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Hydrolytic and transphosphatidylation activities of phospholipase D from Savoy cabbage towards lysophosphatidylcholine
Chemistry and Physics of Lipids 106 (2000) 41 – 51 www.elsevier.com/locate/chemphyslip
Hydrolytic and transphosphatidylation activities of phospholipase D from Savoy cabbage towards lysophosphatidylcholine Carmen Virto *, Ingemar Svensson, Patrick Adlercreutz Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund Uni6ersity, P.O. Box 124, S-22100 Lund, Sweden Received 15 November 1999; received in revised form 7 February 2000; accepted 8 February 2000
Abstract The hydrolysis and transphosphatidylation of lysophosphatidylcholine (LPC), with a partially purified preparation of phospholipase D (PL D) from Savoy cabbage, was investigated. These reactions were about 20 times slower than the hydrolysis of phosphatidylcholine (PC) in a micellar system. For the transfer reaction, 2 M glycerol was included in the media, which suppressed the hydrolytic reaction. Both reactions presented similar Vmax values, suggesting that the formation of the phosphatidyl–enzyme intermediate is the rate-limiting step. The enzyme had an absolute requirement for Ca2 + , and the optimum concentration was approximately 40 mM CaCl2. KCa(app) was calculated to be 8.6 9 0.74 mM for the hydrolytic and 10 9 0.97 mM for the transphosphatidylation reaction. Both activities reached a maximum at pH 5.5, independent of Ca2 + concentration. Kinetic studies showed that the Km(app) for the glycerol in the transphosphatidylation reaction is 388 937 mM. Km(app) for the lysophosphatidylcholine depended on Ca2 + concentration and fell between 1 and 3 mM at CaCl2 concentrations from 4 to 40 mM. SDS, TX-100, and CTAB did not activate the enzyme as reported for phosphatidylcholine hydrolysis; on the contrary, reaction rates decreased at detergent concentrations at or above that of lysophosphatidylcholine. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phospholipase D; Lysophosphatidylcholine; Lysophosphatidic acid; Lysophosphatidylglycerol; Hydrolysis; Transphosphatidylation
1. Introduction Phospholipase D (PL D; phosphatidylcholine phosphatidohydrolase, E.C.3.1.4.4) is a well-char* Corresponding author. Tel.: +46-46-2224842; fax: + 4646-2224713. E-mail address: [email protected] (C. Virto)
acterised enzyme that catalyses the hydrolysis of the terminal phosphodiester bond on phospholipids. However, this enzyme is also known for its unique transphosphatidylation activity (Heller, 1978). In the presence of an alcoholic nucleophile, PL D is capable of exchanging the polar head of the phospholipid, forming a new phosphoester bond.
0009-3084/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 0 0 ) 0 0 1 3 0 - 4
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PL D is also known to have a broad selectivity, and many phospholipids have proven to be substrates for the enzyme, e.g. phophatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, and lysophosphatidylcholine are readily hydrolysed by the enzyme at different rates depending on the origin of the enzyme (Heller, 1978). However, transphosphatidylation activity has been mostly characterised using phosphatidylcholine as the phosphatidyldonor. It is important to take into consideration the physical state of the substrates when determining the kinetic parameters of a reaction catalysed by a phospholipase (Dennis, 1983; Berg et al., 1997; Zhou et al., 1997). The activity of the enzyme is greatly influenced by whether the substrate aggregates or is in monomer form. Most of the kinetic studies have been done with phosphatidylcholine in aqueous/organic two-phase system (Bruzic and Tsai, 1984; Juneja et al., 1988) or in the form of small unilamellar vesicles (Yamamoto et al., 1993). Detergents such as sodium dodecyl sulphate (SDS) or triton X-100 (TX-100) have also been included, and the phospholipids were thus solubilised in mixed micelles (Lambrecht and Ulbrich-Hofmann, 1992; Carrea et al., 1997). Synthetic phospholipids with short fatty-acid chains are water soluble and have very high critical micelle concentrations (CMCs), which makes possible kinetic studies on monomeric substrates (Allgyer and Wells, 1979; Lambrecht and Ulbrich-Hofmann, 1992). Such studies have demonstrated that the hydrolysis and transphosphatidylation of phosphatidylcholine follow a two-step, ping-pong reaction mechanism with the formation of a phosphatidyl – enzyme intermediate. Lysophospholipids, as the short fatty-acid chain phospholipids, are also water-soluble and have higher CMCs than natural PC, which depend on the fatty-acid chain length of the molecule. Stafford et al., (1989) calculated a CMC of 0.007 mM for a 1-palmitoyl-lysophosphatidylcholine. Above CMC they exist as micelles, and in the presence of detergent they also form mixed micelles. In dispersions of LPC with excess water, there is also evidence for the formation of a more ordered structure, a lamellar phase, below a char-
acteristic transition temperature (Tm) (Wu et al., 1982; Hui and Huang, 1986). This temperature was 26°C for 1-stearoyl-lysophosphatidylcholine (Wu et al., 1982). The transition from lamellar to micellar phase occurs very quickly, in contrast with the opposite transition, which is very slow at temperatures near the Tm. The hydrolysis of lysophosphatidylcholine by a crude preparation of cabbage PL D was first studied by Long et al. (1967a,b). They demonstrated that, besides the formation of lysophosphatidic acid (LPA), the free hydroxyl group in the lysophospholipid competes in the nucleophilic attack of the phosphatidyl intermediate, and a cyclic lysophosphatidic acid is formed. Later studies (Friedman et al., 1996) have shown that this cyclic LPA is hydrolysed by the same enzyme, producing LPA. The transphosphatidylation of the lysophospholipids has not yet been reported. In this paper, we describe the characterisation and comparison of the hydrolytic and transfer activities of PL D towards lysophosphatidylcholine in a micellar system. We demonstrate that, in spite of the similarity of the substrate with phosphatidylcholine, there are significant differences in the mechanism of the transformation of these phospholipids, probably due to the difference in the structure of the lipid phases present in the reaction mixtures.
2. Experimental
2.1. Materials Fresh Savoy cabbage leaves were obtained from a local market. Egg phosphatidylcholine (purity \ 99%) and egg lysophosphatidylcholine (purity \ 99%) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA); Octyl-Sepharose CL4B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden); all buffer salts, SDS, and HPTLC plates silica gel 60 were obtained from Merck (Darmstadt, Germany); glycerol (purity \ 99%) was purchased from Kebo lab AB (Sweden); Triton X-100 (TX-100) was a product of Fluka Chemie AG, (Buchs, Switzerland); and cetyltrimethylamoniumbromide (CTAB) was ob-
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tained from BDH Chemical Ltd, (Poole, England). All reagents for the determination of the protein content on the samples were purchased from Bio-Rad Laboratories (Richmond, California, USA). All solvents were purchased from Merck and were of analytical grade.
2.1.1. Purification of Phospholipase D Crude phospholipase D was prepared from Savoy cabbage by the method of Davidson and Long, (1958). The inner, light-green leaves of the cabbage (500 g) were mixed with 300 ml of water and homogenised. The homogenate was then filtered through filter paper and the filtrate centrifuged at 13 000×g for 30 min at 5°C to remove all insoluble material. After heat treatment of the clear supernatant at 55°C for 10 min, the mixture was centrifuged for 30 min at 13 000 × g at 5°C. The pellet-containing precipitated proteins was discharged, and the supernatant was treated with 2 volumes of cold acetone (− 20°C). The mixture was centrifuged as above and the supernatant discharged. The precipitate containing PL D activity was dried under vacuum overnight and stored at − 20°C. It accounted for 890 mg of powder with a specific activity of 67 U/mg of protein and a purification factor of 5.5. Further purification of the enzyme was achieved by hydrophobic chromatography as described by Lambrecht and Ulbrich-Hofmann, (1992). Typically, 500 mg of the acetone powder was dissolved in 5 ml of 30 mM PIPES buffer containing 50 mM CaCl2; the pH of the solution was set to 6.2. The mixture was centrifuged to remove insoluble material. Octyl-Sepharose CL 4B (20 ml) was washed, packed into a column (190× 17 mm) and equilibrated with the same buffer as above. The enzyme solution was applied to the column and eluted at a flow rate of 20 ml/h. After all the unbound proteins were eluted, the elution was continued with 20 ml of 10 mM PIPES buffer containing 30 mM CaCl2, pH 6.2, and finally with 100 ml of 10 mM PIPES containing 0.1 mM EDTA, pH 6.2. Eluates were collected in 5-ml fractions. Fractions containing PL D activity were pooled and freeze-dried; these accounted for 157 mg of powder with a specific activity of 736 U/mg of protein and a purification factor of 60.
