcholine-specific phospholipase D exhibits both hydrolytic activity and transphosphatidylation activity like the plant phospholipase D, 1 as shown in Fig. 1. Transphosphatidylation activity is a characteristic of the enzyme, and the activity is easily detected because it produces unusual lipids (e.g., phosphatidylethanol) in the presence of primary alcohols (e.g., ethanol). 2 Phospholipase D activity is barely detectable in the absence of appropriate activators in vitro measurement. Miranol H2M and taurodeoxycholate activate phospholipase D to some degree. Phospholipase D is best activated by unsaturated free fatty acids such as sodium oleate, arachidonate, linoleate, and linolenate. 3 Taki and Kanfer 4 first solubilized and partially purified mammalian phospholipase D from rat brain with the detergent Miranol H2M. At that time it was not apparent that unsaturated free fatty acids such as oleic acid 1 j. N. Kanfer, Can. J. Biochem. 58, 1370 (1980). 2 M. Kobayashi and J. N. Kanfer, J. Neurochem. 48, 1597 (1987). 3 R. Chalifour J. N. Kanfer, J. Neurochem. 39, 299 (1982). 4 T. Taki and J. N. Kanfer, J. Biol. Chem. 254, 9761 (1979).

METHODS IN ENZYMOLOGY, VOL. 197

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

576

PHOSPHOLIPASE D

[57]

Hydrolytic Activity Phosphatidylcholine + H20---~.-phosphatidic acid + choline

Transphosphatidylation Activity Phosphatidylcholine + n-alcohol--~-phosphatidyl alcohol + choline FXG. 1. Catalytic schema for hydrolytic and transphosphatidylation activity of phospho-

lipase D.

are the most potent activator of phospholipase D. Miranol H2M is now commercially unavailable; therefore, we examined other detergents to solubilize mammalian phospholipase D.

Assay for Phospholipase D Activity

Assay Principle The assay of phospholipase D is based on [~4C]phosphatidic acid formation from [14C]phosphatidylcholine. The phosphatidic acid produced can be degraded to diacylglycerol by phosphatidic acid phosphatase. Therefore, the assay mixture contains sodium fluoride. Inhibition of phosphatidic acid phosphatase activity by fluoride is incomplete. Transphosphatidylation activity is easier to measure, but in this case we determined the hydrolytic activity since it is unequivocal.

Reagents Mixed micelles of 12.5 mM [~4C]phosphatidylcholine and 25 mM sodium oleate: Egg yolk phosphatidylcholine (Serdary Research Laboratories, London, Ontario, Canada), 9.375 mg, and 5/.LCi 1-palmitoyl-2 [14C]oleoyl-sn-glycerol-3-phosphocholine (54.5 mCi/mmol, New England Nuclear, Boston, MA) are placed in a glass tube to adjust the specific activity to 0.42 Ci/mol, and 1 ml of 25 mM sodium oleate solution is added. The tube and contents are sonicated in a bath-type sonicator for 30 min. The resulting clear solution is used as the substrate. 500 mM 13-dimethylglutaric acid buffer, pH 6.5 200 mM EDTA, pH 6.5 250 mM NaF 1 mg/ml phosphatidic acid solution in chloroform

[57]

PHOSPHOLIPASE D FROM RAT TISSUES

577

Assay Procedure The incubation mixture contains 2.5 mM [~4C]phosphatidylcholine (1.67 Ci/mol) and 5 mM sodium oleate microdispersion, 50 mM/3-dimethylglutaric acid buffer, pH 6.5, 10 mM EDTA, 25 mM NaF, and enzyme preparation in a total volume of 120 ~1. The reaction is initiated by adding enzyme protein. When the enzyme preparation contains Triton X-100, the final concentration of the detergent is adjusted to 0.01% or less. The incubations are carried out at 30° for 90 min in a shaking bath. After the incubation, reactions are terminated by the addition of 2 ml of chloroform followed by 1 ml of methanol. Fifteen microliters of the phosphatidic acid solution in chloroform is added to each tube as a carrier. The samples are processed by the method of Folch et al. 5 Lipids are separated by thinlayer chromatography on 20 × 20 cm silica gel 60 thin-layer chromatography plates (Merck, Darmstadt, FRG) with a solvent system consisting of chloroform/methanol/acetone/acetic acid/water (50 : 15 : 15 : 10 : 5, by volume). The dried plates are exposed to iodine vapor, and the areas cochromatographing with standard phosphatidic acid and at the solvent front are scraped off and collected in scintillation vials. Ten milliliters of Scinti-Verse I (Fisher Scientific Co., Pittsburgh, PA) is added, and the radioactivity is determined.

