226
HYDROLASES
[27]
sionally equivalent to microkatal per gram and in effect makes the working approximation t h a t a solution with an A~so of 1 contains 1 m g / m l of protein. While this approximation is valid only to within a factor of three, 5 it is nonetheless a convenient and useful approximation at all stages of purification. Adjustment of the value of A2so by a factor related to the A~so/A280 ratio 25 is a needless complication during the purification of proteins. The spectrophotometrie determination of material is ideal in t h a t fraetionation with respect to nucleic acids (A280/A2~o) 25 and heine proteins (A2so/A410) is instantly indicated. Those proteins which are insoluble except in the presence of lipids or detergents are not amenable to this technique unless a stage of purity is reached at which the lipid or detergent becomes unnecessary for solubility. It has been noted t h a t A224, 26 A215, 27 and A1942s may be used for determining protein concentration. In certain situations it is necessary to use the low wavelength absorption of pept.ides and proteins, 29 but its use is dangerous in protein purification because m a n y impurities and buffer components absorb appreciably in this region. ~50. Warburg and W. Christian, Biochem. Z. 310, 384 (1941); H. M. Kalekar, J. Biol. Chem. 167, 461 (1947). 56W. E. Groves, F. C. Davis, Jr., and B. H. Sells, Anal. Biochem. 22, 195 (1968). sTW. J. Waddell, J.Lab. Clin. Med. 48, 311 (1956). ~ M. M. Mayer and J. A. Miller, Anal. Biochem. 36, 91 (1970). ~J. R. Spies, D. C. Chambers, and E. J. Coulson, Arch. Biochem. Biophys. 84, 286 (1959); J. B. Murphy and M. W. Kies, Biochim. Biophys. Acta 45, 382 (1960).
[27] P h o s p h o l i p a s e
D from Peanut
Seeds
EC 3.1.4.4 Phosphatidylcholine phosphatidohydrolase B y MICHAEL HELLER, NAVA MOZES, and EDDIE MAES
Phosphatidylcholine -t- R - O H ~-- p h o s p h a t i d y l - - O R + choline Peanut seeds contain an enzyme catalyzing the transfer of a phosphatidyl moiety from glycerophosphatides to a primary alcohol or water. With water as acceptor, hydrolysis occurs with formation of phosphatidic acid, whereas with primary alcohols (e.g., methanol, ethanol, propanol, and glycerol) a transfer reaction occurs resulting in the formation of a
[27]
PHOSPHOLIPASE
D FROM P E A N U T SEEDS
227
phosphatidyl alcohol. The product retains its L-O~ configuration in the phosphatidyl residue. If an acceptor containing an asymmetric carbon (e.g., glycerol) is used, a DL mixture is formed, 1 e.g. : L-a-Phosphatidylcholine + glycerol L-a-phosphatidyl DL-a-glycerol + choline Assay Method 2 Principle. The activity of phospholipase D can be determined by the liberation of choline from phosphatidylcholine in the presence of an acceptor containing a primary hydroxyl group (either water or an alcohol). The products are separated by partitioning between a chloroform-rich phase and an aqueous-methanol phase, a The reaction m a y be monitored by measuring the amount of aqueous-methanol soluble radioactivity arising from the [3H]methyl groups of the choline portion of the substrate. Alternatively, the choline formed may be assayed by any conventional technique for choline determination. 4 Reagents. [3H]Choline-labeled lecithin (phosphatidylcholine) may be obtained from tissue cultures (BSC1 line of monkey kidney cells 5) grown in the presence of [3H]methyl-choline. Alternatively, a synthetic medium containing the tritiated choline may be innoculated with fresh yeast2 Ovolecithin has been isolated from egg yolk according to Pangborn, 7 and the product obtained used directly. Although small amounts of lysolecithin, sphingomyelin, and phosphatidylethanolamine were present, they did not affect the enzyme's assay. The ovolecithin thus obtained could also be purified chromatographically on a column of aluminum oxide 8 yielding a product containing 99% ovolecithin. The labeled lecithin is diluted with the nonradioactive ovolecithin in chloroform to give a 0.1 M solution with a specific radioactivity of 1-3 tLCi/~mole of lecithin (based on phosphate content).
Acetate buffer, 0.2 M, pH 5.6 CaCl.,, 0.5 M Sodium dodecyl sulfate (SDS), 25 m M S. F. Yang, S. Freer, and A. A. Benson, J. Biol. Chem. 242, 477 (1967). 2R. Tzur and B. Shapiro, Biochim. Biophys. Acta 280, 290 (1972). aJ. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497' (1957). 4j. C. Dittmer and M. A. Wells, Vol. 14, Sect. 53, p. 482. 5y. Ascher, M. Heller, and Y. Becker, Y. Gen. Virol. 4, 65 (1969). 6 A. D. Bangham and R. M. C. Dawson, Biochem. J., 75, 133 (1960). 7M. C. Pangborn, J. Biol. Chem. 188, 471 (1951). 8 M. A. Wells and D. J. Hanahan, Vol. 14, Sect. 33, p. 178.