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2.1.2. Enzyme assay PL D activity in the various preparations was determined by a pH-stat method. Substrate solution samples, 5 ml each, containing Na–acetate buffer (0.5 mM, pH 5.6), 40 mM CaCl2, 100 mM NaCl, 1 mM PC, and 0.5 mM SDS were filled into 10 ml reaction vessels and the temperature set to 30°C. The pH of the mixtures was set at 5.6. After equilibration of the pH, the reaction was started by adding 5 ml of the enzyme solution, and the pH was maintained by the addition of 10 mM NaOH. PL D activity was calculated as a mean value of three different measurements. One unit of enzyme activity is defined as one mmole of substrate transformed/min/mg protein. 2.1.3. Protein determination The protein content of PL D preparations was determined by the Bio-Rad protein assay, based in the method of Bradford, (1976) using bovine serum albumin as standard. 2.1.4. Enzyme reactions Unless otherwise stated, hydrolytic reactions were carried out in 1.5 ml capped vials; total sample volumes of 0.2 ml consisted of lysophosphatidylcholine, with a final concentration ranging from 0.5 to 8 mM, in 0.15 M Na–acetate buffer (pH 5.5) containing the desired concentration of CaCl2. For the transphosphatidylation reactions, the media contained glycerol with a final concentration of 2 M. Reaction vessels were warmed until a clear transparent solution was achieved. The reactions were started by adding 0.025 U of the enzyme; the vessels were placed on a rotational shaker (1000 rpm) and incubated at 25°C. Samples of 25 ml were withdrawn at intervals for HPTLC analysis, and the reaction was stopped by adding 5 ml of 1 N HCl and 50 ml of tetrahydrofuran (THF). For the final quantification of the products formed, to the remaining media (typically 125 ml), 25 ml of 1 N HCl and 150 ml of THF were added. The reaction rates were determined from the slopes derived by linear regression of three to four data points below 20% conversion. The calculations are given as mean values of two or three different measurements.
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The reactions carried out at various pHs contained LPC (5 mM), CaCl2 (4, 10, and 40 mM), with or without glycerol (2 M), in 0.1 M Na – acetate buffer (pH range 4.5 – 5.5) or in 0.1 M MES buffer (pH range 5.5 – 7) in a final volume of 0.2 ml.
2.1.5. HPTLC analysis Thin-layer chromatography was performed on 20 × 10 cm silica gel 60 HPTLC plates (Merck). Prior to use, the plates were dipped in a solution containing boric acid (25 g/l) in ethanol and dried at 110°C on a heating plate for at least 1 h. Samples of 1–15 ml volume were applied as 6 mm bands using an autosampler (Camag ATS3, Muttenz, Switzerland). The plates were eluted with chloroform–acetone – methanol – acetic acid – water (14:8:2.5:2:1 6/6) in an unsaturated automatic development chamber (Camag ADC). Developed and dried plates were immersed for 5 s in a dipping tank (Camag chromatogram-immersion device III) containing copper sulphate detection reagent and heated for 10 min at 180°C. Substrates and products were detected as brown narrow bands on a clear background. The bands were densitometrically evaluated with a Camag TLC Scanner 3 in absorbance/reflection mode. The slit dimensions were set at 5× 0.3 mm, and the wavelength was set at 450 nm (tungsten lamp). The relative amounts of the substrates and the products formed were calculated in each chro-
matographic plate from the peak areas. Calibration curves for lysophosphatidylcholine, lysophosphatidylglycerol (LPG) and lysophosphatidic acid showed that the detector response was similar for all of them and linear in the concentration range used.
2.1.6. Determination of kinetic constants Initial reaction rates were calculated at various substrate concentrations (specified below), and the data obtained were fitted to a Michaelis–Menten equation — 6= Vmax × [S]/(Km + [S]) — by non-linear regression using Kaleidagraph (Abelbeck Software). Alternatively, data were replotted on Hanes plots ([S]/6 against [S]), which give similar results. The kinetic parameters thus calculated are given as mean value9 S.D.
3. Results
3.1. Hydrolytic and transphosphatidylation reactions Scheme 1 illustrates the possible mechanism of the enzymatic reactions catalysed by PL D. In the action of the enzyme towards the lysophospholipid, a phosphatidyl-enzyme intermediate is formed. The concentration of the nucleophiles, an alcohol or water, will determine the extent of the transphosphatidylation and hydrolysis. In general,
Scheme 1. Possible mechanism for the PL D catalysed hydrolysis and transphosphatidylation reactions towards lysophosphatidylcholine. R1 =fatty acid chain; R2 = choline; R3 = glycerol.
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demonstrated that, at glycerol concentration of 2 M or more, the enzyme is very selective for the reaction, and almost no hydrolytic product is found even when the substrate is completely consumed.
3.2. Calcium dependence
Fig. 1. Effect of CaCl2 concentration on the transphosphatidylation ( ) and hydrolysis ( ) of 5 mM LPC, with or without 2 M glycerol, in 0.15 M Na-acetate buffer, pH 5.5 at different CaCl2 concentrations and 0.025 unit PL D in a final volume of 0.2 ml. Reaction temperature 25°C.
the transphosphatidylation reaction is favoured even at low alcohol/water ratios. Furthermore, Long et al. (1967a,b), showed that the hydrolysis of lysophosphatidylcholine, in the absence of an alcohol, proceeds in two ways: one in which LPA is formed, and another in which an intermediate, cyclic LPA is formed before being converted to LPA. The formation of this intermediate proceeded via an intramolecular phosphoester bond with the free hydroxyl group in the sn-2 position. This cyclic LPA was finally hydrolysed to LPA by the same enzyme in a hypothetically different binding site (Friedman et al., 1996). However, the cyclic intermediate was not detected in our case, probably because of the acidic conditions of the sample treatment (Serdarevich, 1967) which would catalyse quickly the hydrolysis of the phosphoester bond. When the concentration of the HCl added was decreased to 0.25 N (5 ml), a new band appeared on the TLC plates, with a relative Rf similar to the one reported by Long et al. (1967b); this band could correspond to the cyclic LPA. The intensity of the band increased with time, as did the one for LPA. After treatment with 1 N HCl, only the band corresponding to LPA could be detected. Under otherwise identical conditions, when 2 M glycerol was present in the media no cyclic LPA was detected. Kinetic studies on the transphosphatidylation of LPC and glycerol (see below)
As for the action on PC, PL D depends strictly on divalent calcium ions for optimum activity towards LPC. Long et al. (1967a) reported the optimal concentration of Ca2 + ions to be about 25 mM for the cabbage enzyme acting on LPC, and Strauss et al. (1976) showed values between 30 and 50 mM in their investigations on PL D from peanut seed. Fig. 1 illustrates the effect of CaCl2 on hydrolysis and transphosphatidylation. The results agree well with those reported previously. The saturating concentration of Ca2 + was reached between 20 and 40 mM, and the rates were basically the same for both reactions, suggesting that the rate-limiting step in the transformation of lysophosphatidylcholine by PL D is the formation of the phosphatidyl–enzyme complex. The obtained data for a substrate concentration of 5 mM were fitted to a Michaelis–Menten equation for the calculation of KCa(app), which was found to be 8.6 90.75 mM for hydrolysis and 109 0.97 mM for transphosphatidylation. A calcium-ion concentration of 40 mM was used in the rest of the experiments unless otherwise stated.
3.3. pH and calcium dependence Several authors have reported that the pH optimum of the hydrolysis of PC depends on the concentration of Ca2 + ions. In general, a decrease in calcium concentration results in a shift in the apparent optimum of two units towards higher pH (Heller 1978; Allgyer and Wells, 1979; Abousalham et al., 1993). This pH dependence was investigated for hydrolysis and transphosphatidylation towards lysophosphatidylcholine at three different Ca2 + concentrations — 4, 10, and 40 mM — and the results are illustrated in Fig. 2 for the hydrolytic reaction. Clearly, varying the concentration of calcium ions did not promote a clear shift in the pH optimum for the enzyme; the
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pH optimum was found to be 5.5 in all the cases. At lower and higher pH values, the rates decreased considerably and almost no activity was detected at pH over 7. The activities and the profiles were essentially the same for the transphosphatidylation reaction, in which 2 M glycerol was included in the reaction media (data not shown).
