[56]
GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PLD
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[56] G l y c o s y l p h o s p h a t i d y l i n o s i t o l - S p e c i f i c P h o s p h o l i p a s e D
By Kuo-SEN HUANG, SHIRLEY LI, and MARTIN G. LOW Introduction The glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was first observed as a result of its ability to degrade the GPI anchor of alkaline phosphatase during extraction from mammalian tissues with butanol, l This anchor-degrading activiy was initially thought to be due to the action of inositol phospholipid-specific phospholipases C, which are very active in most mammalian tissues. However, more detailed studies distinguished these two activities and indicated that a novel and highly specific phospholipase D was involved. 2 The activity was subsequently found to be abundant in mammalian plasma and serum, suggesting that the activity observed in the tissues may be, to a large extent, the result of blood contamination. 3-5 The GPI-PLD has recently been purified from human 6 and bovine 7 serum. The physiological function of this enzyme has not been determined, but it is proposed to play a role in the regulation of cell surface expression of GPI-anchored proteins (reviewed in Refs. 8-10).
Assay Methods The GPI-PLD hydrolyzes the phosphodiester linkagc of the phosphatidylinositol moiety in the GPI anchor of several membrane proteins. The products of this reaction are phosphatidic acid and an inositol residue which is attached to the C terminus of the protein through the glycan (for details of the structure of the GPI anchor, see Refs. 9 and 10).Thc assay I M. G. Low and D. B. Zilversmit, Biochemistry 19, 3913 (1980). 2 A. S. Malik and M. G. Low, Biochem. J. 2411, 519 (1986). 3 M. A. Davitz, D. Hereld, S. Shak, J. Krakow, P. T. Englund, and V. Nussenzweig, Science 238, 81 (1987). 4 M. G. Low and A. R. S. Prasad, Proc. Natl. Acad. Sci.U.S.A. 85, 980 (1988). 5 M. L. Cardoso de Almeida, M. J. Turner, B. B. Stambuk, and S. Schenkman, Biochem. Biophys. Res. Commun. 150, 476 (1988). 6 M. A. Davitz, J. Horn, and S. Schenkman, J. Biol. Chem. 264, 13760 (1989). 7 K.-S. Huang, S. Li, W.-J.C. Fung, J. D. Hulmes, L. Reik, Y.E. Pan, and M. G. Low, J. Biol. Chem. 265, 17738 (1990). a M. G. Low and A. R. Saltiel, Science 239, 268 (1988). 9 M. A. J. Ferguson and A. F. Williams, Annu. Rev. Biochem. 57, 285 (1988). to M. G. Low, Biochim. Biophys. Acta, 988, 427 (1989).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
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PHOSPHOLIPASED
[56]
methods that have been used in the purification procedure described here monitor production of either of these products.
Assay I: Alkaline Phosphatase as Substrate In the following assay the loss of the hydrophobic part of the anchor from the protein is determined by partitioning in Triton X-114 H and measuring the distribution of the protein between the hydrophilic, detergentpoor phase and the hydrophobic, detergent-rich phase. In principle, any GPI-anchored protein whose distribution could be determined easily would be an appropriate substrate. In the assay to be described here the substrate is human placental alkaline phosphatase, and its distribution in Triton X-114 is determined by measuring its ability to hydrolyze pnitrophenyl phosphate in a subsequent incubation. The major disadvantage of this type of assay is that it does not distinguish between GPI-PLDmediated cleavage and those catalyzed by other hydrolases especially GPI-PLC. It is also more time consuming since it involves a second incubation to assay for the degraded alkaline phosphatase. However, an advantage over the other assay procedure is the ready availability of the starting material for preparation of the substrate. Purification o f Human Placental Alkaline Phosphatase. Alkaline phosphatase, with the phosphatidylinositol anchor intact, is purified from human placenta by a procedure based on previous studies of anchor degradation in human placenta. 2 Except where specified otherwise, the following procedure is carried out at 00-4 °. Term human placenta frozen after delivery is thawed, membranes and fibrous material discarded, and the remaining tissue (typically about 300 g) homogenized in 100 mM Tris-HCl, pH 8.5 (3 ml/g tissue) containing benzamidine (1 mM final concentration) and phenylmethylsulfonyl fluoride (PMSF, 0.01 mg/ml). The homogenate is mixed with ice-cold butanol (3 ml/g tissue) and shaken vigorously at intervals for 5 min. The mixture is centrifuged at 5000 g for 45 min, and the lower aqueous phase is collected and dialyzed against 3 changes of 12 volumes of distilled water over a period of 48 hr. An alkaline pH is essential during the initial butanol extraction since the endogenous GPI-PLD is activated below pH 7.0 by the butanol. The dialyzate is concentrated 4-fold in an Amicon (Danvers, MA) ultrafiltration cell using a YM30 membrane. Triton X-114 [1% (w/v) final concentration], Tris-HCl, pH 8.5 (10 mM), MgCI2 (0.1 mM), and zinc acetate (10/~M) are added as 10x or 100x solutions and the mixture incubated at 37° for 10 min. The phases are separated by centrifugation at 1500 g for 5 min and the upper, detergentpoor phase removed by aspiration. 11 C. Bordier, J. Biol. Chem. 256, 1604 0981).