228
HYDROLASES
[27]
Chloroform-methanol, 2:1 (by volume) Insta-gel 9
Procedure. F r o m the chloroform solution of the tritiated lecithin, 0.05 ml is pipetted into a 25-ml test tube, the solvent evaporated to dryness under a stream of nitrogen, and 0.1 ml of SDS and 0.25 ml of acetate buffer added. The tube is shaken vigorously on a mixer (Vortex). A homogeneous suspension should be obtained without flakes. Then 0.1 ml of CaCl~ is added and the reaction initiated by addition of a suitable amount of enzyme (5-30 milliunits or 0.5-5 tLg of protein). The final volume is made with water to 1 ml. The enzyme should always be added niter the addition of CaCl2. After incubation at 30 ° for 10 minutes with constant shaking, the reaction is terminated by the addition of 4 ml of chloroform-methanol (2:1); the tube is vigorously mixed (on a Vortex mixer) and kept in ice until it is centrifuged for 5-10 minutes at 500 g. An aliquot of 0.5 ml of the aqueous-methanol upper phase is withdrawn directly into a small glass counting vial; 3.5 ml of Insta-gel scintillation mixture is added and the radioactivity determined in a P a c k a r d T r i - C a r b liquid scintillation counter or other suitable detection systems. Units. Activity i~ expressed in units which represent the amount of enzyme t h a t catalyzes the release of 1 tLmole of choline per minute under the conditions specified above. The amount of choline formed is proportional to the amount of protein added only up to 30 milliunits. 1° Specific activity is defined as enzyme units per milligram of protein. The concentrations of protein are determined by the method of Lowry et al., ~ with bovine serum albumin as standard, or by determining the absorption at 280 nm, assuming that 1 m g / m l of protein has an absorbance of 1.0. E n z y m e P u r i f i c a t i o n ~2
D r y peanut seeds (Arachis hypogea var. virginia) were obtained from a local seeds selection institute following the summer harvest. The seeds A tradename given by Packard Instruments Inc. to a scintillation fluor containing detergents employed to overcome the presence of water in samples for radioactivity determinations by scintillation. ,o The most purified enzyme preparations (following the last step) exhibited a yetunexplained activation which is obtained by the increase of the enzyme concentrations beyond 30 milliunits. The specific activity at this stage may therefore vary depending on the enzyme concentrations. 1~O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
1~M. Heller, N. Mozes, E. Maes, and I. Abramovitz, Biochim. Biophys. Acta (in press).
[27]
PHOSPHOLIPASE D FROM PEANUT SEEDS
229
may be stored in a cool place for several months without losing the enzymatic activity. All operations of the purification scheme described below have been carried out at temperatures below 4 ° unless otherwise noted. Step I. Soluble Extracts o] Peanuts. Two-hundred grams of dry seeds are surface sterilized with a commercial detergent containing approximately 4% (w/v) SDS, rinsed thoroughly with water and soaked overnight in water at 26 ° in a thermostatic incubator. The brown seed cover (testa) is removed and the cotyledons homogenized with 600 ml of a solution containing 0.25 M sucrose, 50 mM Tris, 2 mM EDTA, and 3 mM 2-mercaptoethanol, pH 7.4, in a chilled Waring blender at top speed for 1 minute. The homogenate is filtered through cheesecloth and centrifuged for 15 minutes at 30,000 g in a Sorvall centrifuge. The floating fat and the debris are discarded, and the clear supernatant centrifuged again for 60 minutes at 105,000 g in a Spinco centrifuge using rotor No. 30. The resulting yellowish supernatant is aspirated and the pH adjusted to 7.4. Step 2. Ammonium Sul]ate Fractionation. Solid (NH4)2S04 is added to a final concentration of 20% (w/v), care being taken to maintain the pH at 7.4 or somewhat higher. Following 60 minutes of stirring the mixture is centrifuged at 30,000 g for 15 minutes (Sorvall centrifuge). The precipitate is suspended in a minimal volume of a solution containing 50 mM Tris, 5 mM EDTA, and 3 mM 2-mercaptoethanol, pH 8.0 (abbreviated TEM) and dialyzed against several changes of 50-100 volumes of TEM. The preparation at this stage may either be used immediately for further purification or lyophilized and stored at --20 ° without appreciable loss of activity. [Note: in some batches of peanuts variations in the distribution of protein during the (NH4).