Fig. 2. Effect of pH on the hydrolysis of 5 mM LPC, in 0.1 M Na-acetate buffer (open symbols) or 0.1 M MES buffer (closed symbols), different concentrations of CaCl2 and 0.025 units PL D in a total volume of 0.2 ml. Reaction temperature 25°C. (2, ") 4 mM CaCl2 ( , ) 10 mM CaCl2 and (, ) 40 mM CaCl2.
3.4. Reaction kinetics and Ca 2 + dependence The kinetic parameters for the formation of LPA and LPG were determined at various Ca2 + concentrations. LPC concentration was varied from 0.5 to 8 mM at calcium concentrations of 4, 10, 20, and 40 mM. Glycerol, to a final concentration of 2 M, was added for the transphosphatidylation reaction, which caused hydrolysis to be negligible in the range of conversions measured. To restate, the formation of cyclic LPA was not detected in our system, most probably because of the high concentration of the HCl added to the sample. Thus, all the product formed in the hydrolysis of LPC was detected as LPA. As shown in Fig. 3, the Hanes plots were linear at all calcium concentrations. Table 1 presents the kinetic parameters obtained by fitting the initial rates to the Michaelis– Menten equation by non-linear regression. Km(app) and Vmax(app) increased with Ca2 + concentration both in the hydrolysis and in the transfer reactions. Km(app) varied between 1.139 0.50 and 2.88 90.30 mM, and Vmax(app) was between 0.03690.006 and 0.1029 0.007 (mmol/min/mg protein). However, the specificity constant, that is, Vmax/Km, shows very little variation.
3.5. Kinetics of the transphosphatidylation acti6ity
Fig. 3. Hanes plot for the hydrolysis of lysophosphatidylcholine at different CaCl2 concentrations: () 4 mM; ( ) 10 mM; () 20 mM; ( ) 40 mM. Reaction media composed with different concentrations of LPC in 0.15 M Na-acetate buffer, pH 5.5, 0.025 U of PL D in a final volume of 0.2 ml. Reaction temperature 25°C.
As mentioned earlier, the reaction mechanism could be described as a ping–pong mechanism modified with a hydrolytic branch. However, if the concentrations of LPC and water are kept constant, the velocity of the transphosphatidylation reaction against glycerol concentration can be treated as a single-substrate reaction and will have the typical Michaelis–Menten kinetics profile. The data thus obtained were fitted using non-linear regression (Fig. 4), and the kinetic parameters, Vmax(app) and Km(app), for glycerol were calculated to be 0.13290.036 (mmole/min/ mg protein) and 3889 37 mM, respectively. No comparative data for Km were available for the cabbage enzyme, but Carrea et al. (1997) determined the Km for 3-dimethylamino-1-propanol to be 1.3 M for the Streptomyces sp. enzyme in the transphosphatidylation of PC with the alcohol.
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Table 1 Kinetic parameters for the hydrolysis and transfer activities of PL D towards LPCa CaCl2 (mM)
Km (mM)
Vmax (mmol/min/mg protein)
Vmax/Km (min−1/mg protein)
Hydrolysis
4 10 20 40
1.139 0.50 1.75 9 0.30 2.88 9 0.29 2.449 0.59
0.036 90.006 0.053 9 0.005 0.101 90.006 0.102 90.007
0.1790.01 0.15 9 0.02 0.17 90.01 0.20 90.02
Transphosphatidylation
4 10 20 40
1.39 90.37 1.88 9 0.37 2.689 0.42 2.089 0.17
0.042 90.013 0.067 90.009 0.099 90.008 0.091 90.030
0.17 9 0.03 0.15 90.02 0.17 90.03 0.21 90.03
a Reaction media contained substrate concentration from 0.5 to 8 mM with or without 2 M glycerol in 0.15 M Na-acetate buffer, pH 5.5, CaCl2 concentrations from 4 to 40 mM and 0.025 units PL D in a final volume of 0.2 ml. Reaction temperature 25°C. Data are given as a mean value 9 S.D.
The selectivity of the enzymatic reaction given as the ratio between hydrolysis and transphosphatidylation reaction increases almost linearly as the glycerol/water ratio increases (Fig. 5).
3.6. Detergent effect The inclusion of detergents or solvents for the solubilisation of water-insoluble phosphatidylcholines is a common practice, and the effects are multiple. Diethyl ether and SDS are reported to be the best activators for the enzyme. The solubilisation of the substrate makes it more susceptible to hydrolysis. However, this is not a general rule for all detergents, because others such as CTBA have been shown to inhibit the enzymatic activity (Heller, 1978). Quarles and Dawson (1969) suggested that the positive effect of the amphiphilic anionic compounds as SDS and phosphatidic acid could be due to a neutralisation of the positive charges in the PC. The negatively charged detergent could attract Ca2 + ions to the proximity of the substrate. However, there is no explanation why other anionic substances would not produce the same activation. The effects of SDS, CTAB, and TX-100 concentrations on the activity of the enzyme towards LPC are shown in Fig. 6. There is no increase in activity with any of the detergents tested in the concentration range used; on the contrary, the activity dropped at CTAB and TX-100 concentrations as low as half of the LPC concentration.
Further studies were performed with SDS. Various concentrations of SDS, from 0.5 to 5 mM, were tried at two different Ca2 + concentrations. As seen in Fig. 6(b), SDS seems not to affect the enzymatic activity at concentrations up to 2.5 mM (half the LPC concentration). Higher amounts were shown to lower enzyme activity, but this effect could be due to an interfacial dilution phenomenon (Dennis, 1983). Because the substrate is water soluble, it is unsurprising that the detergent does not affect enzyme kinetics. SDS does not make the enzyme more active at low Ca2 + concentrations (Fig. 6(b)), and the profile of activity versus detergent concentration at 4 mM Ca2 + concentration looks similar to the one for 40 mM.
Fig. 4. Reaction kinetics for the transphosphatidylation of 5 mM LPC and concentrations of glycerol from 50 to 2000 mM, in 0.15 M Na-acetate buffer, pH 5.5, 40 mM CaCl2 and 0.025 units PL D in a total volume of 0.2 ml. Reaction temperature 25°C.
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4. Discussion
Fig. 5. Effect of the molar ratio of glycerol and water on the ratio of hydrolytic and transfer reactions. Reaction media consisted of 5 mM LPC and concentrations of glycerol from 50 to 2000 mM, in 0.15 M Na-acetate buffer, pH 5.5, 40 mM CaCl2 and 0.025 units PL D in a total volume of 0.2 ml. Reaction temperature 25°C.
Fig. 6. (a) Effect of CTAB ( , ) and Triton X-100 (, ) addition on the transphosphatidylation (circles) and hydrolysis (squares) of 5 mM LPC, with or without 2 M glycerol, in 0.15 M Na-acetate buffer, pH 5.5, 40 mM CaCl2 and 0.025 units PL D. Reaction temperature 25°C. (b) Effect of SDS concentration on the transphosphatidylation ( , ) and hydrolysis (2, ") of 5 mM LPC, with or without 2 M glycerol, in 0.15 M Na-acetate buffer, pH 5.5, 4 (open symbols) and 40 mM CaCl2 (closed symbols) and 0.025 units PL D. Reaction temperature 25°C.
