J. Biochem. 112, 224-228 (1992)
Effect of Diolein on Hydrolysis of Phosphatidylcholine by Phospholipase C from Clostridium perfringens Takahiro Tsujita and Hiromichi Okuda Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu, Ehime 791-02 Received for publication, March 16, 1992
The activity of phospholipase C from Clostridium perfringens on l-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pore POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.31,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [I26I] phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DOfilmwas 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.
Phospholipase C [EC 3.1.4.3], a phosphodiesterase that hydrolyzes phosphoglyceride at the ester bond between diglyceride and the phosphoric acid-substituted polar head group, has been found in bacteria and animals. Phospholipase C of Clostridium perfringens is a main component of the bacterial toxin (1), and has lethal, dermo-necrotic, and hemolytic activities (2). On the other hand, mammalian phospholipase C (especially phosphatidylinositol- specific phospholipase C) regulates phosphatidylinositol turnover. Through the breakdown of phosphatidylinositol, it is involved in transmembrane signaling, cell differentiation and tumor promotion (3-5). Many phospholipase Cs are water-soluble and are found in culture media (1) or the cytosolic fraction (6, 7). In contrast, their substrates (membrane phospholipids) are water-insoluble and are separated from the aqueous solution. Therefore, for catalysis to occur, the enzyme and substrate must be partitioned in a common phase. It is thus of interest to know how phospholipase C binds to the substrate-surface and how the bound enzyme acts at the substrate-water interface. To study interfacial reactions such as lipolysis, many investigators have used a monolayer system (8). The advantages of this system are that the surface area, lipid packing density and lipid composition can be controlled. Mixed monolayerfilmahave been used in many studies on lipolytic enzymes (9-11), and results have shown that small changes in film composition can regulate lipolysis. In previous studies, we examined the interaction of porcine pancreatic carboxylester lipase with different lipidAbbreviatdons: DDA, 13,16-docosadienoic acid; 1,3-DO, 1,3-dioleoylglycerol; 1,2-DO, 1,2-dioleoylglycerol; EDTA, ethylenediaminetetraacetic acid; MO, monooleoylglycerol;POPC, l-palinitoyl-2-oleoyl-8n-glycero-3-pho8phocholine.
water interfaces and showed that the interfacial lipid composition, Le. diolein/l-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC) interactions, could be an important regulator of lipid-protein interactions (12, 13). We also reported that small changes in interfacial diolein concentration caused "all-or-none1' changes in the extent of diolein hydrolysis (14). In the present study, we examined the mechanism of action of phospholipase C using a well characterized model consisting of monolayers of 1,2 - dioleo ylglycerol (1,2-DO)/POPC mixtures as substrate and purified phospholipase C from C. perfringens as enzyme. We found that enzyme adsorption decreased linearly with increase in the surface concentration of POPC (a substrate), and increased with increase in the surface concentration of 1,2-DO (a reaction product). The results suggest that the interfacial lipid composition can regulate the adsorption of enzyme to the surface and may also regulate the enzyme activity. These findings may be helpful in understanding how phospholipase C activity is regulated by the properties of its substrate and how the breakdowns of biological important phospholipids such as phosphatidylinositol by phospholipase C are regulated. MATERIALS AND METHODS Materials—l-Palmitoyl-2-[l-14C]oleoyl-sn-glycero-3phosphocholine, 1,2- [ 1 - U C] dioleoyl- sn-glycero-3-phosphocholine and carrier-free Na115I in NaOH solution (15.4 mCi/fjt g of iodine, pH 7-11) were obtained from Amersham Japan (Tokyo). 1,3-Dioleoylglycerol (1,3-DO), 1,2-DO, monooleoylglycerol (MO), and 13,16-docosadienoic acid (DDA) were from Nu-Chek-Prep (Elysian, MN) and were shown to be ^99% pure by thin-layer chromatography. POPC (Serdary Research Laboratories, Ontario, Canada)