~S04 fractionation may cause a lower yield of active enzyme which subsequently affects the chromatographic separation pattern.] Step 3. Chromatography on DEAE-CeUulose. The lyophilized powder from step 2 is dissolved in a minimum volume of TEM, exhaustively dialyzed against TEM, and applied to a 2.5 X 50 cm column containing approximately 20 g of dry ion exchanger packed and equilibrated in T E M (if all of the lyophilized powder does not dissolve, the preparation should be centrifuged for 10 minutes at 30,000 g and the precipitate discarded). Approximately 300 ml of T E M are first applied to the column. A linear salt gradient consisting of 1 liter of TEM buffer and 1 liter 1 M KC1 in the same buffer is then applied and fractions of 15 ml each are collected at a rate of 45 ml/hour. The enzymatic activity is eluted at concentrations of 0.25 M to 0.4 M KC1. The active fractions are pooled and concentrated by ultrafiltration through PM-30 Diaflo membranes (Amicon). The excess KCl is removed by several washes with TEM on
230
HYDROLASES
[27]
the Diaflo membrane. At this stage, concentrated enzyme solutions may be lyophilized and stored either at --20 or 4 °. Alternatively, it may be dialyzed immediately against several changes of 50 m M Tris, 1 mM E D T A , 0.25 m M dithiothreitol ( D T T ) , and 3.5% (w/v) glycerol p H 7.9 (abbreviated buffer G) for the next step. Step 4. Sepharose 6B Chromatography. The appropriately dialyzed preparation obtained in step 3 is applied to a 2.5 ;< 80 cm Sepharose 6B column operated with upward flow and eluted at a rate of 30-50 ml/hour. Fractions of 7 ml are collected during the elution by buffer G. The enzyme was eluted at approximately Ve = 2V0. The handling of the active fractions proceeded similarly to that of the previous step. Step 5. Preparative Acrylamide Disc Gel Electrophoresis. 13'1~ The concentrated protein solution of the previous step is dialyzed against 50 m M Tris-glycine buffer, pH 8.9, and made dense by addition of several grains of solid sucrose or a few drops of glycerol. I t is then layered on top of the 3.5% acrylamide spacer gel, polymerized on a 7.5 or 10% acrylamide which is used as the separating gel. The instructions of the manufacturer (Canalco ~3) should be followed for the preparation of the gels as well as the setting up of the column and the conditions to be used for elution. The latter is regulated by a pump to give a flow rate of 15-20 ml/hour and fractions of 3-5 ml are collected. Runs usually last about 8-10 hours, using a constant current of 10 mA and a voltage of 300-500 V. The activity is monitored and the enzymatically active fractions pooled dialyzed against the same buffer and lyophilized. The amount of material eluted at this stage is small and may have low absorbance values before concentration. It does, however, show one major band on analytical disc gel electrophoresis. Using the above procedure, phospholipase D preparations were obtained with specific activities exceeding 200 units/mg of protein.
Properties Activators and Inhibitors. With lecithin as substrate, no activity was detected in the absence of Ca 2÷. This cation, at optimal concentrations of 40-60 mM, gives maximal hydrolysis rates. It cannot be replaced by Mg2+. 1-°,~'~ Coarse, aqueous dispersions of most phospholipids are either not affected or are hydrolyzed at a very slow rate. ~,~6 Ultrasonic irradiation for about 10 minutes at 2 ° under N., forms leci~3Prep. Disc. Instruction manual, Canal, Industrial Corporation, Rockville. 14L. Shuster, Vol. 22, Sect. 34, p. 412. ~ M. Heller, E. Aladjem, and B. Shapiro, Bull. Soc. Chim. Biol. 50, 1395 (1968). 1~M. Heller and R. Arad, Biochim. Biophys. Acta 210, 276 (1970).