The hydrolytic and transphosphatidylation activities of PL D from Savoy cabbage towards LPC are somewhat different from the same activities towards PC. Both phospholipids are substrates for the enzyme, although LPC seems to be poorer. The hydrolytic activity of the enzyme determined in a PC/SDS mixed-micellar system, at a substrate concentration of 1 mM, was about 20 times higher than with the lysophosphatidylcholine. The physical state of the substrates and the nature of the interface at which reaction takes place are known to play a very important role on the activity of phospholipases (Dennis, 1983; Berg et al., 1997; Zhou et al., 1997). Therefore, it is difficult to make a fair comparison between the kinetics of these substrates with PL D, because LPC occurs in micellar form and PC forms bilayers. Micellation of PC can be achieved by incorporating surfactants or with synthetic phospholipids of short fatty-acid chain length. Under the conditions of our test, the comparison of the transphosphatidylation and hydrolysis reactions showed that the kinetic mechanism of both reactions are similar in the formation of the phosphatidyl–enzyme intermediate, as demonstrated for PC previously (Carrea et al., 1997). The kinetic parameters for hydrolysis and transphosphatidylation are very similar in the conditions tested, demonstrating that the formation of a phosphatidyl intermediate is the ratelimiting step. PL D is a metal-dependent enzyme, and maximum activities are achieved when Ca2 + ions are present in the medium. However, the metal requirement of this enzyme has proven to be complicated, and the concentration for optimal enzyme activity was different if the reaction conditions were varied by pH or by the addition of detergents and solvents (Quarles and Dawson, 1969; Allgyer and Wells, 1979; Abousalham et al., 1993). LPC was best transformed in the presence of more than 40 mM Ca2 + , and no detergent or solvents were necessary. No activity was detected in the absence of CaCl2.
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Investigations of the optimum pH for the activity of PL D from cabbage towards PC are somewhat ambiguous. Although Quarles and Dawson (1969) reported shifts of pH optima of up to 1.5 units in ultrasonically treated large PC particles, resulting from the addition of anionic amphipathic substances such as phosphatidic acid or SDS, pH optimum was unaffected by CaCl2 concentration. However, other authors have reported clear pH optimum shifts when Ca2 + ion concentrations were varied (Abousalham et al., 1993; Yamamoto et al., 1993); higher pH optima were calculated with decreasing concentrations of CaCl2. In the activity of PL D towards LPC, the pH optimum was independent of Ca2 + ion concentration. Recent reports have demonstrated that, in plants, PL D occurs in multiple molecular forms associated with various stages of growth and development (Brauer et al., 1991; Dyer et al., 1994; Young et al., 1996; Pappan et al., 1997; Wang, 1997). At least three different forms were identified in castor bean (Dyer et al., 1994) and two forms in maize root (Brauer et al., 1991), rice (Young et al., 1996), and Arabidopsis (Pappan et al., 1997). In castor bean, these forms present significant differences at the molecular level. The molecular weight and isoelectric points were clearly different, as was substrate selectivity. In Arabidopsis and maize root, the two different forms presented different Ca2 + requirements; one functioned at micromolar ion concentrations, but the optimum level for the other was in millimolar concentrations. These two forms also had different pH optima in Arabidopsis. It is likely that the cabbage PL D is expressed in similar multiple forms, although there is no experimental evidence for this. We propose that the different pH optima at various Ca2 + concentrations could be due to the presence of different isoforms with different functional requirements. Thus, at high Ca2 + concentrations the PL D form requiring millimolar ion concentration and acidic pH would be functional, but activities at low Ca2 + concentrations and high pH would be assigned to a PL D isoform with higher Ca2 + affinity (Wang, 1997). Allgyer and Wells (1979) reported an optimum pH of 7.25 for the cabbage enzyme at 0.5 mM
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CaCl2 concentration; at 50 mM, the optimum shifted to 6.25, but a second optimum (with lower activity) remained at pH 7.25. The KCa determined at pH 7 was very low, 0.21 mM. We believe that these different forms of PL D have different substrate selectivity and, thus, the PL D with high Ca2 + affinity and high pH optimum would not be active with lysophospholipids. This would explain the markedly different effect of pH and CaCl2 in substrates as closely related as PC and LPC. Calcium concentration had an effect on the kinetic parameters, with increasing calcium concentrations producing a slight increase in Km(app) and Vmax(app) but unchanged Vmax/Km ratios. The effect of calcium on the Vmax(app) is probably exclusive to the enzyme level, reflected in its low affinity towards Ca2 + and the subsequent high ion-concentration requirement for optimum activity, as discussed above. On the other hand, the effect on Km may be understood, in terms of a change in the structural properties of the substrate due to the interaction with divalent ions. The interaction of ions with phospholipid bilayers is complicated and depends greatly on the physicochemical state of the membrane (Cevc, 1990). Changes in the structure are reported to be due to changes in the membrane hydration and to the formation of intermolecular bridges. In general, membranes, which bind small inorganic ions at their hydration sites, become less hydrophilic and more rigid. In this line, investigations on Ca2 + ion effects on lysophosphatidylcholine foam films (Cohen and Exerowa, 1994) and phosphatidylcholine bilayers (Ohshima et al., 1982) have shown a progressive decrease of the film thickness and the intermembrane separation at increasing Ca2 + concentrations between 1 and 200 mM. Due to the Ca2 + ions, the attractive forces between membranes increase relative to the repulsive forces, which results in more ordered structures. If calcium would affect LPC micelles in the same way, the better packing of the aggregates could make the substrate less susceptible to attack by the enzyme with the subsequent increase in Km. Finally, it was also observed that the enzyme is not activated by SDS, as has been widely reported for the enzyme hydrolysing PC. The change in the
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ionic environment of the substrate due to the extra charges added with the detergent seem to play no important role, at least at the lowest detergent concentrations. Because LPC is water soluble, the addition of detergents is not necessary. In the same way, the addition of diethylether was reported to be inhibiting for the hydrolysis of this substrate due to its insolubility on the solvent (Davidson and Long, 1958).
Acknowledgements This project was supported by the Swedish National Board for Technical and Industrial Development (NUTEK) and the Swedish Research Council for Engineering Sciences (TFR).
References Abousalham, A., Riviere, M., Teissere, M., Verger, R., 1993. Improved purification and characterization of phospholipase D from cabbage. Biochim. Biophys. Acta 1158, 1– 7. Allgyer, T.T., Wells, M.A., 1979. Phospholipase D from Savoy cabbage: purification and preliminary kinetic characterization. Biochemistry 18, 5348–5353. Berg, O.G., Rogers, J., Yu, B-Z., Yao, J., Romsted, L.S., Jain, M.K., 1997. Thermodynamic and kinetic basis of interfacial activation: Resolution of binding and allosteric effects on pancreatic phospholipase A(2) at zwitterionic interfaces. Biochemistry 36, 14512–14530. Bradford, M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Brauer, D., Schubert, C., Conner, D., Tu, S., 1991. Calcium activation of maize root phospholipase D. J. Plant Nutrition 14, 729 – 740. Bruzic, K., Tsai, M.D., 1984. Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D. Biochemistry 23, 1656 – 1661. Carrea, G., D’Arrigo, P., Mazzotti, M., Secundo, F., Servi, S., 1997. On the kinetic mechanism of phospholipase D from Streptomyces sp. in an emulsion system. Biocat. Biotransf. 15, 251 – 264. Cevc, G., 1990. Membrane electrostatics. Biochim. Biophys. Acta 1031, 311 – 382. Cohen, R., Exerowa, D., 1994. Electrosurface properties of lysophosphatidylcholine foam films: effect of pH and