224
J. Biochem.
225
Effect of Diolein on Phospholipase C Activity
Vol. 112, No. 2, 1992
Relative protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as a standard. RESULTS Results on the percentage hydrolysis of POPC as a function of the initial surface pressure are shown in Fig. 1. With pure POPC films, more than 90% of the POPC was hydrolyzed at initial surface pressures of up to 28 mN/m on addition of 0.9 nM enzyme to the subphase. However, at an initial surface pressure of 30 mN/m or more, hydrolysis of POPC was less than 10%. Thus there was a sharp cut-off of the hydrolysis of POPC at 30 mN/m. On addition of a 0.2 molar fraction of 1,2-DO to the monolayer, the cut-off point of the hydrolysis of POPC was slightly increased. On addition of a 0.3 molar fraction of 1,2-DO to the monolayer, the cut-off of hydrolysis of POPC disappeared and the enzyme hydrolyzed more than 90% of the POPC at initial surface pressures of up to 41 mN/m (near the collapse pressure). With films in which hydrolysis was less than 10%, the surface pressure was essentially constant for 10 min after addition of the enzyme. However, with films in which hydrolysis was more than 90%, the surface pressure decreased slightly (less than 5 mN/m) for 10 min after enzyme addition (data not shown). Figure 2 shows the enzyme concentration dependence of POPC hydrolysis at high (34-36 mN/m) and low (19-21 mN/m) initial surface pressures. With pure POPC films, POPC was hydrolyzed more than 90% on addition of 0.15 nM enzyme at low initial surface pressure but only 15% even with a tenfold higher enzyme concentration (1.5 nM) at a high initial surface pressure (34-35 mN/m) (Fig. 2A). POPC hydrolysis was increased at both high and low surface pressures by addition of a 0.3 mol fraction of 1,2-DO: more than 90% of the POPC was hydrolyzed on addition of 0.1 nM enzyme at low initial surface pressure or 0.6 nM enzyme at high initial surface pressure (Fig. 2B). Figure 3 shows the hydrolysis of POPC as a function of the initial film composition at an initial surface pressure of 34-36 mN/m. On addition 0.9 nM enzyme, the hydrolysis D
100
a
o
80
Q X CJ Cu
60
o
40
ENT
was also ^99% pure. Hydrophobic paper for surface collection, 7-cm diameter, Type IPS, from Whatman was prepared for use by washing with solvent and equilibration with water as described previously {14). Iodo-beada was obtained from Pierce (Rockford, EL). Hexane, methanol, and chloroform (Wako Pure Chemical Industries, Osaka) were of HPLC grade and were used without further purification. Water for interfacial experiments was purified by reverse osmosis, deionization, and filtration (0.2 /*m filter) and had a specific resistance of more than 18Mfl-cm~'. l,2-[l- u C]Dioleoylglycerol was prepared from 1,2-[1U C] dioleoyl- sn-glycero-3-phosphochoUne by phospholipase C treatment as described previously (14). Methods—Usually for measurement of hydrolysis of lipid films at an air-buffer interface, 38 ml of 50 mM Tris-HCl buffer, pH 7.2, at 25°C was placed in a 5.9 cm diameter Teflon trough. The surface was cleaned by compression with a Teflon bar followed by aspiration of the surface. As a result, the volume was reduced to 28 ml. An aliquot of hexane containing the desired lipid was slowly added to give the desired surface pressure, measured by the Wilhelmy method. After 5-10 min to allow complete evaporation of the solvent, the enzyme in a volume of 0.1 ml was injected under the lipid film and stirred at 40 rpm. Then 10 min later, unless otherwise indicated, a disc of hydrophobic paper was placed on the surface. After 2-3 s, the paper was removed by slowly pulling it up parallel to the surface. This process was repeated using the other side of the paper. The adsorbed lipid was then recovered by washing the paper with 20 (10+10) ml of chloroform/ methanol (2 : 1) containing 25 //M unlabeled substrate and products. The solvent was evaporated, the residue was dissolved in 100 fA of elution solvent, and the substrate and products were separated by thin-layer chromatography (Whatman K5) in chloroform/methanol/ammonium hydroxide (90 : 10: 1). The carrier was detected by brief exposure of the plate to iodine vapor, the spots containing labeled lipid were scraped off and their radioactivity was