/. Biochem. 83, 1395-1405 (1978)
and Its Properties Hideo MISAKI1 and Makoto MATSUMOTO Department of Biochemistry, Shizuoka College of Pharmacy, Oshika, Shizuoka, Shizuoka 422 Received for publication, December 6, 1977
Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg1+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1 % SDS or 8 M urea at 37°C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.
Several workers have reported the preparation of lysophospholipase in a homogeneous form from several sources; beef pancreas (/), beef liver (2), Penicillium no tat urn (3), and Escherichia coli (4). Most of these preparations showed fairly broad substrate specificity. The enzyme preparations obtained from P. notatum (3) and beef pancreas ~
7] ^ • TL Present address: Research Laboratory, Toyo Jozo Co., Ltd., Mifuku, Ohitocho, Tagata-gun, Shizuoka 410-23, Japan. Abbreviations: GPC, glycerophosphorylcholine; GPE, glycerophosphorylethanolamine; SDS, sodium dodecylsulfate. 1
Vol. 83, No. 5, 1978
1395
(6, 7) showed the phospholipase A activity as well as lysophospholipase, being the phospholipase B type. Furthermore, two kinds of the enzyme from beef liver (2) and beef pancreas (7) also showed hydrolytic activity on triglycerides with shortchain acyl substituents and on p-nitrophenylacetate. In the present paper, the purification of lysophospholipase of V. parahaemolyticus and its . . . . , ' . P ^ P ^ e s includ.ng substrate specific.ty are described.
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1395/775457 by Stockholm University Library user on 18 January 2019
Purification of Lysophospholipase of Vibrio parahaemolyticus
3396
H. M1SAKI and M. MATSUMOTO
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1395/775457 by Stockholm University Library user on 18 January 2019
One unit(LJ) was defined as the amount of enzyme catalyzing the hydrolysis of 1 ftmol of the substrate MATERIALS AND METHODS in 1 min at 37°C. Cells—V. parahaemolyticus, strain K-128 (0-3, Assay for Ester-Hydrolase Activity—The enX-ll), 1 a virulent one, was cultured at 37°C for zyme activities of hydrolyzing tripalmitin, triolein, 18 h in a medium of pH 7.6 containing 1 % poly- and monopalmitin dispersed in 0.2% Triton X-100 peptone, 1 % meat extract, 3 % NaCI, and a small by sonication (9.83 kHz, 200 W for 10 min) were -amount of NatCO, to adjust the pH. The cultured measured by titration of the liberated fatty acids •cells were harvested by centrifugation at 10,000 with 10 mM NaOH at pH 10 using Radiometer x g for 15 min, and washed three times with 3.5% pH-stat iTijc equipment. In the case of tribuNaCI solution. tyrin, its hydrolysis was estimated similarly at pH Enzyme Preparation—Wet cells of V. para- 9 to avoid spontaneous hydrolysis of the substrate haemolyticus weighing 400 g were homogenized in at pH 10. 