[56]
GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PLD
569
The lower, detergent-rich phase (containing alkaline phosphatase with an intact GPI anchor) is mixed with l volume of l0 mM Tris-HC1/0.1 mM MgCI2/10/zM zinc acetate, pH 8.0 (buffer A) and applied to a column (2.5 x 40 cm) of SM-2 BioBeads (Bio-Rad, Richmond, CA) or Amberlite XAD-2 (Sigma, St. Louis, MO) equilibrated and eluted with buffer A at a flow rate of 25 ml/h. The detergent-depleted eluate (100-170 ml eluted), containing most of the alkaline phosphatase activity, is concentrated in an Amicon ultrafiltration cell using a YMI0 membrane to a volume of approximately 20 ml and centrifuged at 10,000 g for 15 min to remove insoluble material. The supernatant is applied to a column (two 2.5 x 118 cm columns connected together) of Sephacryl S-300 (Pharmacia, Piscataway, NJ) equilibrated with 150 mM NaCl/0.1 mM MgCIJ10 /xM zinc acetate/10 mM HEPES-NaOH, pH 7.4, and eluted at a flow rate of 12 ml/ hr. The high molecular weight, hydrophobic form of alkaline phosphatase (approximate elution volume 450-570 ml) is pooled and stored in aliquots at - 2 0 °. This material is stable for at least 12 months; one placenta produces sufficient purified alkaline phosphatase for approximately I0,000 of the anchor degradation assays described below. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the alkaline phosphatase shows it to be approximately 50% pure with alkaline phosphatase being the single major protein species present. Assay Procedure. The alkaline phosphatase substrate (50/xl containing 1 volume of alkaline phosphatase, purified as described above, 2 volumes of 1% (w/v) Nonidet P-40 (NP-40), and 2 volumes of 200 mM Tris-maleate, pH 7) is incubated with aliquots of supernatant fractions, etc., in a total volume of 0.2 ml for 30 min at 37 °. The incubation mixture is then diluted with 0.8 ml of ice-cold 150 mM NaCI/0.1 mM MgCl2/10/.tM zinc acetate/ l0 mM H E P E S - N a O H , pH 7.0, and a 50-/~1 aliquot is removed and mixed with 0.2 ml of the same buffer and 0.25 ml 2% precondensed Triton X-114. After sampling a 0.1-ml aliquot for assay of total alkaline phosphatase activity, the mixture is incubated at 37° for l0 min, then centrifuged (1500 g) immediately at room temperature* for 2 min to separate the phases, and a 0.1-ml aliquot of the upper phase is sampled. Alkaline phosphatase activity in the samples is determined as described previously 2 except that 0.2% Triton X-100 is included in the alkaline phosphatase assay incubation and 50/zl of 1% sodium deoxycholate is added after the enzyme reaction has been stopped. Anchor degradation is measured by comparing the activity in the upper phase (i.e., the degraded form) with that in the total incubation mixture * When large numbers o f samples are assayed, it is advisable to carry out the centrifugation at 37 ° to avoid the high background due to partial phase mixing at room temperature.
570
PHOSPHOLIPASED
[56]
before phase separation at 37°. One unit is the amount of enzyme hydrolyzing 1% of the alkaline phosphatase per minute. Under the conditions utilized here, approximately 5-10% of the total alkaline phosphatase is found in the upper phase in the absence of added anchor-degrading activity. This does not change substantially during incubations of up to 2 hr, indicating that the alkaline phosphatase purification procedure removes most of the GPI-PLD present in human placenta. 2 Assay H: Variant Surface Glycoprotein as Substrate In the following assay the GPI anchor of the protein substrate is biosynthetically labeled with a 3H-labeled fatty acid, and the released phosphatidic acid can then readily be detected by liquid scintillation counting following extraction with aqueous butanol. Although the routine assay to be described below does not distinguish between GPI-PLD and GPIPLC, this can be checked by taking t h e extracted 3H-labeled product and analyzing it by thin-layer chromatography. For most GPI-anchored proteins, biosynthetic labeling is too low for use as substrates since they are of relatively low abundance. However the variant surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei is abundant and can be labelled with [3H]myristic acid quite readily. Preparation of Substrate. [3H]Myristic acid-labeled VSG is prepared by a modification of the procedure of Hereld et al. 12A rat is infected with T. brucei MITat 117 or 118 until the parasite density reaches approximately 109/ml. The blood is removed by cardiac puncture with 3.8% sodium citrate (1 ml/9 ml of blood) as anticoagulant. The blood is centrifuged at approximately 1000 g for 5 min. The majority of the plasma is removed and discarded. The trypanosomes form a white layer on top of the erythrocytes which is removed into a clean tube and washed at room temperature with phosphate-buffered saline containing 1% (w/v) glucose (PBSG; approximately 1 ml/ml of blood). The pellet of contaminating erythrocytes is removed and discarded after each centrifugation by transferring the trypanosomes into a clean tube. The washing is repeated (3-5 times) until erythrocytes are no longer visible; the yield from one rat is typically about 10j° trypanosomes. Labeling medium is prepared in advance by drying 2 mCi of [9,103H]myristic acid under nitrogen to remove toluene and redissolving it in 40/zl of 95% (v/v) ethanol. One-half milliliter of defatted bovine serum albumin (BSA) (Sigma, St. Louis, MO, Cat. No. A-6003), 20 mg/ml in 150 mM NaC1, 10 mM sodium phosphate buffer (pH 7.0), is added and mixed 12 D. Hereld, J. L. Krakow, J. D. Bangs, G. W. Hart, and P. T. Englund, J. Biol. Chem. 261, 13813 (1986).