Ca2 + . Colloids Surfaces A: Physicochem. Eng. Aspects 85, 271 – 278. Davidson, F., Long, C., 1958. The structure of naturally occurring phosphoglycerides. Action of cabbage-leaf phospholipase D on ovolecithin and related substances. Biochem. J. 69, 458 – 466. Dennis, E.A., 1983. Phospholipases. In: Boyer, P.D. (Ed.), The Enzymes, vol. XVI. Academic Press, New York, pp. 323 – 334. Dyer, J.H., Ryu, S.B., Wang, X., 1994. Multiple forms of phospholipase D following germination and during leaf development of Castor bean. Plant Physiol. 105, 715 – 724. Friedman, P., Haimovitz, R., Markman, O., Roberts, M.F., Shinitzky, M., 1996. Conversion of lysophospholipids to cyclic lysophosphatidic acid by phospholipase D. J. Biol. Chem. 271, 953 – 957. Heller, M., 1978. Phospholipase D. Adv. Lipid. Res. 16, 267 – 326. Hui, S.W., Huang, C-H., 1986. X-ray diffraction evidence for fully interdigitated bilayers of 1-stearoyllysophosphatidylcholine. Biochemistry 25, 1330 – 1335. Juneja, L.R., Kazuoka, T., Yamane, T., Shimizu, S., 1988. Kinetic evaluation of conversion of phosphatidylcholine to phosphatidylethanolamine by phospholipase D from different sources. Biochim. Biophys. Acta 960, 334 – 341. Lambrecht, R., Ulbrich-Hofmann, R., 1992. A facile purification procedure of phospholipase D from cabbage and its characterization. Biol. Chem. Hoppe-Seyler 373, 81 – 88. Long, C., Odavic, R., Sargent, E.J., 1967a. The action of cabbage-leaf phospholipase D upon lysolecithin. Biochem. J. 102, 216 – 220. Long, C., Odavic, R., Sargent, E.J., 1967b. The chemical nature of the products obtained by the action of cabbage-leaf phospholipase D on lysolecithin: the structure of lysolecithin. Biochem. J. 102, 221 – 229. Ohshima, H., Inoko, Y., Mitsui, T., 1982. Hamaker constant and binding constants of Ca2 + and Mg2 + in dipalmitoyl phosphatidylcholine/water system. J. Coll. Interf. Sci. 86, 57 – 72. Pappan, K., Qin, W., Dyer, J.H., Zheng, L., Wang, X., 1997. Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PL Db, from Arabidopsis. J. Biol. Chem. 272, 7055 – 7061. Quarles, R.H., Dawson, R.M.C., 1969. A shift in the optimum pH of phospholipase D produced by activating long-chain anions. Biochem. J. 112, 795 – 799. Serdarevich, B., 1967. Glyceride isomerizations in lipid chemistry. J. Am. Oil Chem. Soc. 44, 381 – 393. Stafford, R.E., Fanni, T., Dennis, E.A., 1989. Interfacial properties and critical micelle concentration of lysophospholipids. Biochemistry 28, 5113 – 5120. Strauss, H., Leibovitz-Ben Gershon, Z., Heller, M., 1976. Enzymatic hydrolysis of 1-monoacyl-sn-glycerol-3-phos-
C. Virto et al. / Chemistry and Physics of Lipids 106 (2000) 41–51 phorylcholine (1-lysolecithin) by phospholipases from peanut seeds. Lipids 11, 442–448. Wang, X., 1997. Molecular analysis of phospholipase D. Trends Plant Sci. 2, 261 – 266. Wu, W-G., Huang, C-H., Conley, T.G., Martin, R.B., Levin, I.W., 1982. Lamellar-micellar transition of 1-stearoyllysophosphatidylcholine assemblies in excess water. Biochemistry 21, 5957 – 5961. Yamamoto, I., Mazumi, T., Handa, T., Miyajima, K., 1993. Effects of 1,2-diacylglycerol and cholesterol on the hydrol-
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ysis activity of phospholipase D in egg-yolk phosphatidylcholine bilayers. Biochim. Biophys. Acta 1145, 293 – 297. Young, S.A., Wang, X., Leach, J.E., 1996. Changes in the plasma membrane distribution of rice phospholipase D during resistant interactions with Xanthomonas oryzae pv oryzae. Plant Cell 8, 1079 – 1090. Zhou, C., Qian, X., Roberts, M.F., 1997. Allosteric activation of phosphatidylinositol-specific phospholipase C: Specific phospholipid binding anchors the enzyme to the interface. Biochemistry 36, 10089 – 10097.
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Hydrolytic and transphosphatidylation activities of phospholipase D from Savoy cabbage towards lysophosphatidylcholine Carmen Virto *, Ingemar Svensson, Patrick Adlercreutz Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund Uni6ersity, P.O. Box 124, S-22100 Lund, Sweden Received 15 November 1999; received in revised form 7 February 2000; accepted 8 February 2000
Abstract The hydrolysis and transphosphatidylation of lysophosphatidylcholine (LPC), with a partially purified preparation of phospholipase D (PL D) from Savoy cabbage, was investigated. These reactions were about 20 times slower than the hydrolysis of phosphatidylcholine (PC) in a micellar system. For the transfer reaction, 2 M glycerol was included in the media, which suppressed the hydrolytic reaction. Both reactions presented similar Vmax values, suggesting that the formation of the phosphatidyl–enzyme intermediate is the rate-limiting step. The enzyme had an absolute requirement for Ca2 + , and the optimum concentration was approximately 40 mM CaCl2. KCa(app) was calculated to be 8.6 9 0.74 mM for the hydrolytic and 10 9 0.97 mM for the transphosphatidylation reaction. Both activities reached a maximum at pH 5.5, independent of Ca2 + concentration. Kinetic studies showed that the Km(app) for the glycerol in the transphosphatidylation reaction is 388 937 mM. Km(app) for the lysophosphatidylcholine depended on Ca2 + concentration and fell between 1 and 3 mM at CaCl2 concentrations from 4 to 40 mM. SDS, TX-100, and CTAB did not activate the enzyme as reported for phosphatidylcholine hydrolysis; on the contrary, reaction rates decreased at detergent concentrations at or above that of lysophosphatidylcholine. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phospholipase D; Lysophosphatidylcholine; Lysophosphatidic acid; Lysophosphatidylglycerol; Hydrolysis; Transphosphatidylation
1. Introduction Phospholipase D (PL D; phosphatidylcholine phosphatidohydrolase, E.C.3.1.4.4) is a well-char* Corresponding author. Tel.: +46-46-2224842; fax: + 4646-2224713. E-mail address: [email protected] (C. Virto)
acterised enzyme that catalyses the hydrolysis of the terminal phosphodiester bond on phospholipids. However, this enzyme is also known for its unique transphosphatidylation activity (Heller, 1978). In the presence of an alcoholic nucleophile, PL D is capable of exchanging the polar head of the phospholipid, forming a new phosphoester bond.
0009-3084/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 0 0 ) 0 0 1 3 0 - 4
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PL D is also known to have a broad selectivity, and many phospholipids have proven to be substrates for the enzyme, e.g. phophatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, and lysophosphatidylcholine are readily hydrolysed by the enzyme at different rates depending on the origin of the enzyme (Heller, 1978). However, transphosphatidylation activity has been mostly characterised using phosphatidylcholine as the phosphatidyldonor. It is important to take into consideration the physical state of the substrates when determining the kinetic parameters of a reaction catalysed by a phospholipase (Dennis, 1983; Berg et al., 1997; Zhou et al., 1997). The activity of the enzyme is greatly influenced by whether the substrate aggregates or is in monomer form. Most of the kinetic studies have been done with phosphatidylcholine in aqueous/organic two-phase system (Bruzic and Tsai, 1984; Juneja et al., 1988) or in the form of small unilamellar vesicles (Yamamoto et al., 1993). Detergents such as sodium dodecyl sulphate (SDS) or triton X-100 (TX-100) have also been included, and the phospholipids were thus solubilised in mixed micelles (Lambrecht and Ulbrich-Hofmann, 1992; Carrea et al., 1997). Synthetic phospholipids with short fatty-acid chains are water soluble and have very high critical micelle concentrations (CMCs), which makes possible kinetic studies on monomeric substrates (Allgyer and Wells, 1979; Lambrecht and Ulbrich-Hofmann, 1992). Such studies have demonstrated that the hydrolysis and transphosphatidylation of phosphatidylcholine follow a two-step, ping-pong reaction mechanism with the formation of a phosphatidyl – enzyme intermediate. Lysophospholipids, as the short fatty-acid chain phospholipids, are also water-soluble and have higher CMCs than natural PC, which depend on the fatty-acid chain length of the molecule. Stafford et al., (1989) calculated a CMC of 0.007 mM for a 1-palmitoyl-lysophosphatidylcholine. Above CMC they exist as micelles, and in the presence of detergent they also form mixed micelles. In dispersions of LPC with excess water, there is also evidence for the formation of a more ordered structure, a lamellar phase, below a char-
acteristic transition temperature (Tm) (Wu et al., 1982; Hui and Huang, 1986). This temperature was 26°C for 1-stearoyl-lysophosphatidylcholine (Wu et al., 1982). The transition from lamellar to micellar phase occurs very quickly, in contrast with the opposite transition, which is very slow at temperatures near the Tm. The hydrolysis of lysophosphatidylcholine by a crude preparation of cabbage PL D was first studied by Long et al. (1967a,b). They demonstrated that, besides the formation of lysophosphatidic acid (LPA), the free hydroxyl group in the lysophospholipid competes in the nucleophilic attack of the phosphatidyl intermediate, and a cyclic lysophosphatidic acid is formed. Later studies (Friedman et al., 1996) have shown that this cyclic LPA is hydrolysed by the same enzyme, producing LPA. The transphosphatidylation of the lysophospholipids has not yet been reported. In this paper, we describe the characterisation and comparison of the hydrolytic and transfer activities of PL D towards lysophosphatidylcholine in a micellar system. We demonstrate that, in spite of the similarity of the substrate with phosphatidylcholine, there are significant differences in the mechanism of the transformation of these phospholipids, probably due to the difference in the structure of the lipid phases present in the reaction mixtures.
