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[41] L y s o p h o s p h o l i p a s e s f r o m E s c h e r i c h i a coli By
K E N K A R A S A W A a n d SHOSHICHI N O J I M A
Introduction lysophospholipaseLt 1-Acyl-sn-glycero-3-phosphoethanolamine + H20 glycerophosphoethanolamine + fatty acid lysophospholipaseL2 2-Acyl-sn-glycero-3-phosphoethanolamine + H20 glycerophosphoethanolamine + fatty acid
A number of enzymes termed lysophospholipases have activity toward diacyl phospholipids and therefore fall into the category of phospholipase B.1'2 In Escherichia coli, there are two kinds of lipolytic enzymes without activities toward the diacyl phospholipids. One is cytosolic, and the other is membrane-associated) ,4 They are termed lysophospholipase L l and lysophospholipase L2, respectively, since the enzymes preferentially hydrolyze 1- and 2-acyllysophospholipids, respectively. However, the enzymes purified from cytoplasm or membrane fractions are not absolutely specific for the acyl ester position. Nevertheless, several lines of genetic and biochemical evidence demonstrate that lysophospholipase L 1 and lysophospholipase L 2 are distinct proteins. Two kinds of mutants of lysophospholipase L2 (one with an elevated level of the enzymes and another defective in it) were isolated, and the relative specific activities oflysophospholipase L~ and lysophospholipase L 2 were found to be variable in a series of mutant colonies. 5In addition, lysophospholipase L1 and lysophospholipase L 2 were purified from E. coli strains each of which contained a hybrid plasmid bearing the structure genes and overproduced the enzymatic activities. The homogeneous lysophospholipase L1 and lysophospholipase L2 preparations exhibited distinct physical properties (e.g., molecular weight) and substrate specificity.6,7 1 H. van den Bosch, in "Phospholipids" (J. N. Hawthorn and G. B. Ansell, eds.), p. 313. Elsevier, Amsterdam, New York, and Oxford, 1982. 2 M. Waite. "The Phospholipases." Plenum, New York, 1987. 3 F. D. Albright, D. A. White, and W. J. Lennarz, J. Biol. Chem. 248, 3968 (1973). 4 0 . Doi and S. Nojima, J. Biol. Chem. 250, 5208 (1975). s T. Kobayashi, H. Homma, Y. Natori, I. Kudo, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) 96, 137 (1984). 6 K. Karasawa, I. Kudo, T. Kobayashi, T. Sa-eki, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) 98, 1117 (1985).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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Assay Method Principle. The assay method is based on the conversion of l- or 2[14C]acyl-sn-glycero-3-phosphoethanolamine to l- or 2- ~4C-labeled fatty acid, extraction of the released labeled fatty acid by a modification of Dole's solvent extraction procedure, 8,9 and determination of the radioactivity of the upper heptane phase. Reagents 1,2-[~4C]Diacylphosphatidylethanolamine [400 disintegrations per minute (dpm)/nmol], biosynthesized using E. coli (see [27] in this volume) Phospholipase A2, prepared prior to use as follows: One milligram of Habu snake (Trimeresurus flaoooiridis Hallowell 1°) venom is dissolved in acetate buffer (5 mM, pH 5.0), followed by heating for 5 min at 100° in a boiling water bath, and denatured proteins are removed by centrifugation for 15 min at 3000 rpm and 4°. The resulting supernatant is used as phospholipase A2. Rhizopus delemar lipase (Seikagaku Kogyo, Tokyo, Japan) Tris-HCl buffer (pH 8.0), 100 mM Tris-maleate buffer (pH 5.6), 50 mM Petroleum ether/diethyl ether (I : 1, v/v) Methanol Chloroform Citric acid solution, 0.5 M Tris-HC1 buffer (pH 7.0), 100 mM Dole's extraction medium (2-propanol/n-heptane/1 N H2SO4, 20: 78 : 7, v/v) Wakogel C-200 (Wako, Tokyo, Japan) EDTA, 50 mM ACS liquid scintillation fluid (Amersham Corp., Arlington Heights, IL) Preparation of Substrate. 11 1-[14C]Acyl-sn-glycero-3-phosphoethanolamine, the substrate for the assay of lysophospholipase L~, is prepared as follows. Four micromoles of E. coli phosphatidylethanolamine (400 dpm/ 7 K. Karasawa, I. Kudo, T. Kobayashi, H. Homma, N. Chiba, H. Mizushima, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) in press (1991). 8 V. P. Dole and H. Meinertz, J. Biol. Chem. 235, 2595 (1960). 9 G. S. Sundaram, K. M. M. Shakir, G. Barnes, and S. Margolis, J. Biol. Chem. 253, 7703 (1978). 10 M. Matsumoto and Y. Suzuki, J. Biochem. (Tokyo) 73, 793 (1973). 11 M. Nishijima, Y. Akamatsu, and S. Nojima, J. Biol. Chem. 249, 5658 (1974).