[56]
GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PLD
571
vigorously to form a BSA-fatty acid complex. The mixture is then added to 30 ml minimal essential medium (MEM) buffered with 25 mM HEPES (pH 7.4) and containing 0.5 mg/ml defatted BSA. The washed trypanosomes are incubated in this medium at 37° for 60 min in a tissue culture flask with occasional gentle shaking to maintain suspension. An additional 20 ml of MEM/HEPES/BSA is added after about 40 min. Incubation can be continued for longer periods (up to 90 min), but in this situation, it is vital to monitor viability (i.e., maintenance of slender shape and rapid motility) of the trypanosomes frequently since lysis leads to activation of the endogenous GPI-specific phospholipase C (GPI-PLC) and rapid degradation of the GPI anchor of VSG. After incubation the flask is placed on ice for approximately 5 min, and the cell suspension is transferred to centrifuge tubes, centrifuged, and washed with ice-cold PBSG. The washed cells are lysed by resuspension in 8 ml ice-cold hypotonic lysis buffer (10 mM sodium phosphate buffer, 1/xg/ml leupeptin, 0. I mM tosyllysine chloromethyl ketone, pH 7.0). After 5 min, 0.42 ml of 100 mM p-chloromercuriphenylsulfonic acid (the sodium salt dissolved in 0.1 M NaOH) is added to inhibit the GPI-PLC, and the suspension is transferred to a 30-ml Corex tube and centrifuged at approximately 12,000 g for 15 min at 4°. The pellet is washed in an additional 8 ml of hypotonic lysis buffer and 0.42 ml of p-chloromercuriphenylsulfonic acid. The washed pellet is dissolved in 2 ml of 1% (w/v) SDS by heating in a 100° water bath with intermittent shaking. The solution is cooled to room temperature, 20 ml of water-saturated 1-butanol is added, and the solution is mixed thoroughly and centrifuged at 12,000 g for 20 min at room temperature. The upper phase is removed and discarded, and the remaining material (interface and lower phase) is reextracted with additional 20-ml portions of water-saturated butanol until the radioactivity in the aqueous phase is less than 104 counts/min (cpm)/ml (usually requires about 10 extractions). The lower aqueous phase is finally eliminated and the VSG precipitated by adding 20 ml anhydrous butanol. The precipitate is extracted 3 times with 5 ml of diethyl ether for 10 min to remove the butanol, and the diethyl ether is removed by evaporation. The dry precipitate is finally redissolved in 2-3 ml of 1% SDS by heating in a boiling water bath. The procedure described above produces sufficient substrate for approximately 5000 of the GPI-PLD assays described below; it is stable at - 2 0 ° for at least 1 year. The procedure can be interrupted after the membranes are dissolved in SDS; however, in order to minimize degradation of the VSG by endogenous GPI-PLC, it is advisable to carry out the first part of the procedure with minimal delay. A s s a y P r o c e d u r e . The assay is based on the procedures described by Hereld et al. 12 and Davitz et al. 3 [3H]Myristate-labeled VSG (2000-3500
572
PHOSPHOLIPASED
[56]
cpm, 2/zg; approximately 0.4/zl of the VSG solution prepared as described above) is mixed with 20 ttl of 200 mM Tris-maleate, pH 7.0, 20/xl of 1% (w/v) NP-40, and 60 /xl of water. The substrate-detergent mixture is incubated with aliquots of GPI-PLD or buffer (0.1 ml) in microcentrifuge tubes for 30 min at 37°. The reaction is stopped by the addition of 0.5 ml of butanol that has been saturated with water. After vortexing, the phases are separated by centrifugation at 1500 g for 3 min. The upper phase (0.3 ml) is sampled, mixed with scintillation fluid, and counted. One unit of GPIPLD activity is arbitrarily defined as the amount of enzyme hydrolyzing 1% of the [3H]myristate-labeled VSG per minute. During the course of these studies it was observed that blanks containing GPI-PLD but not incubated often showed substantial hydrolysis. Preliminary investigations indicate that this may be due to a transient activation of the GPI-PLD by the butanol used for the extraction of the [3H]phosphatidic acid. This may be related to the pronounced activation of GPI-PLD by butanol previously observed in several mammalian tissues.l'2 This problem can be prevented by using butanol saturated with 1 M NH4OH for extraction of the [3H]phosphatidic acid. Under the conditions described here blank values are typically approximately 5% of the total counts. Purification of GPI-PLD The procedure given below is essentially as described by Huang et al. 7 PEG-5000 Precipitation. Two and one-half liters of bovine serum are thawed at 4 ° in the presence of 0.5 mM PMSF and 0.02% NaN 3. With stirring at 4°, PEG-5000 is gradually added to a final concentration of 9%. The mixture is stirred for an additional 1 hr and centrifuged at 10,000 g for 25 min. The supematant is collected and diluted with an equal volume of 50 mM Tris, pH 7.5, 100 mM NaC1, 0.02% NaN 3 (buffer B) containing 0.5 mM PMSF. All subsequent purification steps are performed at 4 ° except where noted. Q Sepharose Chromatography. The diluted supernatant is loaded at a flow rate of 30 ml/min onto a Q Sepharose (Pharmacia, Piscataway, NJ) column (9 × 10 cm) equilibrated in buffer B plus 0.5 mM PMSF. Following a 10-bed volume wash with the equilibration buffer, GPI-PLD activity is eluted with a linear gradient of 0 . l - l . 0 M NaC1 in 4 liters of 50 mM Tris, pH 7.5, 0.02% NaN 3, and 0.5 mM PMSF. The activity-containing fractions, which are eluted after approximately 1.1 liters, are pooled and concentrated by Amicon YMI0 filtration to about 200 ml. Sephacryl S-300 Chromatography. The YM10 concentrate is loaded onto two (10 × 53 cm) S-300 Sephacryl columns, equilibrated in buffer B,