10 volumes (v/w) of 10 m i Tris-HCl buffer (pH Assay for Phosphatidylcholine Hydrolysis—A 7.6) containing 0.5% Triton X-100 by a Waring 10 ml test tube with a plastic screw-cap contained blendor for 5 min in an ice-bath. The homog- a mixture of 20 nmol of l-acyl-2-(l-14C-oleoyl>•enates (E-l) were centrifuged at 10,000 xg for GPC (1,150 cpm/nmol) and 230 nmol of egg yolk 15 min. The precipitates were homogenized again phosphatidylcholine dispersed by sonication in in 1,000 ml of the same solution, and centrifuged 0.4 ml of 40 mM Britton-Robinson buffer (pH 10) jn the same way as described above. Solid ammo- containing 0.1 % Triton X-100 and 0.1 ml of 0.1 M nium sulfate was added to the combined super- CaCl,. The reaction was started by addition of natants until the salt concentration was raised to 0.1 ml of the enzyme solution, incubated at 37°C •60% saturation, and then the mixture was cen- for 1 h, extracted with 2.4 ml of a mixture of trifuged at 77,000 x g for 30 min, resulting in the chloroform-methanoi ( 2 : 1 , v/v) and centrifuged. separation of floating material. The material An aliquot of the chloroform extract was chrothus obtained was dissolved in 800 ml of distilled matographed on a thin layer plate of Silica gel H •water and dialyzed against distilled water. The with chloroform-methanol-water (65 : 25 : 3, insoluble material which appeared in the dialyzed v/v/v) as the developing solvent. The spots coinsolution was removed by centrifugation at 77,000 ciding with fatty acid, phosphatidylcholine, and x g for 1 h, and the supernatant was Iyophilized. lysophosphatidylcholine were scraped off the plate, The crude enzyme preparation thus obtained was and the radioactivity of each spot was measured -dissolved in 200 ml of 5 mM Tris-HCl buffer (pH by a liquid scintillation spectrophotometer (Aloka 7.6), and the insoluble material was removed by model LSC-601, Tokyo). The extent of hydrol•centrifugation at 77,000 X g for 1 h. The enzyme ysis was calculated on the basis of radioactivity solution (E-2) was stored for further purification remaining in phosphatidylcholine. study. Electrophoresis—Polyacrylamide gel disk elecAssay for Lysophospholipase Activity—The trophoresis of the enzyme was performed on a standard reaction mixture contained 3.5 ^mol of 7.5% polyacrylamide gel at pH 7.0 based on the 1-acyl-GPC in 0.5 ml 40 mM Britton-Robinson method of Davis (10). Electrophoresis was carried buffer (pH 10) (8). The reaction was started by out at 5 mA per gel at room temperature for about addition of 0.1 ml of enzyme solution (containing 1.5 h. Fixing and staining were performed within 0-1.2 units per ml), incubated for 20 min at 37°C about 1 h in 0.5% amido black dissolved in 7% and stopped by the addition of 2 ml of ethanol. acetic acid, and then the gels were destained by The decrease in acyl ester bonds was estimated by washing in 7% acetic acid. the method described by Augustyn and Elliott (9). SDS-polyacrylamide gel electrophoresis for molecular weight estimation of the lysophos* The strain was kindly supplied by Dr. Y. Asakawa pholipase was carried out at pH 7 according to the and his associates (Institute for Public Health, Shizuoka) method of Weber and Osborn (11). Isoelectric focusing electrophoresis was carried -who isolated the strain from a patient suffering from out on an LKB 8101 electro-focusing column food-poisoning. J. Biochem.
PURIFICATION OF LYSOPHOSPHOLIPASE
1397
l-Acyl-2-(l-14C-oleoyl>GPe was obtained by the acylation of 1-acyl-GPC with l-14C-oleic acid in the presence of ATP and CoA using hemolysates of bovine erythrocytes as acyltransferase according to the method described by Robertson and Lands Tripalmitin, triolein, monopalmitin, and tributyrin were purchased from Sigma Chemical Co., St. Louis.