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nmol) in chloroform solution is added to a round-bottomed test tube (1 x 10.5 cm). Chloroform is evaporated under N 2 , and the labeled phospholipid is redissolved in ethanol/diethyl ether (5 : 95, v/v), followed by the addition of 0.2 ml of 100 mM Tris-HC1 (pH 8.0), 0.1 ml of 100 mM CaC12, and 0.2 ml of phospholipase A2 in 5 mM acetate buffer (pH 5.0). The reaction mixture is incubated at room temperature for 2 hr, then shaken vigorously with 0.5 ml of water, 1 ml of methanol, and 4 ml of petroleum ether/diethyl ether (1 : 1, v/v). The supernatant is discarded, and the lower phase is extracted twice more with petroleum ether/diethyl ether. The lower phase is combined with 0.2 ml of 0.5 M citric acid, and the mixture is extracted with a mixture of 0.5 ml of methanol and 2.2 ml of chloroform. The resulting chloroform layer contained 1-[14C]acyl-sn-glycero-3-phosphoethanolamine with a specific radioactivity of 200 dpm/nmol. 2-[14C]Acyl-sn-glycero-3-phosphoethanolamine, the substrate for the assay of lysophospholipase L2, is prepared as follows. Four micromoles of E. coli phosphatidylethanolamine (400 dpm/nmol) in chloroform solution is added to a round-bottomed test tube, and the chloroform is evaporated in vacuo under N2; then 0.6 ml of 50 mM Tris-maleate (pH 5.6) and 0.1 ml of 100 mM CaCI2 are added to the tube. The phospholipid is suspended in a bath-type sonicator, then 0.3 ml of Rhizopus delemar lipase (1 mg protein/ml) in a solution of 50 mM Tris-maleate (pH 5.6) and 0.1 ml of diethyl ether are added to the tube. The removal of fatty acid and extraction of lysophospholipid are accomplished by the same method as described above. At alkaline pH, 2-[14C]acyl-sn-glycero3-phosphoethanolamine rapidly undergoes migration of the fatty acyl group to the 1-position of glycerol, so it should be used soon after preparation. Procedure. Lysophospholipase L1 activity is determined as follows. For convenience, a 3 mM stock solution of 1-[14C]acyl-sn-glycero-3-phosphoethanolamine in water is prepared by sonication for approximately 30 sec, and then I0/~1 of the substrate sonicate is transferred to a test tube. After addition of 25/zl of 100 mM Tris-HCl (pH 7.0) and 5/xl of 50 mM EDTA, enzyme is added to give a final volume of 50/~1. After rapid mixing, the tube is incubated at 30 min for 5-10 min. The reaction is stopped by addition of 1.25 ml of Dole's reagent, and the mixture is incubated at 60° for 1 min. The mixture is cooled to room temperature, then 0.7 ml of water and 0.75 ml of n-heptane are added. The mixture is vortexed for 30 sec and centrifuged at 3000 rpm for 5 min. An aliquot of 0.8 ml of the upper heptane phase is transferred using a Pasteur pipette (Corning Glass Works, Corning, NY) into a tube containing 0.8 ml of n-heptane. Then approximately I00 mg of Wakogel C-200 is added, and the tube is vigorously vortexed for 30 sec and centrifuged for 5 min at 3000 rpm. The entire
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heptane phase is transferred to a scintillation vial containing 10 ml of ACS scintillation fluid. For the assay of lysophospholipase L2, 2-[14C]acyl-sn-glycero-3-phosphoethanolamine is used as the substrate; otherwise the enzyme assay is performed according to the same procedure as described above.
Lysophospholipase L 2 from Escherichia coli Membranes The structure gene, pldB, coding for lysophospholipase L 2 w a s cloned on the plasmid pKO1.12 The E. coli strain transformed by pB1071,13 a subclone of pKO1, overproduced lysophospholipase L 2 activity at an approximately 45 times higher level than that of the wild type. 6 For purification purposes it is advantageous to start with a hybrid plasmid-bearing strain, KL1699/pB1071, since relatively few steps are required to obtain a near-homogeneous preparation. Purification Procedure 6 Step 1: Cell-Free Extract. Escherichia coli strain KL1699/pB1071, which overproduces lysophospholipase L2, is grown at 37° to the late log phase in 9 liters of Luria broth. The medium contains, per liter, Bactotryptone (10 g), yeast extract (5 g), and NaC1 (5 g). Harvested cells (wet weight 21 g) are suspended in 90 ml of l0 mM Tris-HCl (pH 7.5)/5 mM MgC12 and disrupted at a pressure of 400 kg/cm 2 in a French press. Then, 1 mg/ml of pancreatic DNase I dissolved in 1 M MgCl 2 solution is added to give a final concentration of 15/.~g/ml, and the suspension is incubated at room temperature for 1 hr. Step 2: Membrane Extract. Lysophospholipase L 2 is a membranebound enzyme. In order to isolate the membrane fraction, the cell-free extract is centrifuged at 105,000 g for 1 hr at 4°. The precipitate fraction is resuspended in 94 ml of 50 mM Tris-HC1 (pH 7.4) containing 1 M KCI and extracted for 1 hr at 4° with gentle stirring. Unextracted material is removed by centrifugation for 1 hr at 105,000 g and 4°. Step 3: Ammonium Sulfate Fractionation. The KC1 extract (84 ml) is brought to 40% saturation with solid ammonium sulfate (20.4 g). After 30 min of stirring, the precipitate is collected by centrifugation at I0,000 g for 15 rain. The ammonium sulfate precipitate is redissolved in 18 ml of 50 12H. Homma, T. Kobayashi, Y. Ito, I. Kudo, K. Inoue, H. Ikeda, M. Sekiguchi, and S. Nojima, J. Biochem. (Tokyo) 94, 2079 (1983). 13T. Kobayashi, I. Kudo, H. Homma, K. Karasawa, K. Inoue, H. Ikeda, and S. Nojima, J. Biochem. (Tokyo) 98, 1007 (1985).
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TABLE I PURIFICATION OF LYSOPHOSPHOLIPASE L 2 FROM Escherichia coli KL1699 BEARING PLASMID pB1071
Step 1. 2. 3. 4. 5. 6.
Cell-free homogenate Membrane fraction KCI extract (NH4)2SO4 (0-40%) Chromatofocusing Heparin-Sepharose CL-6B
Total protein (mg) 2140 1230 287 76.1 ND a 0.615
Total Specific activity Yield activity Purification (nmol/min) (%) (nmol/min/mg) (-fold) 71,000 75,200 26,500 16,700 30,900 14,500
100 106 37.3 23.6 43.5 20.3
33.2 61.1 92.3 220 -23,500
1 1.84 2.78 6.63 -708
Not determined owing to the inhibitory effect of Polybuffer in the method of Lowry et al. 14
mM Tris-HCl (pH 7.4) containing 0.5% (w/v) CHAPS and dialyzed against the same buffer for 3 hr at 4°. Step 4: Chromatofocusing. The dialyzed fraction is applied to a column (1.5 x 22.5 cm) of Polybuffer 94 exchanger (Pharmacia Fine Chemicals, Piscataway, NJ) previously equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.5% CHAPS. The column is eluted with Polybuffer 74, generating a pH gradient from 7.4 to 5.0. The lysophospholipase L2 activity is eluted as a single peak at approximately pH 7.2. The enzymatic activity is recovered at a volume of 42 ml. Step 5: Heparin-Sepharose CL-6B Affinity Chromatography. The pooled fraction is applied to a column (1.0 x 27 cm) of heparin-Sepharose CL-6B (Pharmacia) previously equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.2 M NaC1. The column is washed with 3 column volumes of the same buffer and then eluted with an 80-ml linear gradient of NaC1 from 0.2 to 1.0 M in 50 mM Tris-HC1 (pH 7.4). The activity is recovered as a single peak at a volume of 31 ml. Purity. The purification scheme summarized in Table 1TMindicates an overall purification of 708-fold from the original strain (KL 1699/pB 1071). The final preparation exhibits a single protein band on sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The yield is approximately 20.3% (specific activity 23,500 nmol/min/mg).