Preparation of Enzyme Rat brain and lung are used as tissues for solubilizing phospholipase D. Twenty rats are decapitated, and the forebrains and lungs are quickly removed and weighed. Tissues are minced with a pair of scissors and homogenized with 7 strokes in 0.32 M sucrose, 5 mM HEPES, and 1 mM dithiothreitol (SMD) to make a 20% homogenate using a Teflon homogenizer. The homogenate is diluted with SMD to a final tissue weight concentration of 10%. The homogenate is centrifuged at 1000 g for 10 min at 4 °, the supernatant is centrifuged at 12,000 g for 20 min, and the resultant supernatant is removed and centrifuged at I00,000 g for 60 min. The 100,000 g pellet is suspended in SMD, washed once with SMD, suspended in a minimal volume of SMD, and used as the microsomal fraction. A cruder particulate fraction is obtained by centrifugation of the 1000 g at 100,000 g for 60 min at 4 °. The pellet recovered is washed once with SMD, suspended in a minimal volume of SMD, and used as the crude particulate fraction.

s j. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497 (1957).

578

PHOSPHOLIPASED Miranol H2M

{~:

501

£3

0

"-//

•

[57]

Na taurodeoxycholate Octyl-13-gtucopyranoside

.

0 0~5 0.1

0.013 0.05 0.2 TRITON X-100

.-Z,'

. . . .

0 0063 0.25

0 0031 0.125 0£)16 0.063 025

0 0Q25 0.1 0.013 0.05 Q2

0006 0.025 CHAPS

0.1

•

0.031 0.125 Na cholate

.-//

0 0.004 ODI5 0.002 (&008 0.03

o

0 0.~3 0.o5

.

0.5

_ _

DETERGENT CONCENTRATION (%) FIG. 2. Effect of various detergents on phospholipase D activity in the presence of the optimal concentration of sodium oleate. Phospholipase D activity was assayed with 2.5 mM [14C]phosphatidylcholine and 5 mM sodium oleate. The phospholipase D activity was 89.3 nmol/mg/hr in the absence of detergent.

Effects of Various Detergents on Phospholipase D Activity Effects of several concentrations of various detergents on rat brain microsomal phospholipase D activity are examined in the standard assay method. All the detergents examined inhibited the phospholipase D activity in a concentration-dependent manner (Fig. 2).

Effects of Various Treatments of Rat Brain Microsome on Phospholipase D Activity The rat brain microsome fraction is mixed with 0.1 M NaHCO3, 5 mM HEPES, 1 mM DTT, pH 7.3 (HD buffer), with or without 1 M NaCI or with or without 10 mM EGTA at 4° for 60 min with constant stirring. The final protein concentration is 4 mg/ml. The mixtures are centrifuged at 150,000 g for 60 min, and the phospholipase D activity is measured in both the supernatant and the pellet. The specific activity in the pellet is increased by approximately 25% with these treatments. The presence of NaHCO3 increases the specific activity slightly better than the others. Phospholipase D is inactivated when this fraction is treated with acetone.

[57]

PHOSPHOLIPASE D FROM RAT TISSUES

100

OM KCI

z~]&

a.

579

0.4M KCI

L

I,-or" W t~ "'*--...................

0

0.1

0.2

0.3

0.4

0

0.1

0.2

0.3

0.4

TRITON X-IO0 CONCENTRATION ( % ) FIG. 3. Solubilization of phospholipase D activity with Triton X-100 in the absence and presence of 0.4 M NaC1. The total phospholipase D activity is shown in the curve with open circles, total protein recovered in the supernatant with the filled circles. The total phospholipase D activity remaining in the pellet is represented with open triangle. Solubilization was performed in the absence (left) or presence (right) of 0.4 M NaC1.