[56]
GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PLD
573
and linked in tandem. Proteins are eluted at a flow rate of 3.8 ml/min, and fractions of 23 ml are collected. After 3.9 liters active fractions are eluted and pooled. Wheat Germ Lectin Sepharose Chromatography. NaCI and CHAPS are added to the Sephacryl S-300 pool to give final concentrations of 0.2 M and 0.6% (w/v), respectively. The sample is divided in half for two runs on a 40-ml (2.5 cm diameter) wheat germ lectin-Sepharose (Pharmacia) column equilibrated in 50 mM Tris, pH 7.5, 0.2 M NaCI, 0.02% NAN3, and 0.6% CHAPS. The sample is loaded overnight at 4° at a flow rate of 17 ml/hr. The flow rate is then increased to 26 ml/hr, the column washed with 5 volumes of equilibration buffer, and the GPI-PLD activity eluted with 0.3 M N-acetylglucosamine. Hydroxyapatite Ultrogel Chromatography. The wheat germ lectin eluates from two runs are combined (75 ml) and concentrated to approximately 10 ml. Nine volumes of 5 mM sodium phosphate, pH 6.8, 0.4% CHAPS, 0.02% NaN 3 is added, and the sample is loaded at room temperature (flow rate 3 ml/min) onto a 4.2 × 22 cm column of hydroxyapatite Ultrogel (IBF Biotechnics, Savage, MD) equilibrated in 5 mM sodium phosphate, pH 6.8, 0.6% CHAPS, and 0.02% NAN3. GPI-PLD activity is collected in the unbound fractions, and the bound, contaminating proteins are eluted with 500 mM sodium phosphate, pH 6.8, 0.6% CHAPS, and 0.02% NaN 3. Zinc Chelate Matrix Chromatography. GPI-PLD active fractions from hydroxyapatite agarose chromatography are pooled, concentrated by YM10 filtration to 21 ml, and the pH adjusted with the addition of a 20fold dilution of 1 M Tris-HCl, pH 7.5. The sample is loaded onto a column (1.5 x 5.0 cm) of iminodiacetic acid on Fractogel TSK HW-65F (Pierce, Rockford, IL) chelated with zinc and equilibrated in 50 mM Tris, pH 7.5, 100 mM NaC1, 0.6% CHAPS (buffer C) containing 0.02% NAN3. The first peak of activity is collected in 10-15 bed volumes of wash with equilibration buffer, and a sharper second peak of activity is eluted with 10 mM histidine in equilibration buffer. Mono Q Chromatography. The two zinc chelate pools of activity are concentrated by YM10 filtration. Each sample (5 ml) is injected onto a Mono Q (HR5/5, Pharmacia) column equilibrated in buffer C at room temperature. GPI-PLD activities are eluted at a flow rate of I ml/min with a gradient of 0.1-0.19 M NaCI in 50 mM Tris, pH 7.5, and 0.6% CHAPS in 6 min, followed by isocratic elution at 0.19 M NaC1 for 5 min and a gradient of 0.19-0.4 M NaCI in 14 min. Under these conditions, the first zinc chelate pool eluted as a single peak of activity at 0.19 M NaCI, and the second Zn-chelate pool is resolved into two peaks of activity at 0.19 and 0.25 M NaC1.
574
PHOSPHOLIPASED
[56]
The procedure results in an overall purification in excess of 20oo-fold. Since multiple peaks (due to the presence of aggregates with low specific activities) are revealed by the last two steps it is difficult to estimate recovery precisely. However, the combined activity recovered in the two peaks from the zinc chelate column is about I% overall. The final Mono Q column is useful for reducing the levels of higher molecular weight contaminants from the aggregated GPI-PLD but gives relatively little increase in specific activity. Properties of GPI-PLD The GPI-PLD purified from bovine serum by the procedure described above has a molecular weight of approximately IOO,OO0 according to SDS-gel electrophoresis 7 and a pl of about 5.6 as determined by twodimensional electrophoresis (K.-S. Huang and S. Li, unpublished work, 1989). The purified enzyme also shows a unique amino-terminal sequence for 15 residues [H2N-X-G-I-S-T-(H)-I-E-I-G-X-(R)-A-L-E-F-L] with no strong homology to that of any other known protein. GPI-PLD has also been purified from human serum by a different procedure and has a molecular weight of approximately 110,000. 6 To further characterize the bovine GPI-PLD and confirm that the 100K protein is GPI-PLD, the purified enzyme was immunized in mice. The antiserum was shown to neutralize GPI-PLD activity in both assay systems and to react with the 100K protein on Western blots. 7 Monoclonal antibodies were generated, and several of these precipitated both GPI-PLD activity and the 100K protein in the presence of anti-mouse IgG. One of these antibodies was used to develop an immunopurification procedure which also yielded a 100K protein. 7 The bovine GPI-PLD has a molecular weight of approximately 200K when analyzed by gel-filtration high-performance liquid chromatography under nondenaturing conditions, suggesting that it is a dimer. A 400K form (presumably tetrameric) and other higher molecular weight aggregates are also resolved by the final Mono Q column. Amino acid composition, N-terminal sequence analysis, two-dimensional gel electrophoresis, and Western blotting analysis of the various forms of GPI-PLD isolated during this purification procedure also support the conclusion that they consist of aggregates of a common 100K subunit. 7 Aggregation of the GPI-PLD may also account for the previous estimate of a molecular weight of approximately 5OOK (by gel filtration) for the enzyme in plasma. 4 The purified bovine enzyme is inhibited by EGTA and 1,10-phenanthroline, suggesting a requirement for divalent metal ions. 7 The purified enzyme from human plasma is also sensitive to inhibition by chelators, the inhibitory effect of EGTA being blocked by addition of Ca 2÷ .6 The GPI-
[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.
GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PLD
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[56] G l y c o s y l p h o s p h a t i d y l i n o s i t o l - S p e c i f i c P h o s p h o l i p a s e D
By Kuo-SEN HUANG, SHIRLEY LI, and MARTIN G. LOW Introduction The glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was first observed as a result of its ability to degrade the GPI anchor of alkaline phosphatase during extraction from mammalian tissues with butanol, l This anchor-degrading activiy was initially thought to be due to the action of inositol phospholipid-specific phospholipases C, which are very active in most mammalian tissues. However, more detailed studies distinguished these two activities and indicated that a novel and highly specific phospholipase D was involved. 2 The activity was subsequently found to be abundant in mammalian plasma and serum, suggesting that the activity observed in the tissues may be, to a large extent, the result of blood contamination. 3-5 The GPI-PLD has recently been purified from human 6 and bovine 7 serum. The physiological function of this enzyme has not been determined, but it is proposed to play a role in the regulation of cell surface expression of GPI-anchored proteins (reviewed in Refs. 8-10).
Assay Methods The GPI-PLD hydrolyzes the phosphodiester linkagc of the phosphatidylinositol moiety in the GPI anchor of several membrane proteins. The products of this reaction are phosphatidic acid and an inositol residue which is attached to the C terminus of the protein through the glycan (for details of the structure of the GPI anchor, see Refs. 9 and 10).Thc assay I M. G. Low and D. B. Zilversmit, Biochemistry 19, 3913 (1980). 2 A. S. Malik and M. G. Low, Biochem. J. 2411, 519 (1986). 3 M. A. Davitz, D. Hereld, S. Shak, J. Krakow, P. T. Englund, and V. Nussenzweig, Science 238, 81 (1987). 4 M. G. Low and A. R. S. Prasad, Proc. Natl. Acad. Sci.U.S.A. 85, 980 (1988). 5 M. L. Cardoso de Almeida, M. J. Turner, B. B. Stambuk, and S. Schenkman, Biochem. Biophys. Res. Commun. 150, 476 (1988). 6 M. A. Davitz, J. Horn, and S. Schenkman, J. Biol. Chem. 264, 13760 (1989). 7 K.-S. Huang, S. Li, W.-J.C. Fung, J. D. Hulmes, L. Reik, Y.E. Pan, and M. G. Low, J. Biol. Chem. 265, 17738 (1990). a M. G. Low and A. R. Saltiel, Science 239, 268 (1988). 9 M. A. J. Ferguson and A. F. Williams, Annu. Rev. Biochem. 57, 285 (1988). to M. G. Low, Biochim. Biophys. Acta, 988, 427 (1989).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
568
PHOSPHOLIPASED
[56]
methods that have been used in the purification procedure described here monitor production of either of these products.
Assay I: Alkaline Phosphatase as Substrate In the following assay the loss of the hydrophobic part of the anchor from the protein is determined by partitioning in Triton X-114 H and measuring the distribution of the protein between the hydrophilic, detergentpoor phase and the hydrophobic, detergent-rich phase. In principle, any GPI-anchored protein whose distribution could be determined easily would be an appropriate substrate. In the assay to be described here the substrate is human placental alkaline phosphatase, and its distribution in Triton X-114 is determined by measuring its ability to hydrolyze pnitrophenyl phosphate in a subsequent incubation. The major disadvantage of this type of assay is that it does not distinguish between GPI-PLDmediated cleavage and those catalyzed by other hydrolases especially GPI-PLC. It is also more time consuming since it involves a second incubation to assay for the degraded alkaline phosphatase. However, an advantage over the other assay procedure is the ready availability of the starting material for preparation of the substrate. Purification o f Human Placental Alkaline Phosphatase. Alkaline phosphatase, with the phosphatidylinositol anchor intact, is purified from human placenta by a procedure based on previous studies of anchor degradation in human placenta. 2 Except where specified otherwise, the following procedure is carried out at 00-4 °. Term human placenta frozen after delivery is thawed, membranes and fibrous material discarded, and the remaining tissue (typically about 300 g) homogenized in 100 mM Tris-HCl, pH 8.5 (3 ml/g tissue) containing benzamidine (1 mM final concentration) and phenylmethylsulfonyl fluoride (PMSF, 0.01 mg/ml). The homogenate is mixed with ice-cold butanol (3 ml/g tissue) and shaken vigorously at intervals for 5 min. The mixture is centrifuged at 5000 g for 45 min, and the lower aqueous phase is collected and dialyzed against 3 changes of 12 volumes of distilled water over a period of 48 hr. An alkaline pH is essential during the initial butanol extraction since the endogenous GPI-PLD is activated below pH 7.0 by the butanol. The dialyzate is concentrated 4-fold in an Amicon (Danvers, MA) ultrafiltration cell using a YM30 membrane. Triton X-114 [1% (w/v) final concentration], Tris-HCl, pH 8.5 (10 mM), MgCI2 (0.1 mM), and zinc acetate (10/~M) are added as 10x or 100x solutions and the mixture incubated at 37° for 10 min. The phases are separated by centrifugation at 1500 g for 5 min and the upper, detergentpoor phase removed by aspiration. 11 C. Bordier, J. Biol. Chem. 256, 1604 0981).