14 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
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Properties Stability and Storage. The purified enzyme is stable for 1 month at - 8 0 ° in 50 mM Tris-HC1 (pH 7.4) containing 0.5-1.0 NaCI. Repeated freezing and thawing of the enzyme solution, however, cause a significant loss of the activity. It is recommended that the enzyme solution be divided into small aliquots and stored at - 8 0 °. Physical Properties. The molecular weight of the purified enzyme is estimated to be 38,500 by SDS-polyacrylamide gel electrophoresis, although the same preparation gives an apparent molecular weight of 14,000 in gel filtration on Sephacryl S-200. The abnormal behavior of the enzyme on gel filtration may be due to its specific interaction with the resin. The pI of the enzyme is 7.2. Substrate Specificity. The reaction velocities (nmol/min/mg) with various substrates are as follows: 2-acyl-sn-glycero-3-phosphoethanolamine, 19,900; 2-acyl-sn-glycero-3-phosphocholine, 12,910; 2-acyl-sn-glycero-3phosphoglycerol, 2123. The purified lysophospholipase L2 hydrolyzes 2acyl lysophospholipids approximately 2 to 3 times faster than the 1-acyl isomers. Other Reactions. The purified enzyme catalyzes the transacylation reaction to yield acyl phosphatidylglycerol from phosphatidylglycerol and 2-acyl(or 1-acyl)lysophospholipid. As in the hydrolysis reaction, the 2acyl isomer of lysophospholipid is utilized as an acyl donor to form acylphosphatidylglycerol more effectively than the 1-acyl isomer. Kinetics. An apparent Km of 142/zM and an apparent Vmax of 16,000 nmol/min/mg are obtained from Lineweaver-Burk plots for the purified enzyme. No abrupt change in the activity is seen at concentration above and below the critical micellar concentration. This result indicates that E. coli lysophospholipase L 2 is active toward substrates in the monomer state as well as in the micellar state. Inhibitors. The E. coli lysophospholipase L2 is inhibited by such detergents as Triton X-100 and deoxycholate. This inhibition is presumably attributable to dilution of the substrate in the micelles. The purified enzyme is irreversibly inactivated by diisopropyl fluorophosphate. Ca 2÷ and EDTA do not affect the enzyme activity at all. Primary Structure of Lysophospholipase L 2 The DNA sequence of the pldB gene coding for lysophospholipase Lz ofE. coli was determined, and the amino acid sequence was deduced 15to 15 T. Kobayashi, I. Kudo, K. Karasawa, H. Mizushima, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) 98, 1017 (1985).
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i I0 20 30 40 ~ET PHE GLNGLMC,LN LYS ASP TRP GLUTHRARGGLUASN ALAP~ ALAALAPI~ THRMETGL¥ PROLEOTHRASP PIlETRP ARGGLNALGASP GLUALAGLUP~ T~ GLYVALASP ASP
ILE PRO V~ A~ PIE V~ ~O ~
2LY ~
50 60 ~O ~ G~ HIS HIS ASP ~ 6 VAL VAL V~ l~ ~S PRO GLY ~ O ILE GLU S~ ~
70 V~ LYS ~ ~
GLU ~
~
~
ASP LEU ~
80 HIS L~
90 I00 ii0 120 ASP VAL LEU ILE ILE ASP HIS ARO OLY GIN GLY ARG SER GLY ARG LEU LEU ALA ASP PRO HIS LEU GLY HIS V^L ASN ARG ~ ASN ASP TYR VAL ASP ASP LEU ALA ALA PHE
130 r~ OLN GLN GLU V~ G~ PRO GLY PRO T~ ~O LYS ~G 'firlie ~
~
170 OLY ILE VAL IL~ ARG ~
PRO SER ~
140 A~ HIS S~ MET GLY GLY ~
L~ ~
150 LEIJOLN ~G HIS PRO GLY VAL ~S ASP ~
180 190 BE~TALA ARG OLN ILE LEU ASM TRP ALA GLU ALA HIS pRO A~ PIE ARG ASP GLY TYR ~
L~ ~
ALA PRO ~
T@ ~
~
L~ PRO ~
~
210 ILE ASH V~ L~ ~
HIS S~ ~ G GLN ARG ~
V~ ~
G~ SER ILE ~
~
250 GLY O~ OLM V ~ ~
A~ GLY ~
P~ CYS G~ L~ ~G ~
~
~
220 ARG ARO ~N L~ ~O P~ ~
260 GLY ASP ASP A~ ~ PRO ~
~
290 300 GLY HIS PRO V~ OLU GLY GLY ~G PRO L~ V~ I~ LYS GLY ~ 330
ILE SER ~
L~ ~
~
ILE GLY ~
230 ASP ASP PXO ~ l~ ~G V~ GLY OLY PRO ~
~
160 ILE 200 GLY ARG 240 HIS T~
270 280 GIN A~ GiJlGLU GLU ARG V~ V~ ASP ~N ~O ~ ){ISASP AR6
310 ~R HIS GLIJILE I~ PIE GLU LYS ASP ~
~
~
SER V~ ~
320 LEU HIS
340
Au [LSWEASPP~ P~ AS,AS0H~SAS,S~ PROS~ 0LY~S~~0 SU ~ 0LOV~L FIG. 1. A m i n o a c i d s e q u e n c e o f l y s o p h o s p h o l i p a s e L 2 f r o m E. coli.
be as shown in Fig. 1. The deduced amino acid sequence of lysophospholipase L 2 contains 340 amino acid residues, corresponding to a protein with a molecular weight of 38,934, and coincides at its NH2-terminal region with the sequence determined for the 15 NH2-terminal residues of the purified preparation. Escherichia coli lysophospholipase L 2 has a high content of arginine residues (36 out of 340 residues). It is postulated that detergent-resistant phospholipase A in the outer membrane of E. coli is synthesized as a precursor with a typical signal peptide composed of 20 amino acids. In contrast, no DNA sequence corresponding to a signal peptide has been found near the N H 2 terminus of lysophospholipase L 2 . The hydropathy profile, calculated according to Kyte and Doolittle, ~6 indicates that the hydrophobic segments of lysophospholipase L 2 a r e relatively short compared with those of other membrane-bound proteins. This is consistent with the fact that E. coli lysophospholipase L 2 is a peripheral protein which is easily solubilized from the inner membrane by 1 M KCI.