Analytical Methods—Protein was routinelydetermined by the method of Lowry et al. (77> with bovine serum albumin as the standard. For monitoring protein in the column fractions, the= absorbance at 280 nm was measured. RESULTS Purification of Lysophospholipase—The lysophospholipase was solubilized from cells of V. parahaemolyticus with 0.5% Triton X-100 and purified through five steps by the methods described, in " MATERIALS AND METHODS." The enzyme purification process is summarized in TableI with regard to the enzyme activity. The cell homogenate (E-l) contained not only lysophospholipase activity but also phospholipase A activity. The specific activity of solubilized enzyme (E-2> was 0.64 unit/mg protein, and this enzyme preparation showed not only lysophospholipase activity but also weak phospholipase A activity (0.003/imol/min/mg protein). The enzyme preparation (E-2) thus obtained was used for further purification, study. 7. DEAE-Cellulose column chromatographyr The solubilized enzyme solution (E-2, 200 ml> containing approximately 4.5 g of protein in 5 n u t Tris-HCl buffer (pH 7.6) was applied to a DEAEcellulose column (4.5 x 75 cm) equilibrated with the; same buffer. Stepwise elution was carried out with buffer containing 0, 0.1, 0.2, 0.3, and 0.5 M NaCl. The lysophospholipase was eluted with the buffer solution containing 0.3 M NaCl, whereasphospholipase A was not eluted from the column with this experimental condition. The fractions
TABLE I. Purification of lysophospholipase from V. parahaemolyticus. The lysophospholipase was solubilized from 400 g of fresh cells of V. parahaemolyticus with 0.5% Triton X-100 by the method described in " MATERIALS AND METHODS," and then purified by precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose by the methods described in the text. Fraction
Protein (mg)
Cell homogenate (E-l) 41,810 Solubilized enzyme (E-2) 4,535 DEAE-cellulose (E-3) 1,592 pH 5.2 Supernatant (E-4) 1,220 DEAE-Sephadex A-50 (E-5) 588 CM-cellulose (E-6) 42.5
Vol. 83, No. 5, 1978
Sp. activity (U/mg) 0.080 0.64 1.65 2.09 3.82 47.7
Total activity (U) 3,350 2,920 • 2,630 2,470 2,250 2,030
Purification (-fold)
Recovery (%)
1.0 8.0 20.6 26.0 47.8 592
100 87.2 78.6 73.8 67.3 60.7
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1395/775457 by Stockholm University Library user on 18 January 2019
(110 ml) using LKB carrier ampholites with a pH range of 3-6 according to the method of Vesterberg and Svensson (72) at 700 V for 48 h at 2°C. Substrates—Phosphatidylcholine and phosphatidylethanolamine were prepared from egg yolk, and purified chromatographically on a silicic acid column by the method of Hanahan et al. (13). 1-Acyl-GPC, 1-acyl-GPE, and lysocardiolipin were prepared from phosphatidylcholine and phosphatidylethanolamine of egg yolk and cardiolipin of bovine heart, respectively, by treatment with phospholipase A t of Trimeresurus flavoviridis venom (14) and subsequent column chromatographic purification of the lyso derivatives (75). 2-Acyl-GPE was prepared from ethanolamine plasmalogen of Clostridium butyricum by treatment with iodine in the presence of boric acid according to the.method of Lands et al. (IS). The fatty acid compositions of these lysophospholipids are as follows: 68% palmitic acid, 29% stearic acid, and 3-% oleic acid in 1-acyl-GPC; 0.2% myristitic acid, 36.3% palmitic acid, 0.9% palmitoleic acid, 58% stearic acid, and 2.9% oleic acid in-1-acyl-GPE; 19.6% myristiric acid, 75.5% palmitic acid, 2.6% palmitoleic acid, and 2.3% oleic acid in 2-acyl-GPE.
1398