[56]
GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PLD
569
The lower, detergent-rich phase (containing alkaline phosphatase with an intact GPI anchor) is mixed with l volume of l0 mM Tris-HC1/0.1 mM MgCI2/10/zM zinc acetate, pH 8.0 (buffer A) and applied to a column (2.5 x 40 cm) of SM-2 BioBeads (Bio-Rad, Richmond, CA) or Amberlite XAD-2 (Sigma, St. Louis, MO) equilibrated and eluted with buffer A at a flow rate of 25 ml/h. The detergent-depleted eluate (100-170 ml eluted), containing most of the alkaline phosphatase activity, is concentrated in an Amicon ultrafiltration cell using a YMI0 membrane to a volume of approximately 20 ml and centrifuged at 10,000 g for 15 min to remove insoluble material. The supernatant is applied to a column (two 2.5 x 118 cm columns connected together) of Sephacryl S-300 (Pharmacia, Piscataway, NJ) equilibrated with 150 mM NaCl/0.1 mM MgCIJ10 /xM zinc acetate/10 mM HEPES-NaOH, pH 7.4, and eluted at a flow rate of 12 ml/ hr. The high molecular weight, hydrophobic form of alkaline phosphatase (approximate elution volume 450-570 ml) is pooled and stored in aliquots at - 2 0 °. This material is stable for at least 12 months; one placenta produces sufficient purified alkaline phosphatase for approximately I0,000 of the anchor degradation assays described below. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the alkaline phosphatase shows it to be approximately 50% pure with alkaline phosphatase being the single major protein species present. Assay Procedure. The alkaline phosphatase substrate (50/xl containing 1 volume of alkaline phosphatase, purified as described above, 2 volumes of 1% (w/v) Nonidet P-40 (NP-40), and 2 volumes of 200 mM Tris-maleate, pH 7) is incubated with aliquots of supernatant fractions, etc., in a total volume of 0.2 ml for 30 min at 37 °. The incubation mixture is then diluted with 0.8 ml of ice-cold 150 mM NaCI/0.1 mM MgCl2/10/.tM zinc acetate/ l0 mM H E P E S - N a O H , pH 7.0, and a 50-/~1 aliquot is removed and mixed with 0.2 ml of the same buffer and 0.25 ml 2% precondensed Triton X-114. After sampling a 0.1-ml aliquot for assay of total alkaline phosphatase activity, the mixture is incubated at 37° for l0 min, then centrifuged (1500 g) immediately at room temperature* for 2 min to separate the phases, and a 0.1-ml aliquot of the upper phase is sampled. Alkaline phosphatase activity in the samples is determined as described previously 2 except that 0.2% Triton X-100 is included in the alkaline phosphatase assay incubation and 50/zl of 1% sodium deoxycholate is added after the enzyme reaction has been stopped. Anchor degradation is measured by comparing the activity in the upper phase (i.e., the degraded form) with that in the total incubation mixture * When large numbers o f samples are assayed, it is advisable to carry out the centrifugation at 37 ° to avoid the high background due to partial phase mixing at room temperature.
570
PHOSPHOLIPASED
[56]
before phase separation at 37°. One unit is the amount of enzyme hydrolyzing 1% of the alkaline phosphatase per minute. Under the conditions utilized here, approximately 5-10% of the total alkaline phosphatase is found in the upper phase in the absence of added anchor-degrading activity. This does not change substantially during incubations of up to 2 hr, indicating that the alkaline phosphatase purification procedure removes most of the GPI-PLD present in human placenta. 2 Assay H: Variant Surface Glycoprotein as Substrate In the following assay the GPI anchor of the protein substrate is biosynthetically labeled with a 3H-labeled fatty acid, and the released phosphatidic acid can then readily be detected by liquid scintillation counting following extraction with aqueous butanol. Although the routine assay to be described below does not distinguish between GPI-PLD and GPIPLC, this can be checked by taking t h e extracted 3H-labeled product and analyzing it by thin-layer chromatography. For most GPI-anchored proteins, biosynthetic labeling is too low for use as substrates since they are of relatively low abundance. However the variant surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei is abundant and can be labelled with [3H]myristic acid quite readily. Preparation of Substrate. [3H]Myristic acid-labeled VSG is prepared by a modification of the procedure of Hereld et al. 12A rat is infected with T. brucei MITat 117 or 118 until the parasite density reaches approximately 109/ml. The blood is removed by cardiac puncture with 3.8% sodium citrate (1 ml/9 ml of blood) as anticoagulant. The blood is centrifuged at approximately 1000 g for 5 min. The majority of the plasma is removed and discarded. The trypanosomes form a white layer on top of the erythrocytes which is removed into a clean tube and washed at room temperature with phosphate-buffered saline containing 1% (w/v) glucose (PBSG; approximately 1 ml/ml of blood). The pellet of contaminating erythrocytes is removed and discarded after each centrifugation by transferring the trypanosomes into a clean tube. The washing is repeated (3-5 times) until erythrocytes are no longer visible; the yield from one rat is typically about 10j° trypanosomes. Labeling medium is prepared in advance by drying 2 mCi of [9,103H]myristic acid under nitrogen to remove toluene and redissolving it in 40/zl of 95% (v/v) ethanol. One-half milliliter of defatted bovine serum albumin (BSA) (Sigma, St. Louis, MO, Cat. No. A-6003), 20 mg/ml in 150 mM NaC1, 10 mM sodium phosphate buffer (pH 7.0), is added and mixed 12 D. Hereld, J. L. Krakow, J. D. Bangs, G. W. Hart, and P. T. Englund, J. Biol. Chem. 261, 13813 (1986).