Lysophospholipase L 1 from Escherichia coli Cytoplasm The structural gene, pldC, coding for lysophospholipase L~ was cloned on the ColE1 hybrid plasmid after screening of the Clarke and Carbon E. coli collection for lysophospholipase L~ activity. An E. coli strain transformed by a subcloned plasmid, pC124, overproduces the enzyme activity by approximately 11.4 times compared with the parental strain. 16 j . K y t e a n d R . F . D o o l i t t l e , J. Mol. Biol. 157, 105 (1982).
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Purification Procedure 7 Step 1: Preparation of Cytosol. Escherichia coli strain KL 1699/pC 124, which overproduces lysophospholipase L1, is grown at 37° to the late log phase in 9 liters of Luria broth. Harvested cells are resuspended in 90 ml of I0 mM Tris-HC1 (pH 7.5)/5 mM MgCI2 and disrupted at a pressure of 400 kg/cm 2 in a French press. Then 1 mg/ml of pancreatic DNase I dissolved in 1 M MgCl 2 is added t o give a final concentration of 15/zg/ml, and the suspension is incubated at room temperature for 1 hr. The cytosol fraction is obtained by centrifugation at 105,000 g for 1 hr at 4°. Step 2: Streptomycin Treatment and (NH4)2SO4 Fractionation. An aqueous 40% (w/v) streptomycin sulfate solution (3.93 ml) is added dropwise to 105 ml of the cytosol fraction with gentle stirring, and stirring is continued for an additional 40 min. The mixture is centrifuged at 10,000 g for 50 min at 4°. The supernatant (100 ml) is mixed with 12.9 g of (NH4)2SO4 (55% saturation) with gentle stirring at 4°. After centrifugation, the precipitate is dissolved in 10.5 ml of 50 mM potassium phosphate buffer (pH 7.5) and dialyzed against the same buffer for 3 hr at 4°. Step 3: Sephacryl S-300 Column Chromatography. The dialyzed fraction is applied to a column (3.0 x 50 cm) of Sephacryl S-300 (Pharmacia) previously equilibrated with 50 mM potassium phosphate buffer (pH 7.5). Fractions with lysophospholipase L~ activity (52 ml) are pooled, and the pooled fraction is dialyzed against 50 mM Tris-HC1 buffer (pH 7.5). Step 4: DEAE-cellulose Column Chromatography. The dialyzed fraction is applied to a column of DEAE-cellulose (1.5 × 16.9 cm) which is developed with a 140-ml salt gradient from 0 to 0.3 M KC1 in 50 mM TrisHC1 (pH 7.5). Fractions with lysophospholipase LI activity are eluted with 0.1 M KCI and pooled. The pooled fraction (32 ml) is brought to 70% saturation with solid ammonium sulfate (15.1 g). After 30 min of stirring, the precipitate is collected by centrifugation for 15 min at 10,000 g and 4°. The ammonium sulfate precipitate is dissolved in 5 ml of 1 mM potassium phosphate buffer (pH 7.5) and dialyzed against the same buffer for 3 hr at 4°. Step 5: Hydroxyapatite Column Chromatography. The dialyzed fraction is applied to a column (1.0 x 12.7 cm) of hydroxyapatite, previously equilibrated with 1 mM potassium phosphate buffer (pH 7.5). The column is eluted with a linear gradient of 1 to 100 mM phosphate buffer (pH 7.5). Fractions with lysophospholipase L~ activity are eluted with 70 mM potassium phosphate and pooled. The pooled fraction (10.5 ml) is concentrated to 2 ml with a Centricon 10 (Amicon, Danvers, MA). Step 6: Sephacryl S-200 Column Chromatography. The concentrated solution is applied to a column (1.5 x 45 cm) of Sephacryl S-200, equili-
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TABLE II PURIFICATIONOF LYSOPHOSPHOLIPASEL1 FROMEscherichia coli KL1699BEARXNGPLASMIDpC124 Protein Step
Total (rag)
1. 105,000g supernatant 1180 2. Streptomycin 956 fractionation supernatant 3. (NH4)2SO4(35-55%) 311 4. SephacrylS-300 340 5. DEAE-cellulose 8 6. Hydroxyapatite 1.05 7. SephacrylS-200 0.059
Lysophospholipase Lt activity
Recovery Total Recovery Specific Purification (%) (nmol/min) (%) (nmol/min/mg) (-fold) 100 81 26 28.8 0.67 0.089 0.005
11,100 10,300 5380 5240 1980 873 612
100 92.8 48.4 47.2 17.8 7.87 5,51
9.4 10.8
1.0 1.14
17.3 15.4 247 831 10,380
1.84 1.63 26.3 88.4 1104
brated with 10 mM potassium phosphate buffer (pH 7.5). Fractions with lysophospholipase L~ activity are pooled. Purity. As shown by the summary of the purification in Table II, the overall purification is 1104-fold from the plasmid-containing strain. The final yield is approximately 5.51%, with a specific activity of 10,380 nmol/ min/mg. The final enzyme preparation contains a major band with an apparent molecular weight of 20,500 and a minor band of 22,000 by SDS-polyacrylamide gel electrophoresis.
Properties Stability and Storage. The purified enzyme is stable for at least 1 month at - 80°.
Physical Properties. Gel-permeation chromatography on TSK G 3000 SW (Tosoh, Tokyo, Japan) indicates that the lysophospholipase Ll has a molecular weight of approximately 21,000. This value is in good agreement with that estimated by SDS-polyacrylamide gel electrophoresis as described above, suggesting that lysophospholipase L1 is a monomeric polypeptide. Substrate Specificity. The purified lysophospholipase L~ hydrolyzes lacyl-sn-glycero-3-phosphoethanolamine approximately 2 times faster than the 2-acyl isomer. The purified lysophospholipase does not hydrolyze the acyl linkage of diacylglycerophospholipid and shows no acylphosphatidylglycerol synthase activity.