H. MISAKI and M. MATSUMOTO ity were combined, dialyzed against distilled water and lyophilized (E-5 in Table I). 4. CM-cellulose column chromatography: The lyophilized powder (E-5, 401 mg protein) was dissolved in 5 mM sodium acetate buffer (pH 4.5) and applied to a CM-cellulose column (3.2 x 60 cm) previously equilibrated with 5 mM sodium acetate buffer (pH 4.5). Stepwide elution was carried out with the same buffer containing various NaCl concentrations. In order to avoid inactivation of the enzyme in an acidic pH, each 10 ml fraction was collected in a test tube to which 1 ml of 1 M Tris-HCl buffer (pH 7.6) had previously been added. Most of the lysophospholipase activity was eluted with the acetate buffer without NaCl, and the fractions possessing the enzyme activity were combined, dialyzed against distilled water and finally lyophilized (E-6 in Table I). The enzyme was purified about 600-fold as highly as the cell homogenate (E-l) with about 60% recovery. Properties of the Lysophospholipase Purity—The lysophospholipase thus obtained (E-6) was homogeneous as shown on Sephadex G-100 gel filtration (Fig. 1) which was carried out using 10 mM Tris-HCl buffer (pH7.6) as eluant and also on isoelectric focusing electrophoresis (Fig. 2). Further more, the enzyme showed a single band of
300
10
15 Fraction
20 Number
30
Fig. 1. Gel filtration of the purified lysophospholipase on a Sephadex G-100 column. The purified enzyme preparation (E-6, 1.92 mg protein) was dissolved in 2 ml of 10 mM Tris-HCl buffer (pH 7.6), and applied to a Sephadex G-100 column (1.1x35 cm) equilibrated with the same buffer. Elution was carried out with the same buffer at a flow rate of 7 ml/h. 2 ml of fractions were collected. O, Protein; • , lysophospholipase activity (LPLase). J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1395/775457 by Stockholm University Library user on 18 January 2019
showing the enzyme activity were combined, dialyzed against distilled water and lyophilized (E-3 in Table I). 2. Acidification of the enzyme solution to pH 5.2: The lyophilized powder (E-3, 1.57 g protein) was dissolved in 30 ml of distilled water, and the pH of the solution was adjusted to 5.2 with 0.3 ml of 1 M sodium acetate buffer (pH 5.2). The solution was left to stand in an ice-bath for 20 min, precipitates forming were then removed by centrifugation at 77,000 x g for 30 min. The supernatant thus obtained was dialyzed against distilled water after adjusting its pH to 7.6 with 0.1 M NaOH solution. Ninety-four percent of the enzyme activity of the E-3 preparation was recovered in the supernatant fraction (E-4 in Table I). 3. DEAE-Sephadex A-50 column chromatography: The enzyme solution (E-4, 38 ml, 992 mg protein) obtained in the previous step was added to 2 ml of 0.1 M Tris-HCl buffer (pH 7.6) and applied to DEAE-Sephadex A-50 column (3.2 x 60 cm) previously equilibrated with 5 HIM Tris-HQ buffer (pH 7.6). Stepwise elution was carried out with the same buffer containing 0, 0.1, 0.2, 0.25, 0.3, and 0.5 M NaCl. The lysophospholipase was eluted with the buffer containing 0.25-0.3 M NaCl. The fractions showing the lysophospholipase activ-
PURIFICATION OF LYSOPHOSPHOLIPASE
•
0-20
- 5
^ 0-16
- 4
5 &12
•*3
- 4-0
6
c
- 20'
|ao8 - 2
and Its Properties Hideo MISAKI1 and Makoto MATSUMOTO Department of Biochemistry, Shizuoka College of Pharmacy, Oshika, Shizuoka, Shizuoka 422 Received for publication, December 6, 1977
Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg1+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1 % SDS or 8 M urea at 37°C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.
Several workers have reported the preparation of lysophospholipase in a homogeneous form from several sources; beef pancreas (/), beef liver (2), Penicillium no tat urn (3), and Escherichia coli (4). Most of these preparations showed fairly broad substrate specificity. The enzyme preparations obtained from P. notatum (3) and beef pancreas ~
7] ^ • TL Present address: Research Laboratory, Toyo Jozo Co., Ltd., Mifuku, Ohitocho, Tagata-gun, Shizuoka 410-23, Japan. Abbreviations: GPC, glycerophosphorylcholine; GPE, glycerophosphorylethanolamine; SDS, sodium dodecylsulfate. 1
Vol. 83, No. 5, 1978
1395
(6, 7) showed the phospholipase A activity as well as lysophospholipase, being the phospholipase B type. Furthermore, two kinds of the enzyme from beef liver (2) and beef pancreas (7) also showed hydrolytic activity on triglycerides with shortchain acyl substituents and on p-nitrophenylacetate. In the present paper, the purification of lysophospholipase of V. parahaemolyticus and its . . . . , ' . P ^ P ^ e s includ.ng substrate specific.ty are described.