[27]
PHOSPHOLIPASE
D FROM
PEANUT
SEEDS
231
thin dispersions which are susceptible to the enzyme. 16,17 Organic solvents are capable of activating the hydrolysis of phospholipids. The most useful were diethyl ether or acetone (but not chloroform or petroleum-ether) which exert maximal effect at about 25-50% (v/v). Primary alcohols, e.g., methanol, ethanol, or glycerol, are excellent activators of the reaction catalyzed by phospholipase D. However, by virtue of their ability to replace water as acceptor of the phosphatidyl moiety, the alcohols are, in fact, acting both as substrates and as activators (probably affecting the physical state of the phospholipids). 1~,16 Detergents such as SDS, sodium taurocholate (STC), or deoxycholate (DOC) activate the enzymatic hydrolysis, whereas Triton X-100 or cetyltrimethyl ammonium bromide have only very weak effects. 15,1s The negatively charged SDS was found superior to the others as an activator, and its maximal effect was obtained at a "molar" ratio of lecithin to SDS of about 2:1. ~,18 The molar ratios are calculated on the basis of formula weights. A similar activation by STC was obtained with a "molar" ratio of 10:8.18 The products of the hydrolytic or transphosphatidyl reactions (e.g., calcium salts of phosphatidic acid, phosphatidylglycerol, or phosphatidylmethanol) increase the rate of the lecithin hydrolysis or lecithin-methanol transfer reactions. TM Vesicles of biologic membranes (e.g., rat liver microsomes) also activate the hydrolysis of lecithin. TM fl-Lipoprotein from bovine or rat serum, at a range of concentrations of 1-5 mg of protein per milliliter, were found to be potent inhibitots. 16 Similarly, bovine serum albumin inhibited the hydrolysis of lecithin at a range of concentration of 0.5-2 mg of albumin per milliliter. 2 Specificity and Some Practical Applications. Phospholipase D has a broad specificity and with aqueous dispersions, and in the presence of the appropriate activators, it catalyzed hydrolysis or transfer reactions of all phospholipids tested, with the exception of sphingomyelin-~,1~-16,19,2°: lecithin (activation by ether, acetone, SDS, STC, DOC, ultrasonic irradiation, or when membrane-bound), phosphatidylethanolamine (activation when membrane-bound), phosphatidylserine (activation when membranebound), phosphatidylglycerol (activation by ether), diphosphatidylglycerol (cardiolipin; activation by STC, DOC, or even without activator). The phospholipids bound to natural membranes, e.g., erythrocyte plasma membranes (ghosts) 19,2° or rat liver endoplasmic reticulum (microsomes),2,16 are readily hydrolyzed even in the absence of an acti,7 M. Heller and I. Abramovitz, unpublished observations (1973). ,8 E. Aladjem, M.Sc. Thesis, Hebrew University, Jerusalem (1969). 19M. Heller, B. •oelofsen, R. F. A. Zwaal, C. Woodward, and L. L. M. van Deenen, unpublished observations (1971). ~oB. Roelofsen and L. L. M. van Deenen, Eur. J. Biochem. 40, 245 (1973).
232
HYDROLASES
[27]
vator. The phospholipids of serum fl-lipoproteins are resistant to the enzyme.16 The hydrolytie as well as the transphosphatidyl activities of phospholipase D seem to be associated with the same protein. The ratio of their activities remain eonstant throughout purifieationY The transfer reactions to alcohols yield 95% conversion of lecithin to phosphatidylmethanol with 20 to 25% (v/v) of methanol as aeeeptor. Similar concentrations of ethanol were optimal for the transfer, while higher concentrations were inhibitory. Transfer of the phosphatidyl moiety from lecithin to glyeerol requires the presenee of SDS and about 4 M of the aeeeptor. Total conversion was about 50%, of which one-third to one-half was phosphatidylglyeerol, the rest being phosphatidie acid. Large-scale preparations of a variety of phospholipids is thus possible with any starting phospholipid, exeept sphingomyelin, having the desired fatty acid eomposition.15,16 pH Optimum and Kin. Phospholipase D has a pH optimum of 5.5-6.0 with SDS or diethyl ether as activators, and 5.45 for ultrasonically irradiated lecithin. 1"-1s These values were obtained with the following buffers: acetate, maleate, pyridine, eollidine, and dimethylglutarate, l~,ims The apparent K,, values for the hydrolysis of lecithin (ex ovo) were as follows: with ether as activator; 1.25 X 10-2 M; with ultrasonieally irradiated substrate: 3.38 ;4 10-3 M. 1~'1T Stability. A. STORAGE.~'~2'1s Solutions of the enzyme at various stages of purification are more stable at 4 than at --20 °. Dilute enzyme solutions (approximately 0.1 mg of protein per milliliter) could be kept on the laboratory shelf for a few days, provided the pH was maintained above 7.4, in the presence of DTT. Addition of glyeerol to a final eoneentration of 0.5 M or, better yet, higher, extended the stability period up to about 3 weeks. Dry powders (after dialysis and lyophilization) are stable at 4 or --20 ° for long periods, provided the freeze-drying was done on rather eoneentrated enzyme solutions. Enzyme suspensions in 3 M or 4 M (NII~) ~SO, are very useful, and may be stored at 4 °. B. TEMPEaATURE.TM Enzyme solutions at pH above 7.4 could be warmed for 10 minutes at 45 ° without losing activity, but 50 and 100% losses were obtained at 55 and 65 °, respectively. C. ACID.~2 Isoeleetrie focusing and pH gradient elution on DEAEcellulose revealed an isoeleetrie point (pI) at 4.65. At this pH or at the optimal pH of around 5.6 or any other acid pH for that matter, the enzyme is unstable and undergoes irreversible loss of activity.