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[41] L y s o p h o s p h o l i p a s e s f r o m E s c h e r i c h i a coli By
K E N K A R A S A W A a n d SHOSHICHI N O J I M A
Introduction lysophospholipaseLt 1-Acyl-sn-glycero-3-phosphoethanolamine + H20 glycerophosphoethanolamine + fatty acid lysophospholipaseL2 2-Acyl-sn-glycero-3-phosphoethanolamine + H20 glycerophosphoethanolamine + fatty acid
A number of enzymes termed lysophospholipases have activity toward diacyl phospholipids and therefore fall into the category of phospholipase B.1'2 In Escherichia coli, there are two kinds of lipolytic enzymes without activities toward the diacyl phospholipids. One is cytosolic, and the other is membrane-associated) ,4 They are termed lysophospholipase L l and lysophospholipase L2, respectively, since the enzymes preferentially hydrolyze 1- and 2-acyllysophospholipids, respectively. However, the enzymes purified from cytoplasm or membrane fractions are not absolutely specific for the acyl ester position. Nevertheless, several lines of genetic and biochemical evidence demonstrate that lysophospholipase L 1 and lysophospholipase L 2 are distinct proteins. Two kinds of mutants of lysophospholipase L2 (one with an elevated level of the enzymes and another defective in it) were isolated, and the relative specific activities oflysophospholipase L~ and lysophospholipase L 2 were found to be variable in a series of mutant colonies. 5In addition, lysophospholipase L1 and lysophospholipase L 2 were purified from E. coli strains each of which contained a hybrid plasmid bearing the structure genes and overproduced the enzymatic activities. The homogeneous lysophospholipase L1 and lysophospholipase L2 preparations exhibited distinct physical properties (e.g., molecular weight) and substrate specificity.6,7 1 H. van den Bosch, in "Phospholipids" (J. N. Hawthorn and G. B. Ansell, eds.), p. 313. Elsevier, Amsterdam, New York, and Oxford, 1982. 2 M. Waite. "The Phospholipases." Plenum, New York, 1987. 3 F. D. Albright, D. A. White, and W. J. Lennarz, J. Biol. Chem. 248, 3968 (1973). 4 0 . Doi and S. Nojima, J. Biol. Chem. 250, 5208 (1975). s T. Kobayashi, H. Homma, Y. Natori, I. Kudo, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) 96, 137 (1984). 6 K. Karasawa, I. Kudo, T. Kobayashi, T. Sa-eki, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) 98, 1117 (1985).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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Assay Method Principle. The assay method is based on the conversion of l- or 2[14C]acyl-sn-glycero-3-phosphoethanolamine to l- or 2- ~4C-labeled fatty acid, extraction of the released labeled fatty acid by a modification of Dole's solvent extraction procedure, 8,9 and determination of the radioactivity of the upper heptane phase. Reagents 1,2-[~4C]Diacylphosphatidylethanolamine [400 disintegrations per minute (dpm)/nmol], biosynthesized using E. coli (see [27] in this volume) Phospholipase A2, prepared prior to use as follows: One milligram of Habu snake (Trimeresurus flaoooiridis Hallowell 1°) venom is dissolved in acetate buffer (5 mM, pH 5.0), followed by heating for 5 min at 100° in a boiling water bath, and denatured proteins are removed by centrifugation for 15 min at 3000 rpm and 4°. The resulting supernatant is used as phospholipase A2. Rhizopus delemar lipase (Seikagaku Kogyo, Tokyo, Japan) Tris-HCl buffer (pH 8.0), 100 mM Tris-maleate buffer (pH 5.6), 50 mM Petroleum ether/diethyl ether (I : 1, v/v) Methanol Chloroform Citric acid solution, 0.5 M Tris-HC1 buffer (pH 7.0), 100 mM Dole's extraction medium (2-propanol/n-heptane/1 N H2SO4, 20: 78 : 7, v/v) Wakogel C-200 (Wako, Tokyo, Japan) EDTA, 50 mM ACS liquid scintillation fluid (Amersham Corp., Arlington Heights, IL) Preparation of Substrate. 11 1-[14C]Acyl-sn-glycero-3-phosphoethanolamine, the substrate for the assay of lysophospholipase L~, is prepared as follows. Four micromoles of E. coli phosphatidylethanolamine (400 dpm/ 7 K. Karasawa, I. Kudo, T. Kobayashi, H. Homma, N. Chiba, H. Mizushima, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) in press (1991). 8 V. P. Dole and H. Meinertz, J. Biol. Chem. 235, 2595 (1960). 9 G. S. Sundaram, K. M. M. Shakir, G. Barnes, and S. Margolis, J. Biol. Chem. 253, 7703 (1978). 10 M. Matsumoto and Y. Suzuki, J. Biochem. (Tokyo) 73, 793 (1973). 11 M. Nishijima, Y. Akamatsu, and S. Nojima, J. Biol. Chem. 249, 5658 (1974).