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1395/775457 by Stockholm University Library user on 18 January 2019
Purification of Lysophospholipase of Vibrio parahaemolyticus
3396
H. M1SAKI and M. MATSUMOTO
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1395/775457 by Stockholm University Library user on 18 January 2019
One unit(LJ) was defined as the amount of enzyme catalyzing the hydrolysis of 1 ftmol of the substrate MATERIALS AND METHODS in 1 min at 37°C. Cells—V. parahaemolyticus, strain K-128 (0-3, Assay for Ester-Hydrolase Activity—The enX-ll), 1 a virulent one, was cultured at 37°C for zyme activities of hydrolyzing tripalmitin, triolein, 18 h in a medium of pH 7.6 containing 1 % poly- and monopalmitin dispersed in 0.2% Triton X-100 peptone, 1 % meat extract, 3 % NaCI, and a small by sonication (9.83 kHz, 200 W for 10 min) were -amount of NatCO, to adjust the pH. The cultured measured by titration of the liberated fatty acids •cells were harvested by centrifugation at 10,000 with 10 mM NaOH at pH 10 using Radiometer x g for 15 min, and washed three times with 3.5% pH-stat iTijc equipment. In the case of tribuNaCI solution. tyrin, its hydrolysis was estimated similarly at pH Enzyme Preparation—Wet cells of V. para- 9 to avoid spontaneous hydrolysis of the substrate haemolyticus weighing 400 g were homogenized in at pH 10. 10 volumes (v/w) of 10 m i Tris-HCl buffer (pH Assay for Phosphatidylcholine Hydrolysis—A 7.6) containing 0.5% Triton X-100 by a Waring 10 ml test tube with a plastic screw-cap contained blendor for 5 min in an ice-bath. The homog- a mixture of 20 nmol of l-acyl-2-(l-14C-oleoyl>•enates (E-l) were centrifuged at 10,000 xg for GPC (1,150 cpm/nmol) and 230 nmol of egg yolk 15 min. The precipitates were homogenized again phosphatidylcholine dispersed by sonication in in 1,000 ml of the same solution, and centrifuged 0.4 ml of 40 mM Britton-Robinson buffer (pH 10) jn the same way as described above. Solid ammo- containing 0.1 % Triton X-100 and 0.1 ml of 0.1 M nium sulfate was added to the combined super- CaCl,. The reaction was started by addition of natants until the salt concentration was raised to 0.1 ml of the enzyme solution, incubated at 37°C •60% saturation, and then the mixture was cen- for 1 h, extracted with 2.4 ml of a mixture of trifuged at 77,000 x g for 30 min, resulting in the chloroform-methanoi ( 2 : 1 , v/v) and centrifuged. separation of floating material. The material An aliquot of the chloroform extract was chrothus obtained was dissolved in 800 ml of distilled matographed on a thin layer plate of Silica gel H •water and dialyzed against distilled water. The with chloroform-methanol-water (65 : 25 : 3, insoluble material which appeared in the dialyzed v/v/v) as the developing solvent. The spots coinsolution was removed by centrifugation at 77,000 ciding with fatty acid, phosphatidylcholine, and x g for 1 h, and the supernatant was Iyophilized. lysophosphatidylcholine were scraped off the plate, The crude enzyme preparation thus obtained was and the radioactivity of each spot was measured -dissolved in 200 ml of 5 mM Tris-HCl buffer (pH by a liquid scintillation spectrophotometer (Aloka 7.6), and the insoluble material was removed by model LSC-601, Tokyo). The extent of hydrol•centrifugation at 77,000 X g for 1 h. The enzyme ysis was calculated on the basis of radioactivity solution (E-2) was stored for further purification remaining in phosphatidylcholine. study. Electrophoresis—Polyacrylamide gel disk elecAssay for Lysophospholipase Activity—The trophoresis of the enzyme was performed on a standard reaction mixture contained 3.5 ^mol of 7.5% polyacrylamide gel at pH 7.0 based on the 1-acyl-GPC in 0.5 ml 40 mM Britton-Robinson method of Davis (10). Electrophoresis was carried buffer (pH 10) (8). The reaction was started by out at 5 mA per gel at room temperature for about addition of 0.1 ml of enzyme solution (containing 1.5 h. Fixing and staining were performed within 0-1.2 units per ml), incubated for 20 min at 37°C about 1 h in 0.5% amido black dissolved in 7% and stopped by the addition of 2 ml of ethanol. acetic acid, and then the gels were destained by The decrease in acyl ester bonds was estimated by washing in 7% acetic acid. the method described by Augustyn and Elliott (9). SDS-polyacrylamide gel electrophoresis for molecular weight estimation of the lysophos* The strain was kindly supplied by Dr. Y. Asakawa pholipase was carried out at pH 7 according to the and his associates (Institute for Public Health, Shizuoka) method of Weber and Osborn (11). Isoelectric focusing electrophoresis was carried -who isolated the strain from a patient suffering from out on an LKB 8101 electro-focusing column food-poisoning. J. Biochem.