determined in a liquid scintillation counter. Recovery of labeled lipid in the solvent extract from the paper was 89 ± 2% and was the same for POPC and 1,2-DO. All data were corrected to 100% recovery using this value. Protein adsorption was measured using 125I-labeled phospholipase C in the same manner except that the paper was not eluted but cut into pieces and its radioactivity was determined in an Aloka Auto Well Gamma System (Aloka, Tokyo). Carry-over of the aqueous phase on the hydrophobic paper was calibrated in separate experiments using [S2P]phosphate as described previously (14). Results showed that the average subphase carry-over was 29.1 +1.4/^I/paper. Phospholipase C (Type I from C. perfringens; Sigma, St. Louis, MO) was purified by affinity chromatography on Agarose-linked egg-yolk lipoprotein, and then gel nitration on Sephadex G-100 (15). The purified enzyme appeared homogeneous on SDS-polyacrylamide gel electrophoresis and its specific activity was 210 U/mg protein/min, when 1 enzyme unit was defined as the amount required to hydrolyze 1 //mol of lecithin per min. Purified phospholipase C was iodinated by the method of Markwell (16). Residual iodide was removed by gel filtration on Sephadex G-100. The specific activity of iodinated enzyme (691 protein) was about 90% of that of unlabeled enzyme.
20
U OS
w a,
0
10
20
30
INITIAL SURFACE PRESSURE (mN/m) Fig. 1. Surface-pressure dependence of POPC hydrolysis by phospholipase C in 1,2-DO/POPC mixed films. Enzyme was added at 0.9 nM to the gubphase of films initially containing 0 (O), 0.2 (•), and 0.3 (A) molar fraction of 1,2-DO. The buffer was 50 mM Tris-HCl, pH 7.2, containing 0.1 M NaCl, 5 mM CaCl,, and 0.5 mM ZnCl,.
226
T. Tsujita and H. Okuda
u
= 60
CM
O
9 40 u u a. « 0
0.3
0.6
0.9
1.2
1.5
0
ENZYME CONCENTRATION
Effect of Diolein on Hydrolysis of Phosphatidylcholine by Phospholipase C from Clostridium perfringens Takahiro Tsujita and Hiromichi Okuda Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu, Ehime 791-02 Received for publication, March 16, 1992
The activity of phospholipase C from Clostridium perfringens on l-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pore POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.31,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [I26I] phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DOfilmwas 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.
Phospholipase C [EC 3.1.4.3], a phosphodiesterase that hydrolyzes phosphoglyceride at the ester bond between diglyceride and the phosphoric acid-substituted polar head group, has been found in bacteria and animals. Phospholipase C of Clostridium perfringens is a main component of the bacterial toxin (1), and has lethal, dermo-necrotic, and hemolytic activities (2). On the other hand, mammalian phospholipase C (especially phosphatidylinositol- specific phospholipase C) regulates phosphatidylinositol turnover. Through the breakdown of phosphatidylinositol, it is involved in transmembrane signaling, cell differentiation and tumor promotion (3-5). Many phospholipase Cs are water-soluble and are found in culture media (1) or the cytosolic fraction (6, 7). In contrast, their substrates (membrane phospholipids) are water-insoluble and are separated from the aqueous solution. Therefore, for catalysis to occur, the enzyme and substrate must be partitioned in a common phase. It is thus of interest to know how phospholipase C binds to the substrate-surface and how the bound enzyme acts at the substrate-water interface. To study interfacial reactions such as lipolysis, many investigators have used a monolayer system (8). The advantages of this system are that the surface area, lipid packing density and lipid composition can be controlled. Mixed monolayerfilmahave been used in many studies on lipolytic enzymes (9-11), and results have shown that small changes in film composition can regulate lipolysis. In previous studies, we examined the interaction of porcine pancreatic carboxylester lipase with different lipidAbbreviatdons: DDA, 13,16-docosadienoic acid; 1,3-DO, 1,3-dioleoylglycerol; 1,2-DO, 1,2-dioleoylglycerol; EDTA, ethylenediaminetetraacetic acid; MO, monooleoylglycerol;POPC, l-palinitoyl-2-oleoyl-8n-glycero-3-pho8phocholine.