2. Experimental
2.1. Materials Fresh Savoy cabbage leaves were obtained from a local market. Egg phosphatidylcholine (purity \ 99%) and egg lysophosphatidylcholine (purity \ 99%) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA); Octyl-Sepharose CL4B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden); all buffer salts, SDS, and HPTLC plates silica gel 60 were obtained from Merck (Darmstadt, Germany); glycerol (purity \ 99%) was purchased from Kebo lab AB (Sweden); Triton X-100 (TX-100) was a product of Fluka Chemie AG, (Buchs, Switzerland); and cetyltrimethylamoniumbromide (CTAB) was ob-
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tained from BDH Chemical Ltd, (Poole, England). All reagents for the determination of the protein content on the samples were purchased from Bio-Rad Laboratories (Richmond, California, USA). All solvents were purchased from Merck and were of analytical grade.
2.1.1. Purification of Phospholipase D Crude phospholipase D was prepared from Savoy cabbage by the method of Davidson and Long, (1958). The inner, light-green leaves of the cabbage (500 g) were mixed with 300 ml of water and homogenised. The homogenate was then filtered through filter paper and the filtrate centrifuged at 13 000×g for 30 min at 5°C to remove all insoluble material. After heat treatment of the clear supernatant at 55°C for 10 min, the mixture was centrifuged for 30 min at 13 000 × g at 5°C. The pellet-containing precipitated proteins was discharged, and the supernatant was treated with 2 volumes of cold acetone (− 20°C). The mixture was centrifuged as above and the supernatant discharged. The precipitate containing PL D activity was dried under vacuum overnight and stored at − 20°C. It accounted for 890 mg of powder with a specific activity of 67 U/mg of protein and a purification factor of 5.5. Further purification of the enzyme was achieved by hydrophobic chromatography as described by Lambrecht and Ulbrich-Hofmann, (1992). Typically, 500 mg of the acetone powder was dissolved in 5 ml of 30 mM PIPES buffer containing 50 mM CaCl2; the pH of the solution was set to 6.2. The mixture was centrifuged to remove insoluble material. Octyl-Sepharose CL 4B (20 ml) was washed, packed into a column (190× 17 mm) and equilibrated with the same buffer as above. The enzyme solution was applied to the column and eluted at a flow rate of 20 ml/h. After all the unbound proteins were eluted, the elution was continued with 20 ml of 10 mM PIPES buffer containing 30 mM CaCl2, pH 6.2, and finally with 100 ml of 10 mM PIPES containing 0.1 mM EDTA, pH 6.2. Eluates were collected in 5-ml fractions. Fractions containing PL D activity were pooled and freeze-dried; these accounted for 157 mg of powder with a specific activity of 736 U/mg of protein and a purification factor of 60.
43
2.1.2. Enzyme assay PL D activity in the various preparations was determined by a pH-stat method. Substrate solution samples, 5 ml each, containing Na–acetate buffer (0.5 mM, pH 5.6), 40 mM CaCl2, 100 mM NaCl, 1 mM PC, and 0.5 mM SDS were filled into 10 ml reaction vessels and the temperature set to 30°C. The pH of the mixtures was set at 5.6. After equilibration of the pH, the reaction was started by adding 5 ml of the enzyme solution, and the pH was maintained by the addition of 10 mM NaOH. PL D activity was calculated as a mean value of three different measurements. One unit of enzyme activity is defined as one mmole of substrate transformed/min/mg protein. 2.1.3. Protein determination The protein content of PL D preparations was determined by the Bio-Rad protein assay, based in the method of Bradford, (1976) using bovine serum albumin as standard. 2.1.4. Enzyme reactions Unless otherwise stated, hydrolytic reactions were carried out in 1.5 ml capped vials; total sample volumes of 0.2 ml consisted of lysophosphatidylcholine, with a final concentration ranging from 0.5 to 8 mM, in 0.15 M Na–acetate buffer (pH 5.5) containing the desired concentration of CaCl2. For the transphosphatidylation reactions, the media contained glycerol with a final concentration of 2 M. Reaction vessels were warmed until a clear transparent solution was achieved. The reactions were started by adding 0.025 U of the enzyme; the vessels were placed on a rotational shaker (1000 rpm) and incubated at 25°C. Samples of 25 ml were withdrawn at intervals for HPTLC analysis, and the reaction was stopped by adding 5 ml of 1 N HCl and 50 ml of tetrahydrofuran (THF). For the final quantification of the products formed, to the remaining media (typically 125 ml), 25 ml of 1 N HCl and 150 ml of THF were added. The reaction rates were determined from the slopes derived by linear regression of three to four data points below 20% conversion. The calculations are given as mean values of two or three different measurements.
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The reactions carried out at various pHs contained LPC (5 mM), CaCl2 (4, 10, and 40 mM), with or without glycerol (2 M), in 0.1 M Na – acetate buffer (pH range 4.5 – 5.5) or in 0.1 M MES buffer (pH range 5.5 – 7) in a final volume of 0.2 ml.
2.1.5. HPTLC analysis Thin-layer chromatography was performed on 20 × 10 cm silica gel 60 HPTLC plates (Merck). Prior to use, the plates were dipped in a solution containing boric acid (25 g/l) in ethanol and dried at 110°C on a heating plate for at least 1 h. Samples of 1–15 ml volume were applied as 6 mm bands using an autosampler (Camag ATS3, Muttenz, Switzerland). The plates were eluted with chloroform–acetone – methanol – acetic acid – water (14:8:2.5:2:1 6/6) in an unsaturated automatic development chamber (Camag ADC). Developed and dried plates were immersed for 5 s in a dipping tank (Camag chromatogram-immersion device III) containing copper sulphate detection reagent and heated for 10 min at 180°C. Substrates and products were detected as brown narrow bands on a clear background. The bands were densitometrically evaluated with a Camag TLC Scanner 3 in absorbance/reflection mode. The slit dimensions were set at 5× 0.3 mm, and the wavelength was set at 450 nm (tungsten lamp). The relative amounts of the substrates and the products formed were calculated in each chro-
matographic plate from the peak areas. Calibration curves for lysophosphatidylcholine, lysophosphatidylglycerol (LPG) and lysophosphatidic acid showed that the detector response was similar for all of them and linear in the concentration range used.
2.1.6. Determination of kinetic constants Initial reaction rates were calculated at various substrate concentrations (specified below), and the data obtained were fitted to a Michaelis–Menten equation — 6= Vmax × [S]/(Km + [S]) — by non-linear regression using Kaleidagraph (Abelbeck Software). Alternatively, data were replotted on Hanes plots ([S]/6 against [S]), which give similar results. The kinetic parameters thus calculated are given as mean value9 S.D.
3. Results
3.1. Hydrolytic and transphosphatidylation reactions Scheme 1 illustrates the possible mechanism of the enzymatic reactions catalysed by PL D. In the action of the enzyme towards the lysophospholipid, a phosphatidyl-enzyme intermediate is formed. The concentration of the nucleophiles, an alcohol or water, will determine the extent of the transphosphatidylation and hydrolysis. In general,
Scheme 1. Possible mechanism for the PL D catalysed hydrolysis and transphosphatidylation reactions towards lysophosphatidylcholine. R1 =fatty acid chain; R2 = choline; R3 = glycerol.
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45
demonstrated that, at glycerol concentration of 2 M or more, the enzyme is very selective for the reaction, and almost no hydrolytic product is found even when the substrate is completely consumed.