HYDROLASES
[27]
sionally equivalent to microkatal per gram and in effect makes the working approximation t h a t a solution with an A~so of 1 contains 1 m g / m l of protein. While this approximation is valid only to within a factor of three, 5 it is nonetheless a convenient and useful approximation at all stages of purification. Adjustment of the value of A2so by a factor related to the A~so/A280 ratio 25 is a needless complication during the purification of proteins. The spectrophotometrie determination of material is ideal in t h a t fraetionation with respect to nucleic acids (A280/A2~o) 25 and heine proteins (A2so/A410) is instantly indicated. Those proteins which are insoluble except in the presence of lipids or detergents are not amenable to this technique unless a stage of purity is reached at which the lipid or detergent becomes unnecessary for solubility. It has been noted t h a t A224, 26 A215, 27 and A1942s may be used for determining protein concentration. In certain situations it is necessary to use the low wavelength absorption of pept.ides and proteins, 29 but its use is dangerous in protein purification because m a n y impurities and buffer components absorb appreciably in this region. ~50. Warburg and W. Christian, Biochem. Z. 310, 384 (1941); H. M. Kalekar, J. Biol. Chem. 167, 461 (1947). 56W. E. Groves, F. C. Davis, Jr., and B. H. Sells, Anal. Biochem. 22, 195 (1968). sTW. J. Waddell, J.Lab. Clin. Med. 48, 311 (1956). ~ M. M. Mayer and J. A. Miller, Anal. Biochem. 36, 91 (1970). ~J. R. Spies, D. C. Chambers, and E. J. Coulson, Arch. Biochem. Biophys. 84, 286 (1959); J. B. Murphy and M. W. Kies, Biochim. Biophys. Acta 45, 382 (1960).
[27] P h o s p h o l i p a s e
D from Peanut
Seeds
EC 3.1.4.4 Phosphatidylcholine phosphatidohydrolase B y MICHAEL HELLER, NAVA MOZES, and EDDIE MAES
Phosphatidylcholine -t- R - O H ~-- p h o s p h a t i d y l - - O R + choline Peanut seeds contain an enzyme catalyzing the transfer of a phosphatidyl moiety from glycerophosphatides to a primary alcohol or water. With water as acceptor, hydrolysis occurs with formation of phosphatidic acid, whereas with primary alcohols (e.g., methanol, ethanol, propanol, and glycerol) a transfer reaction occurs resulting in the formation of a
[27]
PHOSPHOLIPASE
D FROM P E A N U T SEEDS
227
phosphatidyl alcohol. The product retains its L-O~ configuration in the phosphatidyl residue. If an acceptor containing an asymmetric carbon (e.g., glycerol) is used, a DL mixture is formed, 1 e.g. : L-a-Phosphatidylcholine + glycerol L-a-phosphatidyl DL-a-glycerol + choline Assay Method 2 Principle. The activity of phospholipase D can be determined by the liberation of choline from phosphatidylcholine in the presence of an acceptor containing a primary hydroxyl group (either water or an alcohol). The products are separated by partitioning between a chloroform-rich phase and an aqueous-methanol phase, a The reaction m a y be monitored by measuring the amount of aqueous-methanol soluble radioactivity arising from the [3H]methyl groups of the choline portion of the substrate. Alternatively, the choline formed may be assayed by any conventional technique for choline determination. 4 Reagents. [3H]Choline-labeled lecithin (phosphatidylcholine) may be obtained from tissue cultures (BSC1 line of monkey kidney cells 5) grown in the presence of [3H]methyl-choline. Alternatively, a synthetic medium containing the tritiated choline may be innoculated with fresh yeast2 Ovolecithin has been isolated from egg yolk according to Pangborn, 7 and the product obtained used directly. Although small amounts of lysolecithin, sphingomyelin, and phosphatidylethanolamine were present, they did not affect the enzyme's assay. The ovolecithin thus obtained could also be purified chromatographically on a column of aluminum oxide 8 yielding a product containing 99% ovolecithin. The labeled lecithin is diluted with the nonradioactive ovolecithin in chloroform to give a 0.1 M solution with a specific radioactivity of 1-3 tLCi/~mole of lecithin (based on phosphate content).
Acetate buffer, 0.2 M, pH 5.6 CaCl.,, 0.5 M Sodium dodecyl sulfate (SDS), 25 m M S. F. Yang, S. Freer, and A. A. Benson, J. Biol. Chem. 242, 477 (1967). 2R. Tzur and B. Shapiro, Biochim. Biophys. Acta 280, 290 (1972). aJ. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497' (1957). 4j. C. Dittmer and M. A. Wells, Vol. 14, Sect. 53, p. 482. 5y. Ascher, M. Heller, and Y. Becker, Y. Gen. Virol. 4, 65 (1969). 6 A. D. Bangham and R. M. C. Dawson, Biochem. J., 75, 133 (1960). 7M. C. Pangborn, J. Biol. Chem. 188, 471 (1951). 8 M. A. Wells and D. J. Hanahan, Vol. 14, Sect. 33, p. 178.