water interfaces and showed that the interfacial lipid composition, Le. diolein/l-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC) interactions, could be an important regulator of lipid-protein interactions (12, 13). We also reported that small changes in interfacial diolein concentration caused "all-or-none1' changes in the extent of diolein hydrolysis (14). In the present study, we examined the mechanism of action of phospholipase C using a well characterized model consisting of monolayers of 1,2 - dioleo ylglycerol (1,2-DO)/POPC mixtures as substrate and purified phospholipase C from C. perfringens as enzyme. We found that enzyme adsorption decreased linearly with increase in the surface concentration of POPC (a substrate), and increased with increase in the surface concentration of 1,2-DO (a reaction product). The results suggest that the interfacial lipid composition can regulate the adsorption of enzyme to the surface and may also regulate the enzyme activity. These findings may be helpful in understanding how phospholipase C activity is regulated by the properties of its substrate and how the breakdowns of biological important phospholipids such as phosphatidylinositol by phospholipase C are regulated. MATERIALS AND METHODS Materials—l-Palmitoyl-2-[l-14C]oleoyl-sn-glycero-3phosphocholine, 1,2- [ 1 - U C] dioleoyl- sn-glycero-3-phosphocholine and carrier-free Na115I in NaOH solution (15.4 mCi/fjt g of iodine, pH 7-11) were obtained from Amersham Japan (Tokyo). 1,3-Dioleoylglycerol (1,3-DO), 1,2-DO, monooleoylglycerol (MO), and 13,16-docosadienoic acid (DDA) were from Nu-Chek-Prep (Elysian, MN) and were shown to be ^99% pure by thin-layer chromatography. POPC (Serdary Research Laboratories, Ontario, Canada)
224
J. Biochem.
225
Effect of Diolein on Phospholipase C Activity
Vol. 112, No. 2, 1992
Relative protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as a standard. RESULTS Results on the percentage hydrolysis of POPC as a function of the initial surface pressure are shown in Fig. 1. With pure POPC films, more than 90% of the POPC was hydrolyzed at initial surface pressures of up to 28 mN/m on addition of 0.9 nM enzyme to the subphase. However, at an initial surface pressure of 30 mN/m or more, hydrolysis of POPC was less than 10%. Thus there was a sharp cut-off of the hydrolysis of POPC at 30 mN/m. On addition of a 0.2 molar fraction of 1,2-DO to the monolayer, the cut-off point of the hydrolysis of POPC was slightly increased. On addition of a 0.3 molar fraction of 1,2-DO to the monolayer, the cut-off of hydrolysis of POPC disappeared and the enzyme hydrolyzed more than 90% of the POPC at initial surface pressures of up to 41 mN/m (near the collapse pressure). With films in which hydrolysis was less than 10%, the surface pressure was essentially constant for 10 min after addition of the enzyme. However, with films in which hydrolysis was more than 90%, the surface pressure decreased slightly (less than 5 mN/m) for 10 min after enzyme addition (data not shown). Figure 2 shows the enzyme concentration dependence of POPC hydrolysis at high (34-36 mN/m) and low (19-21 mN/m) initial surface pressures. With pure POPC films, POPC was hydrolyzed more than 90% on addition of 0.15 nM enzyme at low initial surface pressure but only 15% even with a tenfold higher enzyme concentration (1.5 nM) at a high initial surface pressure (34-35 mN/m) (Fig. 2A). POPC hydrolysis was increased at both high and low surface pressures by addition of a 0.3 mol fraction of 1,2-DO: more than 90% of the POPC was hydrolyzed on addition of 0.1 nM enzyme at low initial surface pressure or 0.6 nM enzyme at high initial surface pressure (Fig. 2B). Figure 3 shows the hydrolysis of POPC as a function of the initial film composition at an initial surface pressure of 34-36 mN/m. On addition 0.9 nM enzyme, the hydrolysis D
100
a
o
80
Q X CJ Cu
60
o
40
ENT