PURIFICATION OF LYSOPHOSPHOLIPASE
1397
l-Acyl-2-(l-14C-oleoyl>GPe was obtained by the acylation of 1-acyl-GPC with l-14C-oleic acid in the presence of ATP and CoA using hemolysates of bovine erythrocytes as acyltransferase according to the method described by Robertson and Lands Tripalmitin, triolein, monopalmitin, and tributyrin were purchased from Sigma Chemical Co., St. Louis.
Analytical Methods—Protein was routinelydetermined by the method of Lowry et al. (77> with bovine serum albumin as the standard. For monitoring protein in the column fractions, the= absorbance at 280 nm was measured. RESULTS Purification of Lysophospholipase—The lysophospholipase was solubilized from cells of V. parahaemolyticus with 0.5% Triton X-100 and purified through five steps by the methods described, in " MATERIALS AND METHODS." The enzyme purification process is summarized in TableI with regard to the enzyme activity. The cell homogenate (E-l) contained not only lysophospholipase activity but also phospholipase A activity. The specific activity of solubilized enzyme (E-2> was 0.64 unit/mg protein, and this enzyme preparation showed not only lysophospholipase activity but also weak phospholipase A activity (0.003/imol/min/mg protein). The enzyme preparation (E-2) thus obtained was used for further purification, study. 7. DEAE-Cellulose column chromatographyr The solubilized enzyme solution (E-2, 200 ml> containing approximately 4.5 g of protein in 5 n u t Tris-HCl buffer (pH 7.6) was applied to a DEAEcellulose column (4.5 x 75 cm) equilibrated with the; same buffer. Stepwise elution was carried out with buffer containing 0, 0.1, 0.2, 0.3, and 0.5 M NaCl. The lysophospholipase was eluted with the buffer solution containing 0.3 M NaCl, whereasphospholipase A was not eluted from the column with this experimental condition. The fractions
TABLE I. Purification of lysophospholipase from V. parahaemolyticus. The lysophospholipase was solubilized from 400 g of fresh cells of V. parahaemolyticus with 0.5% Triton X-100 by the method described in " MATERIALS AND METHODS," and then purified by precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose by the methods described in the text. Fraction
Protein (mg)
Cell homogenate (E-l) 41,810 Solubilized enzyme (E-2) 4,535 DEAE-cellulose (E-3) 1,592 pH 5.2 Supernatant (E-4) 1,220 DEAE-Sephadex A-50 (E-5) 588 CM-cellulose (E-6) 42.5
Vol. 83, No. 5, 1978
Sp. activity (U/mg) 0.080 0.64 1.65 2.09 3.82 47.7
Total activity (U) 3,350 2,920 • 2,630 2,470 2,250 2,030
Purification (-fold)
Recovery (%)
1.0 8.0 20.6 26.0 47.8 592
100 87.2 78.6 73.8 67.3 60.7
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1395/775457 by Stockholm University Library user on 18 January 2019
(110 ml) using LKB carrier ampholites with a pH range of 3-6 according to the method of Vesterberg and Svensson (72) at 700 V for 48 h at 2°C. Substrates—Phosphatidylcholine and phosphatidylethanolamine were prepared from egg yolk, and purified chromatographically on a silicic acid column by the method of Hanahan et al. (13). 1-Acyl-GPC, 1-acyl-GPE, and lysocardiolipin were prepared from phosphatidylcholine and phosphatidylethanolamine of egg yolk and cardiolipin of bovine heart, respectively, by treatment with phospholipase A t of Trimeresurus flavoviridis venom (14) and subsequent column chromatographic purification of the lyso derivatives (75). 2-Acyl-GPE was prepared from ethanolamine plasmalogen of Clostridium butyricum by treatment with iodine in the presence of boric acid according to the.method of Lands et al. (IS). The fatty acid compositions of these lysophospholipids are as follows: 68% palmitic acid, 29% stearic acid, and 3-% oleic acid in 1-acyl-GPC; 0.2% myristitic acid, 36.3% palmitic acid, 0.9% palmitoleic acid, 58% stearic acid, and 2.9% oleic acid in-1-acyl-GPE; 19.6% myristiric acid, 75.5% palmitic acid, 2.6% palmitoleic acid, and 2.3% oleic acid in 2-acyl-GPE.