3.2. Calcium dependence
Fig. 1. Effect of CaCl2 concentration on the transphosphatidylation ( ) and hydrolysis ( ) of 5 mM LPC, with or without 2 M glycerol, in 0.15 M Na-acetate buffer, pH 5.5 at different CaCl2 concentrations and 0.025 unit PL D in a final volume of 0.2 ml. Reaction temperature 25°C.
the transphosphatidylation reaction is favoured even at low alcohol/water ratios. Furthermore, Long et al. (1967a,b), showed that the hydrolysis of lysophosphatidylcholine, in the absence of an alcohol, proceeds in two ways: one in which LPA is formed, and another in which an intermediate, cyclic LPA is formed before being converted to LPA. The formation of this intermediate proceeded via an intramolecular phosphoester bond with the free hydroxyl group in the sn-2 position. This cyclic LPA was finally hydrolysed to LPA by the same enzyme in a hypothetically different binding site (Friedman et al., 1996). However, the cyclic intermediate was not detected in our case, probably because of the acidic conditions of the sample treatment (Serdarevich, 1967) which would catalyse quickly the hydrolysis of the phosphoester bond. When the concentration of the HCl added was decreased to 0.25 N (5 ml), a new band appeared on the TLC plates, with a relative Rf similar to the one reported by Long et al. (1967b); this band could correspond to the cyclic LPA. The intensity of the band increased with time, as did the one for LPA. After treatment with 1 N HCl, only the band corresponding to LPA could be detected. Under otherwise identical conditions, when 2 M glycerol was present in the media no cyclic LPA was detected. Kinetic studies on the transphosphatidylation of LPC and glycerol (see below)
As for the action on PC, PL D depends strictly on divalent calcium ions for optimum activity towards LPC. Long et al. (1967a) reported the optimal concentration of Ca2 + ions to be about 25 mM for the cabbage enzyme acting on LPC, and Strauss et al. (1976) showed values between 30 and 50 mM in their investigations on PL D from peanut seed. Fig. 1 illustrates the effect of CaCl2 on hydrolysis and transphosphatidylation. The results agree well with those reported previously. The saturating concentration of Ca2 + was reached between 20 and 40 mM, and the rates were basically the same for both reactions, suggesting that the rate-limiting step in the transformation of lysophosphatidylcholine by PL D is the formation of the phosphatidyl–enzyme complex. The obtained data for a substrate concentration of 5 mM were fitted to a Michaelis–Menten equation for the calculation of KCa(app), which was found to be 8.6 90.75 mM for hydrolysis and 109 0.97 mM for transphosphatidylation. A calcium-ion concentration of 40 mM was used in the rest of the experiments unless otherwise stated.
3.3. pH and calcium dependence Several authors have reported that the pH optimum of the hydrolysis of PC depends on the concentration of Ca2 + ions. In general, a decrease in calcium concentration results in a shift in the apparent optimum of two units towards higher pH (Heller 1978; Allgyer and Wells, 1979; Abousalham et al., 1993). This pH dependence was investigated for hydrolysis and transphosphatidylation towards lysophosphatidylcholine at three different Ca2 + concentrations — 4, 10, and 40 mM — and the results are illustrated in Fig. 2 for the hydrolytic reaction. Clearly, varying the concentration of calcium ions did not promote a clear shift in the pH optimum for the enzyme; the
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pH optimum was found to be 5.5 in all the cases. At lower and higher pH values, the rates decreased considerably and almost no activity was detected at pH over 7. The activities and the profiles were essentially the same for the transphosphatidylation reaction, in which 2 M glycerol was included in the reaction media (data not shown).
Fig. 2. Effect of pH on the hydrolysis of 5 mM LPC, in 0.1 M Na-acetate buffer (open symbols) or 0.1 M MES buffer (closed symbols), different concentrations of CaCl2 and 0.025 units PL D in a total volume of 0.2 ml. Reaction temperature 25°C. (2, ") 4 mM CaCl2 ( , ) 10 mM CaCl2 and (, ) 40 mM CaCl2.
3.4. Reaction kinetics and Ca 2 + dependence The kinetic parameters for the formation of LPA and LPG were determined at various Ca2 + concentrations. LPC concentration was varied from 0.5 to 8 mM at calcium concentrations of 4, 10, 20, and 40 mM. Glycerol, to a final concentration of 2 M, was added for the transphosphatidylation reaction, which caused hydrolysis to be negligible in the range of conversions measured. To restate, the formation of cyclic LPA was not detected in our system, most probably because of the high concentration of the HCl added to the sample. Thus, all the product formed in the hydrolysis of LPC was detected as LPA. As shown in Fig. 3, the Hanes plots were linear at all calcium concentrations. Table 1 presents the kinetic parameters obtained by fitting the initial rates to the Michaelis– Menten equation by non-linear regression. Km(app) and Vmax(app) increased with Ca2 + concentration both in the hydrolysis and in the transfer reactions. Km(app) varied between 1.139 0.50 and 2.88 90.30 mM, and Vmax(app) was between 0.03690.006 and 0.1029 0.007 (mmol/min/mg protein). However, the specificity constant, that is, Vmax/Km, shows very little variation.
3.5. Kinetics of the transphosphatidylation acti6ity
Fig. 3. Hanes plot for the hydrolysis of lysophosphatidylcholine at different CaCl2 concentrations: () 4 mM; ( ) 10 mM; () 20 mM; ( ) 40 mM. Reaction media composed with different concentrations of LPC in 0.15 M Na-acetate buffer, pH 5.5, 0.025 U of PL D in a final volume of 0.2 ml. Reaction temperature 25°C.
As mentioned earlier, the reaction mechanism could be described as a ping–pong mechanism modified with a hydrolytic branch. However, if the concentrations of LPC and water are kept constant, the velocity of the transphosphatidylation reaction against glycerol concentration can be treated as a single-substrate reaction and will have the typical Michaelis–Menten kinetics profile. The data thus obtained were fitted using non-linear regression (Fig. 4), and the kinetic parameters, Vmax(app) and Km(app), for glycerol were calculated to be 0.13290.036 (mmole/min/ mg protein) and 3889 37 mM, respectively. No comparative data for Km were available for the cabbage enzyme, but Carrea et al. (1997) determined the Km for 3-dimethylamino-1-propanol to be 1.3 M for the Streptomyces sp. enzyme in the transphosphatidylation of PC with the alcohol.
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Table 1 Kinetic parameters for the hydrolysis and transfer activities of PL D towards LPCa CaCl2 (mM)
Km (mM)
Vmax (mmol/min/mg protein)
Vmax/Km (min−1/mg protein)
Hydrolysis
4 10 20 40
1.139 0.50 1.75 9 0.30 2.88 9 0.29 2.449 0.59
0.036 90.006 0.053 9 0.005 0.101 90.006 0.102 90.007
0.1790.01 0.15 9 0.02 0.17 90.01 0.20 90.02
Transphosphatidylation
4 10 20 40
1.39 90.37 1.88 9 0.37 2.689 0.42 2.089 0.17
0.042 90.013 0.067 90.009 0.099 90.008 0.091 90.030
0.17 9 0.03 0.15 90.02 0.17 90.03 0.21 90.03
a Reaction media contained substrate concentration from 0.5 to 8 mM with or without 2 M glycerol in 0.15 M Na-acetate buffer, pH 5.5, CaCl2 concentrations from 4 to 40 mM and 0.025 units PL D in a final volume of 0.2 ml. Reaction temperature 25°C. Data are given as a mean value 9 S.D.
The selectivity of the enzymatic reaction given as the ratio between hydrolysis and transphosphatidylation reaction increases almost linearly as the glycerol/water ratio increases (Fig. 5).
3.6. Detergent effect The inclusion of detergents or solvents for the solubilisation of water-insoluble phosphatidylcholines is a common practice, and the effects are multiple. Diethyl ether and SDS are reported to be the best activators for the enzyme. The solubilisation of the substrate makes it more susceptible to hydrolysis. However, this is not a general rule for all detergents, because others such as CTBA have been shown to inhibit the enzymatic activity (Heller, 1978). Quarles and Dawson (1969) suggested that the positive effect of the amphiphilic anionic compounds as SDS and phosphatidic acid could be due to a neutralisation of the positive charges in the PC. The negatively charged detergent could attract Ca2 + ions to the proximity of the substrate. However, there is no explanation why other anionic substances would not produce the same activation. The effects of SDS, CTAB, and TX-100 concentrations on the activity of the enzyme towards LPC are shown in Fig. 6. There is no increase in activity with any of the detergents tested in the concentration range used; on the contrary, the activity dropped at CTAB and TX-100 concentrations as low as half of the LPC concentration.