was also ^99% pure. Hydrophobic paper for surface collection, 7-cm diameter, Type IPS, from Whatman was prepared for use by washing with solvent and equilibration with water as described previously {14). Iodo-beada was obtained from Pierce (Rockford, EL). Hexane, methanol, and chloroform (Wako Pure Chemical Industries, Osaka) were of HPLC grade and were used without further purification. Water for interfacial experiments was purified by reverse osmosis, deionization, and filtration (0.2 /*m filter) and had a specific resistance of more than 18Mfl-cm~'. l,2-[l- u C]Dioleoylglycerol was prepared from 1,2-[1U C] dioleoyl- sn-glycero-3-phosphochoUne by phospholipase C treatment as described previously (14). Methods—Usually for measurement of hydrolysis of lipid films at an air-buffer interface, 38 ml of 50 mM Tris-HCl buffer, pH 7.2, at 25°C was placed in a 5.9 cm diameter Teflon trough. The surface was cleaned by compression with a Teflon bar followed by aspiration of the surface. As a result, the volume was reduced to 28 ml. An aliquot of hexane containing the desired lipid was slowly added to give the desired surface pressure, measured by the Wilhelmy method. After 5-10 min to allow complete evaporation of the solvent, the enzyme in a volume of 0.1 ml was injected under the lipid film and stirred at 40 rpm. Then 10 min later, unless otherwise indicated, a disc of hydrophobic paper was placed on the surface. After 2-3 s, the paper was removed by slowly pulling it up parallel to the surface. This process was repeated using the other side of the paper. The adsorbed lipid was then recovered by washing the paper with 20 (10+10) ml of chloroform/ methanol (2 : 1) containing 25 //M unlabeled substrate and products. The solvent was evaporated, the residue was dissolved in 100 fA of elution solvent, and the substrate and products were separated by thin-layer chromatography (Whatman K5) in chloroform/methanol/ammonium hydroxide (90 : 10: 1). The carrier was detected by brief exposure of the plate to iodine vapor, the spots containing labeled lipid were scraped off and their radioactivity was determined in a liquid scintillation counter. Recovery of labeled lipid in the solvent extract from the paper was 89 ± 2% and was the same for POPC and 1,2-DO. All data were corrected to 100% recovery using this value. Protein adsorption was measured using 125I-labeled phospholipase C in the same manner except that the paper was not eluted but cut into pieces and its radioactivity was determined in an Aloka Auto Well Gamma System (Aloka, Tokyo). Carry-over of the aqueous phase on the hydrophobic paper was calibrated in separate experiments using [S2P]phosphate as described previously (14). Results showed that the average subphase carry-over was 29.1 +1.4/^I/paper. Phospholipase C (Type I from C. perfringens; Sigma, St. Louis, MO) was purified by affinity chromatography on Agarose-linked egg-yolk lipoprotein, and then gel nitration on Sephadex G-100 (15). The purified enzyme appeared homogeneous on SDS-polyacrylamide gel electrophoresis and its specific activity was 210 U/mg protein/min, when 1 enzyme unit was defined as the amount required to hydrolyze 1 //mol of lecithin per min. Purified phospholipase C was iodinated by the method of Markwell (16). Residual iodide was removed by gel filtration on Sephadex G-100. The specific activity of iodinated enzyme (691 protein) was about 90% of that of unlabeled enzyme.
20
U OS
w a,
0
10
20
30
INITIAL SURFACE PRESSURE (mN/m) Fig. 1. Surface-pressure dependence of POPC hydrolysis by phospholipase C in 1,2-DO/POPC mixed films. Enzyme was added at 0.9 nM to the gubphase of films initially containing 0 (O), 0.2 (•), and 0.3 (A) molar fraction of 1,2-DO. The buffer was 50 mM Tris-HCl, pH 7.2, containing 0.1 M NaCl, 5 mM CaCl,, and 0.5 mM ZnCl,.
226
T. Tsujita and H. Okuda
u
= 60
CM
O
9 40 u u a. « 0
0.3
0.6
0.9
1.2
1.5
0
ENZYME CONCENTRATION