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nmol) in chloroform solution is added to a round-bottomed test tube (1 x 10.5 cm). Chloroform is evaporated under N 2 , and the labeled phospholipid is redissolved in ethanol/diethyl ether (5 : 95, v/v), followed by the addition of 0.2 ml of 100 mM Tris-HC1 (pH 8.0), 0.1 ml of 100 mM CaC12, and 0.2 ml of phospholipase A2 in 5 mM acetate buffer (pH 5.0). The reaction mixture is incubated at room temperature for 2 hr, then shaken vigorously with 0.5 ml of water, 1 ml of methanol, and 4 ml of petroleum ether/diethyl ether (1 : 1, v/v). The supernatant is discarded, and the lower phase is extracted twice more with petroleum ether/diethyl ether. The lower phase is combined with 0.2 ml of 0.5 M citric acid, and the mixture is extracted with a mixture of 0.5 ml of methanol and 2.2 ml of chloroform. The resulting chloroform layer contained 1-[14C]acyl-sn-glycero-3-phosphoethanolamine with a specific radioactivity of 200 dpm/nmol. 2-[14C]Acyl-sn-glycero-3-phosphoethanolamine, the substrate for the assay of lysophospholipase L2, is prepared as follows. Four micromoles of E. coli phosphatidylethanolamine (400 dpm/nmol) in chloroform solution is added to a round-bottomed test tube, and the chloroform is evaporated in vacuo under N2; then 0.6 ml of 50 mM Tris-maleate (pH 5.6) and 0.1 ml of 100 mM CaCI2 are added to the tube. The phospholipid is suspended in a bath-type sonicator, then 0.3 ml of Rhizopus delemar lipase (1 mg protein/ml) in a solution of 50 mM Tris-maleate (pH 5.6) and 0.1 ml of diethyl ether are added to the tube. The removal of fatty acid and extraction of lysophospholipid are accomplished by the same method as described above. At alkaline pH, 2-[14C]acyl-sn-glycero3-phosphoethanolamine rapidly undergoes migration of the fatty acyl group to the 1-position of glycerol, so it should be used soon after preparation. Procedure. Lysophospholipase L1 activity is determined as follows. For convenience, a 3 mM stock solution of 1-[14C]acyl-sn-glycero-3-phosphoethanolamine in water is prepared by sonication for approximately 30 sec, and then I0/~1 of the substrate sonicate is transferred to a test tube. After addition of 25/zl of 100 mM Tris-HCl (pH 7.0) and 5/xl of 50 mM EDTA, enzyme is added to give a final volume of 50/~1. After rapid mixing, the tube is incubated at 30 min for 5-10 min. The reaction is stopped by addition of 1.25 ml of Dole's reagent, and the mixture is incubated at 60° for 1 min. The mixture is cooled to room temperature, then 0.7 ml of water and 0.75 ml of n-heptane are added. The mixture is vortexed for 30 sec and centrifuged at 3000 rpm for 5 min. An aliquot of 0.8 ml of the upper heptane phase is transferred using a Pasteur pipette (Corning Glass Works, Corning, NY) into a tube containing 0.8 ml of n-heptane. Then approximately I00 mg of Wakogel C-200 is added, and the tube is vigorously vortexed for 30 sec and centrifuged for 5 min at 3000 rpm. The entire
440
LYSOPHOSPHOLIPASE
[41 ]
heptane phase is transferred to a scintillation vial containing 10 ml of ACS scintillation fluid. For the assay of lysophospholipase L2, 2-[14C]acyl-sn-glycero-3-phosphoethanolamine is used as the substrate; otherwise the enzyme assay is performed according to the same procedure as described above.
Lysophospholipase L 2 from Escherichia coli Membranes The structure gene, pldB, coding for lysophospholipase L 2 w a s cloned on the plasmid pKO1.12 The E. coli strain transformed by pB1071,13 a subclone of pKO1, overproduced lysophospholipase L 2 activity at an approximately 45 times higher level than that of the wild type. 6 For purification purposes it is advantageous to start with a hybrid plasmid-bearing strain, KL1699/pB1071, since relatively few steps are required to obtain a near-homogeneous preparation. Purification Procedure 6 Step 1: Cell-Free Extract. Escherichia coli strain KL1699/pB1071, which overproduces lysophospholipase L2, is grown at 37° to the late log phase in 9 liters of Luria broth. The medium contains, per liter, Bactotryptone (10 g), yeast extract (5 g), and NaC1 (5 g). Harvested cells (wet weight 21 g) are suspended in 90 ml of l0 mM Tris-HCl (pH 7.5)/5 mM MgC12 and disrupted at a pressure of 400 kg/cm 2 in a French press. Then, 1 mg/ml of pancreatic DNase I dissolved in 1 M MgCl 2 solution is added to give a final concentration of 15/.~g/ml, and the suspension is incubated at room temperature for 1 hr. Step 2: Membrane Extract. Lysophospholipase L 2 is a membranebound enzyme. In order to isolate the membrane fraction, the cell-free extract is centrifuged at 105,000 g for 1 hr at 4°. The precipitate fraction is resuspended in 94 ml of 50 mM Tris-HC1 (pH 7.4) containing 1 M KCI and extracted for 1 hr at 4° with gentle stirring. Unextracted material is removed by centrifugation for 1 hr at 105,000 g and 4°. Step 3: Ammonium Sulfate Fractionation. The KC1 extract (84 ml) is brought to 40% saturation with solid ammonium sulfate (20.4 g). After 30 min of stirring, the precipitate is collected by centrifugation at I0,000 g for 15 rain. The ammonium sulfate precipitate is redissolved in 18 ml of 50 12H. Homma, T. Kobayashi, Y. Ito, I. Kudo, K. Inoue, H. Ikeda, M. Sekiguchi, and S. Nojima, J. Biochem. (Tokyo) 94, 2079 (1983). 13T. Kobayashi, I. Kudo, H. Homma, K. Karasawa, K. Inoue, H. Ikeda, and S. Nojima, J. Biochem. (Tokyo) 98, 1007 (1985).
[41]
LYSOPHOSPHOLIPASES FROM E. coli
441
TABLE I PURIFICATION OF LYSOPHOSPHOLIPASE L 2 FROM Escherichia coli KL1699 BEARING PLASMID pB1071
Step 1. 2. 3. 4. 5. 6.
Cell-free homogenate Membrane fraction KCI extract (NH4)2SO4 (0-40%) Chromatofocusing Heparin-Sepharose CL-6B
Total protein (mg) 2140 1230 287 76.1 ND a 0.615
Total Specific activity Yield activity Purification (nmol/min) (%) (nmol/min/mg) (-fold) 71,000 75,200 26,500 16,700 30,900 14,500
100 106 37.3 23.6 43.5 20.3
33.2 61.1 92.3 220 -23,500
1 1.84 2.78 6.63 -708
Not determined owing to the inhibitory effect of Polybuffer in the method of Lowry et al. 14
mM Tris-HCl (pH 7.4) containing 0.5% (w/v) CHAPS and dialyzed against the same buffer for 3 hr at 4°. Step 4: Chromatofocusing. The dialyzed fraction is applied to a column (1.5 x 22.5 cm) of Polybuffer 94 exchanger (Pharmacia Fine Chemicals, Piscataway, NJ) previously equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.5% CHAPS. The column is eluted with Polybuffer 74, generating a pH gradient from 7.4 to 5.0. The lysophospholipase L2 activity is eluted as a single peak at approximately pH 7.2. The enzymatic activity is recovered at a volume of 42 ml. Step 5: Heparin-Sepharose CL-6B Affinity Chromatography. The pooled fraction is applied to a column (1.0 x 27 cm) of heparin-Sepharose CL-6B (Pharmacia) previously equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.2 M NaC1. The column is washed with 3 column volumes of the same buffer and then eluted with an 80-ml linear gradient of NaC1 from 0.2 to 1.0 M in 50 mM Tris-HC1 (pH 7.4). The activity is recovered as a single peak at a volume of 31 ml. Purity. The purification scheme summarized in Table 1TMindicates an overall purification of 708-fold from the original strain (KL 1699/pB 1071). The final preparation exhibits a single protein band on sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The yield is approximately 20.3% (specific activity 23,500 nmol/min/mg).