228
HYDROLASES
[27]
Chloroform-methanol, 2:1 (by volume) Insta-gel 9
Procedure. F r o m the chloroform solution of the tritiated lecithin, 0.05 ml is pipetted into a 25-ml test tube, the solvent evaporated to dryness under a stream of nitrogen, and 0.1 ml of SDS and 0.25 ml of acetate buffer added. The tube is shaken vigorously on a mixer (Vortex). A homogeneous suspension should be obtained without flakes. Then 0.1 ml of CaCl~ is added and the reaction initiated by addition of a suitable amount of enzyme (5-30 milliunits or 0.5-5 tLg of protein). The final volume is made with water to 1 ml. The enzyme should always be added niter the addition of CaCl2. After incubation at 30 ° for 10 minutes with constant shaking, the reaction is terminated by the addition of 4 ml of chloroform-methanol (2:1); the tube is vigorously mixed (on a Vortex mixer) and kept in ice until it is centrifuged for 5-10 minutes at 500 g. An aliquot of 0.5 ml of the aqueous-methanol upper phase is withdrawn directly into a small glass counting vial; 3.5 ml of Insta-gel scintillation mixture is added and the radioactivity determined in a P a c k a r d T r i - C a r b liquid scintillation counter or other suitable detection systems. Units. Activity i~ expressed in units which represent the amount of enzyme t h a t catalyzes the release of 1 tLmole of choline per minute under the conditions specified above. The amount of choline formed is proportional to the amount of protein added only up to 30 milliunits. 1° Specific activity is defined as enzyme units per milligram of protein. The concentrations of protein are determined by the method of Lowry et al., ~ with bovine serum albumin as standard, or by determining the absorption at 280 nm, assuming that 1 m g / m l of protein has an absorbance of 1.0. E n z y m e P u r i f i c a t i o n ~2
D r y peanut seeds (Arachis hypogea var. virginia) were obtained from a local seeds selection institute following the summer harvest. The seeds A tradename given by Packard Instruments Inc. to a scintillation fluor containing detergents employed to overcome the presence of water in samples for radioactivity determinations by scintillation. ,o The most purified enzyme preparations (following the last step) exhibited a yetunexplained activation which is obtained by the increase of the enzyme concentrations beyond 30 milliunits. The specific activity at this stage may therefore vary depending on the enzyme concentrations. 1~O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
1~M. Heller, N. Mozes, E. Maes, and I. Abramovitz, Biochim. Biophys. Acta (in press).
[27]
PHOSPHOLIPASE D FROM PEANUT SEEDS
229
may be stored in a cool place for several months without losing the enzymatic activity. All operations of the purification scheme described below have been carried out at temperatures below 4 ° unless otherwise noted. Step I. Soluble Extracts o] Peanuts. Two-hundred grams of dry seeds are surface sterilized with a commercial detergent containing approximately 4% (w/v) SDS, rinsed thoroughly with water and soaked overnight in water at 26 ° in a thermostatic incubator. The brown seed cover (testa) is removed and the cotyledons homogenized with 600 ml of a solution containing 0.25 M sucrose, 50 mM Tris, 2 mM EDTA, and 3 mM 2-mercaptoethanol, pH 7.4, in a chilled Waring blender at top speed for 1 minute. The homogenate is filtered through cheesecloth and centrifuged for 15 minutes at 30,000 g in a Sorvall centrifuge. The floating fat and the debris are discarded, and the clear supernatant centrifuged again for 60 minutes at 105,000 g in a Spinco centrifuge using rotor No. 30. The resulting yellowish supernatant is aspirated and the pH adjusted to 7.4. Step 2. Ammonium Sul]ate Fractionation. Solid (NH4)2S04 is added to a final concentration of 20% (w/v), care being taken to maintain the pH at 7.4 or somewhat higher. Following 60 minutes of stirring the mixture is centrifuged at 30,000 g for 15 minutes (Sorvall centrifuge). The precipitate is suspended in a minimal volume of a solution containing 50 mM Tris, 5 mM EDTA, and 3 mM 2-mercaptoethanol, pH 8.0 (abbreviated TEM) and dialyzed against several changes of 50-100 volumes of TEM. The preparation at this stage may either be used immediately for further purification or lyophilized and stored at --20 ° without appreciable loss of activity. [Note: in some batches of peanuts variations in the distribution of protein during the (NH4).