[56]
GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PLD
571
vigorously to form a BSA-fatty acid complex. The mixture is then added to 30 ml minimal essential medium (MEM) buffered with 25 mM HEPES (pH 7.4) and containing 0.5 mg/ml defatted BSA. The washed trypanosomes are incubated in this medium at 37° for 60 min in a tissue culture flask with occasional gentle shaking to maintain suspension. An additional 20 ml of MEM/HEPES/BSA is added after about 40 min. Incubation can be continued for longer periods (up to 90 min), but in this situation, it is vital to monitor viability (i.e., maintenance of slender shape and rapid motility) of the trypanosomes frequently since lysis leads to activation of the endogenous GPI-specific phospholipase C (GPI-PLC) and rapid degradation of the GPI anchor of VSG. After incubation the flask is placed on ice for approximately 5 min, and the cell suspension is transferred to centrifuge tubes, centrifuged, and washed with ice-cold PBSG. The washed cells are lysed by resuspension in 8 ml ice-cold hypotonic lysis buffer (10 mM sodium phosphate buffer, 1/xg/ml leupeptin, 0. I mM tosyllysine chloromethyl ketone, pH 7.0). After 5 min, 0.42 ml of 100 mM p-chloromercuriphenylsulfonic acid (the sodium salt dissolved in 0.1 M NaOH) is added to inhibit the GPI-PLC, and the suspension is transferred to a 30-ml Corex tube and centrifuged at approximately 12,000 g for 15 min at 4°. The pellet is washed in an additional 8 ml of hypotonic lysis buffer and 0.42 ml of p-chloromercuriphenylsulfonic acid. The washed pellet is dissolved in 2 ml of 1% (w/v) SDS by heating in a 100° water bath with intermittent shaking. The solution is cooled to room temperature, 20 ml of water-saturated 1-butanol is added, and the solution is mixed thoroughly and centrifuged at 12,000 g for 20 min at room temperature. The upper phase is removed and discarded, and the remaining material (interface and lower phase) is reextracted with additional 20-ml portions of water-saturated butanol until the radioactivity in the aqueous phase is less than 104 counts/min (cpm)/ml (usually requires about 10 extractions). The lower aqueous phase is finally eliminated and the VSG precipitated by adding 20 ml anhydrous butanol. The precipitate is extracted 3 times with 5 ml of diethyl ether for 10 min to remove the butanol, and the diethyl ether is removed by evaporation. The dry precipitate is finally redissolved in 2-3 ml of 1% SDS by heating in a boiling water bath. The procedure described above produces sufficient substrate for approximately 5000 of the GPI-PLD assays described below; it is stable at - 2 0 ° for at least 1 year. The procedure can be interrupted after the membranes are dissolved in SDS; however, in order to minimize degradation of the VSG by endogenous GPI-PLC, it is advisable to carry out the first part of the procedure with minimal delay. A s s a y P r o c e d u r e . The assay is based on the procedures described by Hereld et al. 12 and Davitz et al. 3 [3H]Myristate-labeled VSG (2000-3500
572
PHOSPHOLIPASED
[56]
cpm, 2/zg; approximately 0.4/zl of the VSG solution prepared as described above) is mixed with 20 ttl of 200 mM Tris-maleate, pH 7.0, 20/xl of 1% (w/v) NP-40, and 60 /xl of water. The substrate-detergent mixture is incubated with aliquots of GPI-PLD or buffer (0.1 ml) in microcentrifuge tubes for 30 min at 37°. The reaction is stopped by the addition of 0.5 ml of butanol that has been saturated with water. After vortexing, the phases are separated by centrifugation at 1500 g for 3 min. The upper phase (0.3 ml) is sampled, mixed with scintillation fluid, and counted. One unit of GPIPLD activity is arbitrarily defined as the amount of enzyme hydrolyzing 1% of the [3H]myristate-labeled VSG per minute. During the course of these studies it was observed that blanks containing GPI-PLD but not incubated often showed substantial hydrolysis. Preliminary investigations indicate that this may be due to a transient activation of the GPI-PLD by the butanol used for the extraction of the [3H]phosphatidic acid. This may be related to the pronounced activation of GPI-PLD by butanol previously observed in several mammalian tissues.l'2 This problem can be prevented by using butanol saturated with 1 M NH4OH for extraction of the [3H]phosphatidic acid. Under the conditions described here blank values are typically approximately 5% of the total counts. Purification of GPI-PLD The procedure given below is essentially as described by Huang et al. 7 PEG-5000 Precipitation. Two and one-half liters of bovine serum are thawed at 4 ° in the presence of 0.5 mM PMSF and 0.02% NaN 3. With stirring at 4°, PEG-5000 is gradually added to a final concentration of 9%. The mixture is stirred for an additional 1 hr and centrifuged at 10,000 g for 25 min. The supematant is collected and diluted with an equal volume of 50 mM Tris, pH 7.5, 100 mM NaC1, 0.02% NaN 3 (buffer B) containing 0.5 mM PMSF. All subsequent purification steps are performed at 4 ° except where noted. Q Sepharose Chromatography. The diluted supernatant is loaded at a flow rate of 30 ml/min onto a Q Sepharose (Pharmacia, Piscataway, NJ) column (9 × 10 cm) equilibrated in buffer B plus 0.5 mM PMSF. Following a 10-bed volume wash with the equilibration buffer, GPI-PLD activity is eluted with a linear gradient of 0 . l - l . 0 M NaC1 in 4 liters of 50 mM Tris, pH 7.5, 0.02% NaN 3, and 0.5 mM PMSF. The activity-containing fractions, which are eluted after approximately 1.1 liters, are pooled and concentrated by Amicon YMI0 filtration to about 200 ml. Sephacryl S-300 Chromatography. The YM10 concentrate is loaded onto two (10 × 53 cm) S-300 Sephacryl columns, equilibrated in buffer B,