Further studies were performed with SDS. Various concentrations of SDS, from 0.5 to 5 mM, were tried at two different Ca2 + concentrations. As seen in Fig. 6(b), SDS seems not to affect the enzymatic activity at concentrations up to 2.5 mM (half the LPC concentration). Higher amounts were shown to lower enzyme activity, but this effect could be due to an interfacial dilution phenomenon (Dennis, 1983). Because the substrate is water soluble, it is unsurprising that the detergent does not affect enzyme kinetics. SDS does not make the enzyme more active at low Ca2 + concentrations (Fig. 6(b)), and the profile of activity versus detergent concentration at 4 mM Ca2 + concentration looks similar to the one for 40 mM.
Fig. 4. Reaction kinetics for the transphosphatidylation of 5 mM LPC and concentrations of glycerol from 50 to 2000 mM, in 0.15 M Na-acetate buffer, pH 5.5, 40 mM CaCl2 and 0.025 units PL D in a total volume of 0.2 ml. Reaction temperature 25°C.
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4. Discussion
Fig. 5. Effect of the molar ratio of glycerol and water on the ratio of hydrolytic and transfer reactions. Reaction media consisted of 5 mM LPC and concentrations of glycerol from 50 to 2000 mM, in 0.15 M Na-acetate buffer, pH 5.5, 40 mM CaCl2 and 0.025 units PL D in a total volume of 0.2 ml. Reaction temperature 25°C.
Fig. 6. (a) Effect of CTAB ( , ) and Triton X-100 (, ) addition on the transphosphatidylation (circles) and hydrolysis (squares) of 5 mM LPC, with or without 2 M glycerol, in 0.15 M Na-acetate buffer, pH 5.5, 40 mM CaCl2 and 0.025 units PL D. Reaction temperature 25°C. (b) Effect of SDS concentration on the transphosphatidylation ( , ) and hydrolysis (2, ") of 5 mM LPC, with or without 2 M glycerol, in 0.15 M Na-acetate buffer, pH 5.5, 4 (open symbols) and 40 mM CaCl2 (closed symbols) and 0.025 units PL D. Reaction temperature 25°C.
The hydrolytic and transphosphatidylation activities of PL D from Savoy cabbage towards LPC are somewhat different from the same activities towards PC. Both phospholipids are substrates for the enzyme, although LPC seems to be poorer. The hydrolytic activity of the enzyme determined in a PC/SDS mixed-micellar system, at a substrate concentration of 1 mM, was about 20 times higher than with the lysophosphatidylcholine. The physical state of the substrates and the nature of the interface at which reaction takes place are known to play a very important role on the activity of phospholipases (Dennis, 1983; Berg et al., 1997; Zhou et al., 1997). Therefore, it is difficult to make a fair comparison between the kinetics of these substrates with PL D, because LPC occurs in micellar form and PC forms bilayers. Micellation of PC can be achieved by incorporating surfactants or with synthetic phospholipids of short fatty-acid chain length. Under the conditions of our test, the comparison of the transphosphatidylation and hydrolysis reactions showed that the kinetic mechanism of both reactions are similar in the formation of the phosphatidyl–enzyme intermediate, as demonstrated for PC previously (Carrea et al., 1997). The kinetic parameters for hydrolysis and transphosphatidylation are very similar in the conditions tested, demonstrating that the formation of a phosphatidyl intermediate is the ratelimiting step. PL D is a metal-dependent enzyme, and maximum activities are achieved when Ca2 + ions are present in the medium. However, the metal requirement of this enzyme has proven to be complicated, and the concentration for optimal enzyme activity was different if the reaction conditions were varied by pH or by the addition of detergents and solvents (Quarles and Dawson, 1969; Allgyer and Wells, 1979; Abousalham et al., 1993). LPC was best transformed in the presence of more than 40 mM Ca2 + , and no detergent or solvents were necessary. No activity was detected in the absence of CaCl2.
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Investigations of the optimum pH for the activity of PL D from cabbage towards PC are somewhat ambiguous. Although Quarles and Dawson (1969) reported shifts of pH optima of up to 1.5 units in ultrasonically treated large PC particles, resulting from the addition of anionic amphipathic substances such as phosphatidic acid or SDS, pH optimum was unaffected by CaCl2 concentration. However, other authors have reported clear pH optimum shifts when Ca2 + ion concentrations were varied (Abousalham et al., 1993; Yamamoto et al., 1993); higher pH optima were calculated with decreasing concentrations of CaCl2. In the activity of PL D towards LPC, the pH optimum was independent of Ca2 + ion concentration. Recent reports have demonstrated that, in plants, PL D occurs in multiple molecular forms associated with various stages of growth and development (Brauer et al., 1991; Dyer et al., 1994; Young et al., 1996; Pappan et al., 1997; Wang, 1997). At least three different forms were identified in castor bean (Dyer et al., 1994) and two forms in maize root (Brauer et al., 1991), rice (Young et al., 1996), and Arabidopsis (Pappan et al., 1997). In castor bean, these forms present significant differences at the molecular level. The molecular weight and isoelectric points were clearly different, as was substrate selectivity. In Arabidopsis and maize root, the two different forms presented different Ca2 + requirements; one functioned at micromolar ion concentrations, but the optimum level for the other was in millimolar concentrations. These two forms also had different pH optima in Arabidopsis. It is likely that the cabbage PL D is expressed in similar multiple forms, although there is no experimental evidence for this. We propose that the different pH optima at various Ca2 + concentrations could be due to the presence of different isoforms with different functional requirements. Thus, at high Ca2 + concentrations the PL D form requiring millimolar ion concentration and acidic pH would be functional, but activities at low Ca2 + concentrations and high pH would be assigned to a PL D isoform with higher Ca2 + affinity (Wang, 1997). Allgyer and Wells (1979) reported an optimum pH of 7.25 for the cabbage enzyme at 0.5 mM
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CaCl2 concentration; at 50 mM, the optimum shifted to 6.25, but a second optimum (with lower activity) remained at pH 7.25. The KCa determined at pH 7 was very low, 0.21 mM. We believe that these different forms of PL D have different substrate selectivity and, thus, the PL D with high Ca2 + affinity and high pH optimum would not be active with lysophospholipids. This would explain the markedly different effect of pH and CaCl2 in substrates as closely related as PC and LPC. Calcium concentration had an effect on the kinetic parameters, with increasing calcium concentrations producing a slight increase in Km(app) and Vmax(app) but unchanged Vmax/Km ratios. The effect of calcium on the Vmax(app) is probably exclusive to the enzyme level, reflected in its low affinity towards Ca2 + and the subsequent high ion-concentration requirement for optimum activity, as discussed above. On the other hand, the effect on Km may be understood, in terms of a change in the structural properties of the substrate due to the interaction with divalent ions. The interaction of ions with phospholipid bilayers is complicated and depends greatly on the physicochemical state of the membrane (Cevc, 1990). Changes in the structure are reported to be due to changes in the membrane hydration and to the formation of intermolecular bridges. In general, membranes, which bind small inorganic ions at their hydration sites, become less hydrophilic and more rigid. In this line, investigations on Ca2 + ion effects on lysophosphatidylcholine foam films (Cohen and Exerowa, 1994) and phosphatidylcholine bilayers (Ohshima et al., 1982) have shown a progressive decrease of the film thickness and the intermembrane separation at increasing Ca2 + concentrations between 1 and 200 mM. Due to the Ca2 + ions, the attractive forces between membranes increase relative to the repulsive forces, which results in more ordered structures. If calcium would affect LPC micelles in the same way, the better packing of the aggregates could make the substrate less susceptible to attack by the enzyme with the subsequent increase in Km. Finally, it was also observed that the enzyme is not activated by SDS, as has been widely reported for the enzyme hydrolysing PC. The change in the
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ionic environment of the substrate due to the extra charges added with the detergent seem to play no important role, at least at the lowest detergent concentrations. Because LPC is water soluble, the addition of detergents is not necessary. In the same way, the addition of diethylether was reported to be inhibiting for the hydrolysis of this substrate due to its insolubility on the solvent (Davidson and Long, 1958).
Acknowledgements This project was supported by the Swedish National Board for Technical and Industrial Development (NUTEK) and the Swedish Research Council for Engineering Sciences (TFR).
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