~S04 fractionation may cause a lower yield of active enzyme which subsequently affects the chromatographic separation pattern.] Step 3. Chromatography on DEAE-CeUulose. The lyophilized powder from step 2 is dissolved in a minimum volume of TEM, exhaustively dialyzed against TEM, and applied to a 2.5 X 50 cm column containing approximately 20 g of dry ion exchanger packed and equilibrated in T E M (if all of the lyophilized powder does not dissolve, the preparation should be centrifuged for 10 minutes at 30,000 g and the precipitate discarded). Approximately 300 ml of T E M are first applied to the column. A linear salt gradient consisting of 1 liter of TEM buffer and 1 liter 1 M KC1 in the same buffer is then applied and fractions of 15 ml each are collected at a rate of 45 ml/hour. The enzymatic activity is eluted at concentrations of 0.25 M to 0.4 M KC1. The active fractions are pooled and concentrated by ultrafiltration through PM-30 Diaflo membranes (Amicon). The excess KCl is removed by several washes with TEM on
230
HYDROLASES
[27]
the Diaflo membrane. At this stage, concentrated enzyme solutions may be lyophilized and stored either at --20 or 4 °. Alternatively, it may be dialyzed immediately against several changes of 50 m M Tris, 1 mM E D T A , 0.25 m M dithiothreitol ( D T T ) , and 3.5% (w/v) glycerol p H 7.9 (abbreviated buffer G) for the next step. Step 4. Sepharose 6B Chromatography. The appropriately dialyzed preparation obtained in step 3 is applied to a 2.5 ;< 80 cm Sepharose 6B column operated with upward flow and eluted at a rate of 30-50 ml/hour. Fractions of 7 ml are collected during the elution by buffer G. The enzyme was eluted at approximately Ve = 2V0. The handling of the active fractions proceeded similarly to that of the previous step. Step 5. Preparative Acrylamide Disc Gel Electrophoresis. 13'1~ The concentrated protein solution of the previous step is dialyzed against 50 m M Tris-glycine buffer, pH 8.9, and made dense by addition of several grains of solid sucrose or a few drops of glycerol. I t is then layered on top of the 3.5% acrylamide spacer gel, polymerized on a 7.5 or 10% acrylamide which is used as the separating gel. The instructions of the manufacturer (Canalco ~3) should be followed for the preparation of the gels as well as the setting up of the column and the conditions to be used for elution. The latter is regulated by a pump to give a flow rate of 15-20 ml/hour and fractions of 3-5 ml are collected. Runs usually last about 8-10 hours, using a constant current of 10 mA and a voltage of 300-500 V. The activity is monitored and the enzymatically active fractions pooled dialyzed against the same buffer and lyophilized. The amount of material eluted at this stage is small and may have low absorbance values before concentration. It does, however, show one major band on analytical disc gel electrophoresis. Using the above procedure, phospholipase D preparations were obtained with specific activities exceeding 200 units/mg of protein.
Properties Activators and Inhibitors. With lecithin as substrate, no activity was detected in the absence of Ca 2÷. This cation, at optimal concentrations of 40-60 mM, gives maximal hydrolysis rates. It cannot be replaced by Mg2+. 1-°,~'~ Coarse, aqueous dispersions of most phospholipids are either not affected or are hydrolyzed at a very slow rate. ~,~6 Ultrasonic irradiation for about 10 minutes at 2 ° under N., forms leci~3Prep. Disc. Instruction manual, Canal, Industrial Corporation, Rockville. 14L. Shuster, Vol. 22, Sect. 34, p. 412. ~ M. Heller, E. Aladjem, and B. Shapiro, Bull. Soc. Chim. Biol. 50, 1395 (1968). 1~M. Heller and R. Arad, Biochim. Biophys. Acta 210, 276 (1970).