[56]
GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PLD
573
and linked in tandem. Proteins are eluted at a flow rate of 3.8 ml/min, and fractions of 23 ml are collected. After 3.9 liters active fractions are eluted and pooled. Wheat Germ Lectin Sepharose Chromatography. NaCI and CHAPS are added to the Sephacryl S-300 pool to give final concentrations of 0.2 M and 0.6% (w/v), respectively. The sample is divided in half for two runs on a 40-ml (2.5 cm diameter) wheat germ lectin-Sepharose (Pharmacia) column equilibrated in 50 mM Tris, pH 7.5, 0.2 M NaCI, 0.02% NAN3, and 0.6% CHAPS. The sample is loaded overnight at 4° at a flow rate of 17 ml/hr. The flow rate is then increased to 26 ml/hr, the column washed with 5 volumes of equilibration buffer, and the GPI-PLD activity eluted with 0.3 M N-acetylglucosamine. Hydroxyapatite Ultrogel Chromatography. The wheat germ lectin eluates from two runs are combined (75 ml) and concentrated to approximately 10 ml. Nine volumes of 5 mM sodium phosphate, pH 6.8, 0.4% CHAPS, 0.02% NaN 3 is added, and the sample is loaded at room temperature (flow rate 3 ml/min) onto a 4.2 × 22 cm column of hydroxyapatite Ultrogel (IBF Biotechnics, Savage, MD) equilibrated in 5 mM sodium phosphate, pH 6.8, 0.6% CHAPS, and 0.02% NAN3. GPI-PLD activity is collected in the unbound fractions, and the bound, contaminating proteins are eluted with 500 mM sodium phosphate, pH 6.8, 0.6% CHAPS, and 0.02% NaN 3. Zinc Chelate Matrix Chromatography. GPI-PLD active fractions from hydroxyapatite agarose chromatography are pooled, concentrated by YM10 filtration to 21 ml, and the pH adjusted with the addition of a 20fold dilution of 1 M Tris-HCl, pH 7.5. The sample is loaded onto a column (1.5 x 5.0 cm) of iminodiacetic acid on Fractogel TSK HW-65F (Pierce, Rockford, IL) chelated with zinc and equilibrated in 50 mM Tris, pH 7.5, 100 mM NaC1, 0.6% CHAPS (buffer C) containing 0.02% NAN3. The first peak of activity is collected in 10-15 bed volumes of wash with equilibration buffer, and a sharper second peak of activity is eluted with 10 mM histidine in equilibration buffer. Mono Q Chromatography. The two zinc chelate pools of activity are concentrated by YM10 filtration. Each sample (5 ml) is injected onto a Mono Q (HR5/5, Pharmacia) column equilibrated in buffer C at room temperature. GPI-PLD activities are eluted at a flow rate of I ml/min with a gradient of 0.1-0.19 M NaCI in 50 mM Tris, pH 7.5, and 0.6% CHAPS in 6 min, followed by isocratic elution at 0.19 M NaC1 for 5 min and a gradient of 0.19-0.4 M NaCI in 14 min. Under these conditions, the first zinc chelate pool eluted as a single peak of activity at 0.19 M NaCI, and the second Zn-chelate pool is resolved into two peaks of activity at 0.19 and 0.25 M NaC1.
574
PHOSPHOLIPASED
[56]
The procedure results in an overall purification in excess of 20oo-fold. Since multiple peaks (due to the presence of aggregates with low specific activities) are revealed by the last two steps it is difficult to estimate recovery precisely. However, the combined activity recovered in the two peaks from the zinc chelate column is about I% overall. The final Mono Q column is useful for reducing the levels of higher molecular weight contaminants from the aggregated GPI-PLD but gives relatively little increase in specific activity. Properties of GPI-PLD The GPI-PLD purified from bovine serum by the procedure described above has a molecular weight of approximately IOO,OO0 according to SDS-gel electrophoresis 7 and a pl of about 5.6 as determined by twodimensional electrophoresis (K.-S. Huang and S. Li, unpublished work, 1989). The purified enzyme also shows a unique amino-terminal sequence for 15 residues [H2N-X-G-I-S-T-(H)-I-E-I-G-X-(R)-A-L-E-F-L] with no strong homology to that of any other known protein. GPI-PLD has also been purified from human serum by a different procedure and has a molecular weight of approximately 110,000. 6 To further characterize the bovine GPI-PLD and confirm that the 100K protein is GPI-PLD, the purified enzyme was immunized in mice. The antiserum was shown to neutralize GPI-PLD activity in both assay systems and to react with the 100K protein on Western blots. 7 Monoclonal antibodies were generated, and several of these precipitated both GPI-PLD activity and the 100K protein in the presence of anti-mouse IgG. One of these antibodies was used to develop an immunopurification procedure which also yielded a 100K protein. 7 The bovine GPI-PLD has a molecular weight of approximately 200K when analyzed by gel-filtration high-performance liquid chromatography under nondenaturing conditions, suggesting that it is a dimer. A 400K form (presumably tetrameric) and other higher molecular weight aggregates are also resolved by the final Mono Q column. Amino acid composition, N-terminal sequence analysis, two-dimensional gel electrophoresis, and Western blotting analysis of the various forms of GPI-PLD isolated during this purification procedure also support the conclusion that they consist of aggregates of a common 100K subunit. 7 Aggregation of the GPI-PLD may also account for the previous estimate of a molecular weight of approximately 5OOK (by gel filtration) for the enzyme in plasma. 4 The purified bovine enzyme is inhibited by EGTA and 1,10-phenanthroline, suggesting a requirement for divalent metal ions. 7 The purified enzyme from human plasma is also sensitive to inhibition by chelators, the inhibitory effect of EGTA being blocked by addition of Ca 2÷ .6 The GPI-
[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.