Solubilization with Triton X-IO0 in Presence of Various KCI Concentrations The rat brain microsome fraction at a protein concentration of 2 mg/ml is suspended in HD buffer, and then varying amounts of 10% Triton X-100 solution and I M KCI are added to achieve required concentrations. The samples are mixed for 90 min in an ice bath with gentle stirring and centrifuged at 150,000 g for 60 min at 5°. The protein concentrations and enzyme activity in both the supernatant and the pellet are determined. As shown in Fig. 3, 35-50% of protein and more than 80% of the phospholipase D activity are solubilized by 0.2-0.4% of Triton X-100 in the absence of KCI. The specific activity of solubilized fraction is 160-190 nmol/mg/hr and is increased about 2-fold above the original microsome. The total activity present in the supernatant and pellet is decreased in the presence of KC1.

Solubilization o f Phospholipase D Activity with Other Detergents Miranol H2M, sodium taurodeoxycholate, and CHAPS are also tested for the ability to solubilize phospholipase D. Miranol H2M successfully solubilizes phospholipase D; however, it is not employed because it is no longer commercially accessible. Sodium taurodeoxycholate solubilizes unrelated proteins rather than the enzyme from microsomal fraction and, therefore, is used for enriching the activity in the pellet. CHAPS solubilizes phospholipase D activity but does not increase the specific activity and might be useful for solubilizing phospholipase D.

580

PHOSPHOLIPASE D

[57]

TABLE I PURIFICATION OF BRAIN PHOSPHOLIPASE D ~

Step 1. 2. 3. 4. 5.

Washed membranes 0.1 M NaHCO3 treatment 0.2% Sodium taurodeoxycholate treatment 0.4% Triton X-100 solubilization DEAE-Sephacel column fractions a

Total protein (mg)

Specific activity (nmol/mg/hr)

Total activity (nmoi/hr)

1427 1304 872 153 20

99.6 104.3 137.5 464.1 901.0

142,129 136,007 119,900 71,007 18,200

PurifiYield cation (%) (-fold) 100 96 84 50 13

1.00 1.05 1.38 4.65 9.05

Protein concentration was determined by the method of G. L. Peterson, this series, Vol. 91, p. 95.

Solubilization of Phospholipase D and DEAE-Sephacel Column Chromatography

Reagents 5 mM HEPES, 1 mM dithiothreitol (DTT), pH 7.3 (HD buffer) 0.1 M NaHCO3 1% sodium taurodeoxycholate solution, pH 7.4 10% Triton X-100 Procedure

Sodium Bicarbonate Treatment. Nine volumes of 0.1 M NaHCO3 is added to 1 volume of the membrane fraction at a final protein concentration of 2-4 mg/ml. The mixture is stirred for 60 min at 4 ° and centrifuged at TABLE II PURIFICATION OF LUNG PHOSPHOLIPASE D a

Step 1. 2. 3. 4. 5.

Washed membranes 0.1 M NaHCO 3 treatment 0.2% Sodium taurodeoxycholate treatment Triton X-100 solubilization DEAE-Sephacel column fractions

Total protein (mg)

Specific activity (nmol/mg/hr)

Total activity (nmol/hr)

156.0 120.6 67.1 31.7 5.7

305.9 401.3 375.5 541.7 1151.1

45,932 48,397 25,196 17,172 6591

PurifiYield cation (%) (-fold) 100 105 55 37 14

1.00 1.31 1.23 1.77 3.76

Protein concentration was determined by the method of G. L. Peterson, this series, Vol. 91, p. 95.

[57]

PHOSPHOLIPASE D FROM RAT TISSUES

a

581

..... ~

71 ~5001 0 30 60100

i 130

160

190

220

250

FRACTION NUMBER(10 ml/fraction )

tC-

-5 80

80

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z 40 I.J I---

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v

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[3..

zoo~o // 0 0 20 6O 90 120 150 180 FRACTION NUMBER ( 5 m l / f r a c t i o n ) FIG. 4. DEAE-Sephacel column chromatography of Triton X-100 solubilized protein. Open circles indicate phospholipase D activity, and the dotted line indicates protein concentration. Phospholipase D activity appears at around 0.2 M NaC1 in both brain (a) and lung (b).

150,000 g for 60 min, and the pellet is suspended in minimal volume of l i D buffer. Sodium Taurodeoxycholate Treatment. Sodium taurodeoxycholate (1%) and an appropriate amount of HD buffer are added to the NaHCO3treated pellet to give a final concentration of sodium taurodeoxycholate of 0.2%. The final protein concentration is 4-6 mg/ml. The mixture is stirred for 60 min at 4 ° and centrifuged at 150,000 g for 60 min, after which the pellet is suspended in a minimal volume of HD buffer.