14 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
442
LYSOPHOSPHOLIPASE
[41]
Properties Stability and Storage. The purified enzyme is stable for 1 month at - 8 0 ° in 50 mM Tris-HC1 (pH 7.4) containing 0.5-1.0 NaCI. Repeated freezing and thawing of the enzyme solution, however, cause a significant loss of the activity. It is recommended that the enzyme solution be divided into small aliquots and stored at - 8 0 °. Physical Properties. The molecular weight of the purified enzyme is estimated to be 38,500 by SDS-polyacrylamide gel electrophoresis, although the same preparation gives an apparent molecular weight of 14,000 in gel filtration on Sephacryl S-200. The abnormal behavior of the enzyme on gel filtration may be due to its specific interaction with the resin. The pI of the enzyme is 7.2. Substrate Specificity. The reaction velocities (nmol/min/mg) with various substrates are as follows: 2-acyl-sn-glycero-3-phosphoethanolamine, 19,900; 2-acyl-sn-glycero-3-phosphocholine, 12,910; 2-acyl-sn-glycero-3phosphoglycerol, 2123. The purified lysophospholipase L2 hydrolyzes 2acyl lysophospholipids approximately 2 to 3 times faster than the 1-acyl isomers. Other Reactions. The purified enzyme catalyzes the transacylation reaction to yield acyl phosphatidylglycerol from phosphatidylglycerol and 2-acyl(or 1-acyl)lysophospholipid. As in the hydrolysis reaction, the 2acyl isomer of lysophospholipid is utilized as an acyl donor to form acylphosphatidylglycerol more effectively than the 1-acyl isomer. Kinetics. An apparent Km of 142/zM and an apparent Vmax of 16,000 nmol/min/mg are obtained from Lineweaver-Burk plots for the purified enzyme. No abrupt change in the activity is seen at concentration above and below the critical micellar concentration. This result indicates that E. coli lysophospholipase L 2 is active toward substrates in the monomer state as well as in the micellar state. Inhibitors. The E. coli lysophospholipase L2 is inhibited by such detergents as Triton X-100 and deoxycholate. This inhibition is presumably attributable to dilution of the substrate in the micelles. The purified enzyme is irreversibly inactivated by diisopropyl fluorophosphate. Ca 2÷ and EDTA do not affect the enzyme activity at all. Primary Structure of Lysophospholipase L 2 The DNA sequence of the pldB gene coding for lysophospholipase Lz ofE. coli was determined, and the amino acid sequence was deduced 15to 15 T. Kobayashi, I. Kudo, K. Karasawa, H. Mizushima, K. Inoue, and S. Nojima, J. Biochem. (Tokyo) 98, 1017 (1985).
[41]
LYSOPHOSPHOLIPASES FROME. coli
443
i I0 20 30 40 ~ET PHE GLNGLMC,LN LYS ASP TRP GLUTHRARGGLUASN ALAP~ ALAALAPI~ THRMETGL¥ PROLEOTHRASP PIlETRP ARGGLNALGASP GLUALAGLUP~ T~ GLYVALASP ASP
ILE PRO V~ A~ PIE V~ ~O ~
2LY ~
50 60 ~O ~ G~ HIS HIS ASP ~ 6 VAL VAL V~ l~ ~S PRO GLY ~ O ILE GLU S~ ~
70 V~ LYS ~ ~
GLU ~
~
~
ASP LEU ~
80 HIS L~
90 I00 ii0 120 ASP VAL LEU ILE ILE ASP HIS ARO OLY GIN GLY ARG SER GLY ARG LEU LEU ALA ASP PRO HIS LEU GLY HIS V^L ASN ARG ~ ASN ASP TYR VAL ASP ASP LEU ALA ALA PHE
130 r~ OLN GLN GLU V~ G~ PRO GLY PRO T~ ~O LYS ~G 'firlie ~
~
170 OLY ILE VAL IL~ ARG ~
PRO SER ~
140 A~ HIS S~ MET GLY GLY ~
L~ ~
150 LEIJOLN ~G HIS PRO GLY VAL ~S ASP ~
180 190 BE~TALA ARG OLN ILE LEU ASM TRP ALA GLU ALA HIS pRO A~ PIE ARG ASP GLY TYR ~
L~ ~
ALA PRO ~
T@ ~
~
L~ PRO ~
~
210 ILE ASH V~ L~ ~
HIS S~ ~ G GLN ARG ~
V~ ~
G~ SER ILE ~
~
250 GLY O~ OLM V ~ ~
A~ GLY ~
P~ CYS G~ L~ ~G ~
~
~
220 ARG ARO ~N L~ ~O P~ ~
260 GLY ASP ASP A~ ~ PRO ~
~
290 300 GLY HIS PRO V~ OLU GLY GLY ~G PRO L~ V~ I~ LYS GLY ~ 330
ILE SER ~
L~ ~
~
ILE GLY ~
230 ASP ASP PXO ~ l~ ~G V~ GLY OLY PRO ~
~
160 ILE 200 GLY ARG 240 HIS T~
270 280 GIN A~ GiJlGLU GLU ARG V~ V~ ASP ~N ~O ~ ){ISASP AR6
310 ~R HIS GLIJILE I~ PIE GLU LYS ASP ~
~
~
SER V~ ~
320 LEU HIS
340
Au [LSWEASPP~ P~ AS,AS0H~SAS,S~ PROS~ 0LY~S~~0 SU ~ 0LOV~L FIG. 1. A m i n o a c i d s e q u e n c e o f l y s o p h o s p h o l i p a s e L 2 f r o m E. coli.
be as shown in Fig. 1. The deduced amino acid sequence of lysophospholipase L 2 contains 340 amino acid residues, corresponding to a protein with a molecular weight of 38,934, and coincides at its NH2-terminal region with the sequence determined for the 15 NH2-terminal residues of the purified preparation. Escherichia coli lysophospholipase L 2 has a high content of arginine residues (36 out of 340 residues). It is postulated that detergent-resistant phospholipase A in the outer membrane of E. coli is synthesized as a precursor with a typical signal peptide composed of 20 amino acids. In contrast, no DNA sequence corresponding to a signal peptide has been found near the N H 2 terminus of lysophospholipase L 2 . The hydropathy profile, calculated according to Kyte and Doolittle, ~6 indicates that the hydrophobic segments of lysophospholipase L 2 a r e relatively short compared with those of other membrane-bound proteins. This is consistent with the fact that E. coli lysophospholipase L 2 is a peripheral protein which is easily solubilized from the inner membrane by 1 M KCI.