1398
H. MISAKI and M. MATSUMOTO ity were combined, dialyzed against distilled water and lyophilized (E-5 in Table I). 4. CM-cellulose column chromatography: The lyophilized powder (E-5, 401 mg protein) was dissolved in 5 mM sodium acetate buffer (pH 4.5) and applied to a CM-cellulose column (3.2 x 60 cm) previously equilibrated with 5 mM sodium acetate buffer (pH 4.5). Stepwide elution was carried out with the same buffer containing various NaCl concentrations. In order to avoid inactivation of the enzyme in an acidic pH, each 10 ml fraction was collected in a test tube to which 1 ml of 1 M Tris-HCl buffer (pH 7.6) had previously been added. Most of the lysophospholipase activity was eluted with the acetate buffer without NaCl, and the fractions possessing the enzyme activity were combined, dialyzed against distilled water and finally lyophilized (E-6 in Table I). The enzyme was purified about 600-fold as highly as the cell homogenate (E-l) with about 60% recovery. Properties of the Lysophospholipase Purity—The lysophospholipase thus obtained (E-6) was homogeneous as shown on Sephadex G-100 gel filtration (Fig. 1) which was carried out using 10 mM Tris-HCl buffer (pH7.6) as eluant and also on isoelectric focusing electrophoresis (Fig. 2). Further more, the enzyme showed a single band of
300
10
15 Fraction
20 Number
30
Fig. 1. Gel filtration of the purified lysophospholipase on a Sephadex G-100 column. The purified enzyme preparation (E-6, 1.92 mg protein) was dissolved in 2 ml of 10 mM Tris-HCl buffer (pH 7.6), and applied to a Sephadex G-100 column (1.1x35 cm) equilibrated with the same buffer. Elution was carried out with the same buffer at a flow rate of 7 ml/h. 2 ml of fractions were collected. O, Protein; • , lysophospholipase activity (LPLase). J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/83/5/1395/775457 by Stockholm University Library user on 18 January 2019
showing the enzyme activity were combined, dialyzed against distilled water and lyophilized (E-3 in Table I). 2. Acidification of the enzyme solution to pH 5.2: The lyophilized powder (E-3, 1.57 g protein) was dissolved in 30 ml of distilled water, and the pH of the solution was adjusted to 5.2 with 0.3 ml of 1 M sodium acetate buffer (pH 5.2). The solution was left to stand in an ice-bath for 20 min, precipitates forming were then removed by centrifugation at 77,000 x g for 30 min. The supernatant thus obtained was dialyzed against distilled water after adjusting its pH to 7.6 with 0.1 M NaOH solution. Ninety-four percent of the enzyme activity of the E-3 preparation was recovered in the supernatant fraction (E-4 in Table I). 3. DEAE-Sephadex A-50 column chromatography: The enzyme solution (E-4, 38 ml, 992 mg protein) obtained in the previous step was added to 2 ml of 0.1 M Tris-HCl buffer (pH 7.6) and applied to DEAE-Sephadex A-50 column (3.2 x 60 cm) previously equilibrated with 5 HIM Tris-HQ buffer (pH 7.6). Stepwise elution was carried out with the same buffer containing 0, 0.1, 0.2, 0.25, 0.3, and 0.5 M NaCl. The lysophospholipase was eluted with the buffer containing 0.25-0.3 M NaCl. The fractions showing the lysophospholipase activ-
PURIFICATION OF LYSOPHOSPHOLIPASE
•
0-20
- 5
^ 0-16
- 4
5 &12
•*3
- 4-0
6
c
- 20'
|ao8 - 2