[27]
PHOSPHOLIPASE
D FROM
PEANUT
SEEDS
231
thin dispersions which are susceptible to the enzyme. 16,17 Organic solvents are capable of activating the hydrolysis of phospholipids. The most useful were diethyl ether or acetone (but not chloroform or petroleum-ether) which exert maximal effect at about 25-50% (v/v). Primary alcohols, e.g., methanol, ethanol, or glycerol, are excellent activators of the reaction catalyzed by phospholipase D. However, by virtue of their ability to replace water as acceptor of the phosphatidyl moiety, the alcohols are, in fact, acting both as substrates and as activators (probably affecting the physical state of the phospholipids). 1~,16 Detergents such as SDS, sodium taurocholate (STC), or deoxycholate (DOC) activate the enzymatic hydrolysis, whereas Triton X-100 or cetyltrimethyl ammonium bromide have only very weak effects. 15,1s The negatively charged SDS was found superior to the others as an activator, and its maximal effect was obtained at a "molar" ratio of lecithin to SDS of about 2:1. ~,18 The molar ratios are calculated on the basis of formula weights. A similar activation by STC was obtained with a "molar" ratio of 10:8.18 The products of the hydrolytic or transphosphatidyl reactions (e.g., calcium salts of phosphatidic acid, phosphatidylglycerol, or phosphatidylmethanol) increase the rate of the lecithin hydrolysis or lecithin-methanol transfer reactions. TM Vesicles of biologic membranes (e.g., rat liver microsomes) also activate the hydrolysis of lecithin. TM fl-Lipoprotein from bovine or rat serum, at a range of concentrations of 1-5 mg of protein per milliliter, were found to be potent inhibitots. 16 Similarly, bovine serum albumin inhibited the hydrolysis of lecithin at a range of concentration of 0.5-2 mg of albumin per milliliter. 2 Specificity and Some Practical Applications. Phospholipase D has a broad specificity and with aqueous dispersions, and in the presence of the appropriate activators, it catalyzed hydrolysis or transfer reactions of all phospholipids tested, with the exception of sphingomyelin-~,1~-16,19,2°: lecithin (activation by ether, acetone, SDS, STC, DOC, ultrasonic irradiation, or when membrane-bound), phosphatidylethanolamine (activation when membrane-bound), phosphatidylserine (activation when membranebound), phosphatidylglycerol (activation by ether), diphosphatidylglycerol (cardiolipin; activation by STC, DOC, or even without activator). The phospholipids bound to natural membranes, e.g., erythrocyte plasma membranes (ghosts) 19,2° or rat liver endoplasmic reticulum (microsomes),2,16 are readily hydrolyzed even in the absence of an acti,7 M. Heller and I. Abramovitz, unpublished observations (1973). ,8 E. Aladjem, M.Sc. Thesis, Hebrew University, Jerusalem (1969). 19M. Heller, B. •oelofsen, R. F. A. Zwaal, C. Woodward, and L. L. M. van Deenen, unpublished observations (1971). ~oB. Roelofsen and L. L. M. van Deenen, Eur. J. Biochem. 40, 245 (1973).
232
HYDROLASES
[27]
vator. The phospholipids of serum fl-lipoproteins are resistant to the enzyme.16 The hydrolytie as well as the transphosphatidyl activities of phospholipase D seem to be associated with the same protein. The ratio of their activities remain eonstant throughout purifieationY The transfer reactions to alcohols yield 95% conversion of lecithin to phosphatidylmethanol with 20 to 25% (v/v) of methanol as aeeeptor. Similar concentrations of ethanol were optimal for the transfer, while higher concentrations were inhibitory. Transfer of the phosphatidyl moiety from lecithin to glyeerol requires the presenee of SDS and about 4 M of the aeeeptor. Total conversion was about 50%, of which one-third to one-half was phosphatidylglyeerol, the rest being phosphatidie acid. Large-scale preparations of a variety of phospholipids is thus possible with any starting phospholipid, exeept sphingomyelin, having the desired fatty acid eomposition.15,16 pH Optimum and Kin. Phospholipase D has a pH optimum of 5.5-6.0 with SDS or diethyl ether as activators, and 5.45 for ultrasonically irradiated lecithin. 1"-1s These values were obtained with the following buffers: acetate, maleate, pyridine, eollidine, and dimethylglutarate, l~,ims The apparent K,, values for the hydrolysis of lecithin (ex ovo) were as follows: with ether as activator; 1.25 X 10-2 M; with ultrasonieally irradiated substrate: 3.38 ;4 10-3 M. 1~'1T Stability. A. STORAGE.~'~2'1s Solutions of the enzyme at various stages of purification are more stable at 4 than at --20 °. Dilute enzyme solutions (approximately 0.1 mg of protein per milliliter) could be kept on the laboratory shelf for a few days, provided the pH was maintained above 7.4, in the presence of DTT. Addition of glyeerol to a final eoneentration of 0.5 M or, better yet, higher, extended the stability period up to about 3 weeks. Dry powders (after dialysis and lyophilization) are stable at 4 or --20 ° for long periods, provided the freeze-drying was done on rather eoneentrated enzyme solutions. Enzyme suspensions in 3 M or 4 M (NII~) ~SO, are very useful, and may be stored at 4 °. B. TEMPEaATURE.TM Enzyme solutions at pH above 7.4 could be warmed for 10 minutes at 45 ° without losing activity, but 50 and 100% losses were obtained at 55 and 65 °, respectively. C. ACID.~2 Isoeleetrie focusing and pH gradient elution on DEAEcellulose revealed an isoeleetrie point (pI) at 4.65. At this pH or at the optimal pH of around 5.6 or any other acid pH for that matter, the enzyme is unstable and undergoes irreversible loss of activity.