Lysophospholipase L 1 from Escherichia coli Cytoplasm The structural gene, pldC, coding for lysophospholipase L~ was cloned on the ColE1 hybrid plasmid after screening of the Clarke and Carbon E. coli collection for lysophospholipase L~ activity. An E. coli strain transformed by a subcloned plasmid, pC124, overproduces the enzyme activity by approximately 11.4 times compared with the parental strain. 16 j . K y t e a n d R . F . D o o l i t t l e , J. Mol. Biol. 157, 105 (1982).
444
LYSOPHOSPHOLIPASE
[41]
Purification Procedure 7 Step 1: Preparation of Cytosol. Escherichia coli strain KL 1699/pC 124, which overproduces lysophospholipase L1, is grown at 37° to the late log phase in 9 liters of Luria broth. Harvested cells are resuspended in 90 ml of I0 mM Tris-HC1 (pH 7.5)/5 mM MgCI2 and disrupted at a pressure of 400 kg/cm 2 in a French press. Then 1 mg/ml of pancreatic DNase I dissolved in 1 M MgCl 2 is added t o give a final concentration of 15/zg/ml, and the suspension is incubated at room temperature for 1 hr. The cytosol fraction is obtained by centrifugation at 105,000 g for 1 hr at 4°. Step 2: Streptomycin Treatment and (NH4)2SO4 Fractionation. An aqueous 40% (w/v) streptomycin sulfate solution (3.93 ml) is added dropwise to 105 ml of the cytosol fraction with gentle stirring, and stirring is continued for an additional 40 min. The mixture is centrifuged at 10,000 g for 50 min at 4°. The supernatant (100 ml) is mixed with 12.9 g of (NH4)2SO4 (55% saturation) with gentle stirring at 4°. After centrifugation, the precipitate is dissolved in 10.5 ml of 50 mM potassium phosphate buffer (pH 7.5) and dialyzed against the same buffer for 3 hr at 4°. Step 3: Sephacryl S-300 Column Chromatography. The dialyzed fraction is applied to a column (3.0 x 50 cm) of Sephacryl S-300 (Pharmacia) previously equilibrated with 50 mM potassium phosphate buffer (pH 7.5). Fractions with lysophospholipase L~ activity (52 ml) are pooled, and the pooled fraction is dialyzed against 50 mM Tris-HC1 buffer (pH 7.5). Step 4: DEAE-cellulose Column Chromatography. The dialyzed fraction is applied to a column of DEAE-cellulose (1.5 × 16.9 cm) which is developed with a 140-ml salt gradient from 0 to 0.3 M KC1 in 50 mM TrisHC1 (pH 7.5). Fractions with lysophospholipase LI activity are eluted with 0.1 M KCI and pooled. The pooled fraction (32 ml) is brought to 70% saturation with solid ammonium sulfate (15.1 g). After 30 min of stirring, the precipitate is collected by centrifugation for 15 min at 10,000 g and 4°. The ammonium sulfate precipitate is dissolved in 5 ml of 1 mM potassium phosphate buffer (pH 7.5) and dialyzed against the same buffer for 3 hr at 4°. Step 5: Hydroxyapatite Column Chromatography. The dialyzed fraction is applied to a column (1.0 x 12.7 cm) of hydroxyapatite, previously equilibrated with 1 mM potassium phosphate buffer (pH 7.5). The column is eluted with a linear gradient of 1 to 100 mM phosphate buffer (pH 7.5). Fractions with lysophospholipase L~ activity are eluted with 70 mM potassium phosphate and pooled. The pooled fraction (10.5 ml) is concentrated to 2 ml with a Centricon 10 (Amicon, Danvers, MA). Step 6: Sephacryl S-200 Column Chromatography. The concentrated solution is applied to a column (1.5 x 45 cm) of Sephacryl S-200, equili-
[41]
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445
TABLE II PURIFICATIONOF LYSOPHOSPHOLIPASEL1 FROMEscherichia coli KL1699BEARXNGPLASMIDpC124 Protein Step
Total (rag)
1. 105,000g supernatant 1180 2. Streptomycin 956 fractionation supernatant 3. (NH4)2SO4(35-55%) 311 4. SephacrylS-300 340 5. DEAE-cellulose 8 6. Hydroxyapatite 1.05 7. SephacrylS-200 0.059
Lysophospholipase Lt activity
Recovery Total Recovery Specific Purification (%) (nmol/min) (%) (nmol/min/mg) (-fold) 100 81 26 28.8 0.67 0.089 0.005
11,100 10,300 5380 5240 1980 873 612
100 92.8 48.4 47.2 17.8 7.87 5,51
9.4 10.8
1.0 1.14
17.3 15.4 247 831 10,380
1.84 1.63 26.3 88.4 1104
brated with 10 mM potassium phosphate buffer (pH 7.5). Fractions with lysophospholipase L~ activity are pooled. Purity. As shown by the summary of the purification in Table II, the overall purification is 1104-fold from the plasmid-containing strain. The final yield is approximately 5.51%, with a specific activity of 10,380 nmol/ min/mg. The final enzyme preparation contains a major band with an apparent molecular weight of 20,500 and a minor band of 22,000 by SDS-polyacrylamide gel electrophoresis.
Properties Stability and Storage. The purified enzyme is stable for at least 1 month at - 80°.
Physical Properties. Gel-permeation chromatography on TSK G 3000 SW (Tosoh, Tokyo, Japan) indicates that the lysophospholipase Ll has a molecular weight of approximately 21,000. This value is in good agreement with that estimated by SDS-polyacrylamide gel electrophoresis as described above, suggesting that lysophospholipase L1 is a monomeric polypeptide. Substrate Specificity. The purified lysophospholipase L~ hydrolyzes lacyl-sn-glycero-3-phosphoethanolamine approximately 2 times faster than the 2-acyl isomer. The purified lysophospholipase does not hydrolyze the acyl linkage of diacylglycerophospholipid and shows no acylphosphatidylglycerol synthase activity.