582

PHOSPHOLIPASED

[57]

~j 1000

50(

o

0

2.5 5.0 Na Oleate ( mM )

FIG. 5. Effect of sodium oleate on phospholipase D activity solubilized from brain. The concentration of [14C]phosphatidylcholine was constant at 2.5 raM. Phospholipase D partially purified with DEAE-Sephacel was used for the assay in the absence and presence of the different concentrations of sodium oleate.

Solubilization with Triton X-IO0. Triton X-100 (10%) and appropriate amount of HD buffer are added to taurodeoxycholate-treated pellet to give a final concentration of Triton X-100 of 0.4%. The final protein concentration is 2-4 mg/ml. The mixture is stirred at 4° for 60 min, then centrifuged at 150,000 g for 60 rain, and the supernatant is saved. DEAE-Sephacel Column Chromatography. Solubilized proteins are applied to DEAE-Sephacel column (2.6 x 20 cm for brain and 1.6 x 11 cm for lung) preequilibrated with 5 mM HEPES, 1 mM DTT, pH 7.3, including 0.1% Triton X-100. After washing with 5 column volumes of equilibration buffer, proteins are eluted with a linear gradient of 400 ml each of equilibration buffer and 0.6 M NaC1 in the equilibration buffer. Phospholipase D activity appears as a single peak at around 0.2 M NaCI as shown in Fig. 4a for brain and in Fig. 4b for lung. The fractions which had the specific activity of more than 500 nmol/mg protein/hr for brain or 1,000 nmol/mg protein/hr for lung are pooled. The purification is summarized in Tables I and II. The specific activity was 901 nmol/mg protein/hr for brain or 1151 nmol/mg protein/hr for lung in the pooled fraction obtained from DEAE-Sephacel column chromatography with yields of 12.8 and 14% for brain and lung, respectively. Triton X-100 is removed from the pooled fraction using Extractigel-D (Pierce, Rockford, IL). Further attempts at purification with column chromatography including hydroxyapatite, Con A-Sepharose, AffiGel blue, octyl-Sepharose, phenyl-Sepharose, butyl-Sepharose, and phosphatidylcholineSepharose were unsuccessful.

[58]

LYSOPHOSPHOLIPASE D

583

Dependency on Sodium Oleate of Solubilized Phospholipase D The phospholipase D activity obtained from the column was measured in the presence of various concentrations of sodium oleate. It was seen that the activity was still dependent on the presence of oleate (Fig. 5).

Phosphatidic Acid Phosphatase Activity in the Partially Purified Enzyme Fraction The phospholipase D assay was performed in the absence or presence of the phosphatidic acid phosphatase inhibitor, sodium fluoride. Very little neutral lipid products were detected even in the absence of sodium fluoride, suggesting that phosphatidic acid phosphatase activity was absent or inactivated in the fraction. Summary Rat phospholipase D was successfully solubilized with Triton X-lO0. Solubilized phospholipase D is clearly separated from phosphatidic acid phosphatase and still dependent on sodium oleate for activity. Acknowledgments This work was supported by the grants from MedicalResearch Councilof Canada and the U.S. Associationfor Dementiaand Alzheimer'sDisease.

[58] L y s o p h o s p h o l i p a s e D

By ROBERT L. WYKLE and JAY C. STRUM Introduction Lysophospholipase D (EC 3.1.4.39) was discovered I during studies of plasmalogen biosynthesis in rat brain. When 1-O-[1-14C]alkyl-2-1yso-snglycero-3-phosphoethanolamine (1-O-alkyl-GPE) was used as a substrate with microsomal preparations from rat brain, it was noted that recovery of the label in phospholipids was low and that a significant portion of the added 1-O-alkyl-GPE had been converted to 1-O-alkyl-sn-glycerol. The conversion to 1-O-alkyl-sn-glycerol could be blocked by washing the microsomal preparations with 5 mM EDTA. The studies revealed that the I R. L. Wykleand J. M. Schremmer,J. Biol. Chem. 249, 1742(1974). METHODS IN ENZYMOLOGY, VOL. 197

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