Effect of the lipid hydrolysis products on the phospholipase A2 action towards lipid monolayer

Chemistry and Physics of ELSEVIER SCIENCE

IRELAND

Chemistry and Physicsof Lipids 70 (1994) 75--81

LIPIDS

Effect of the lipid hydrolysis products on the phospholipase A2 action towards lipid monolayer Vladimir M. Mirsky Institute of Physical and Macromolecular Chemistry, University of Regensburg, 93053 Regensburg, Germany

(Received 15 July 1993; accepted 15 October 1993)

Abstract

The effect of laurie acid (LA) and lysolauroyllecithin (LLL) on the hydrolysis of lipid in monolayer by phospholipase A2 from Bee venom was studied. It was found that LLL inhibits phospholipase action under both high (39 mN/m) and low (25 raN/m) surface pressure. On the other hand, LA inhibits phospholipase action under the low surface pressure (15 mN/m or 25 raN/m), but increases enzyme activity under high surface pressure (39 raN/m). This activating effect can be suppressed by high ionic strength of the aqueous subphase. It is suggested that an increase of the negative surface charge of the lipid monolayer, followed by an increase of the local concentrations of the positively charged enzyme and calcium near the monolayer is a coupling factor between fatty acid accumulation and phospholipase activation. Such an autocatalytie process can only occur when the substrate is organised into monolayer, bilayer or micelles, therefore it can be considered as a reason for the substrate activation and induction time before lipid hydrolysis. Key words: Phospholipase A; Lipid monolayer; Lipolysis products; Phospholipase activation

1. Introduction Phospholipase A 2 is widespread in animals and participates in some physiological and pathological processes. There are two essential kinetic features, observed in lipid hydrolysis by phospholipase: substrate activation (drastic increase of the phospholipase activity after organisation of substrate into micelles, lamellas or liposomes); and a considerable induction time required for reaching the maximal hydrolysis rate [1,2]. Activation of the phospholipase by products was suggested to

explain anomalous kinetics of lipolysis. It was proved, that product accumulation in the lipid bilayer is important for the phospholipase binding [3-71 and leads to the decrease of the induction time [3,4], but a role of this process in the lipolysis activation was questioned [8]. A model of lipolysis, accounting for the effect of activation by products, was postulated [9], and a good fitting of a twodimensional image of lipolytic product distribution observed in data by Grainger et al. [10,11], was obtained. The role of ionic interactions in binding of the enzyme to the substrate interface is

0009-3084/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0009-3084(93)02279-Z

76

now doubtless, however direct electrostatical effects of products on the lipolysis are usually ignored, and product influence on the lipid hydrolysis is considered in terms of defect formation [4,6]. In the present work a product effect on the phospholipase action towards lipid monolayer was studied and electrostatically induced lipolysis activation was evaluated. Such an investigation was made here mainly under very high surface pressure and low phospholipase concentration, when a lipolysis kinetics is very slow, and an enzyme distribution between the bulk solution, the lipid monolayer and the surface of the trough can be considered as quasi-equilibrium. 2. Experimental procedures

All the experiments were performed using Langmuir trough, supplied by the Max-Plank Institute of Biophysical Chemistry (Gottingen, Germany). Surface pressure of the monolayers was measured by the Wilhelmy plate method (filter paper was used as the plate). Lipid monolayers were prepared by spreading of dilauroyllecithin solution in chloroform (1 mg/ml). To obtain a zero order kinetic of the lipolysis, the trough was divided into two compartments, connected with a narrow shallow channel (with a cross-section of about 1 x 1 mm and a length of about 3 mm) [12]. The volume of the reaction compartment (in which the enzyme and CaCl2 were added) was about 40 ml with the surface area of 30 cm 2. The volume of the adjacent compartment was about 60 ml with a surface area of about 150 cm 2. Phospholipase activity was measured as the rate of decrease of the lipid monolayer area under fixed surface pressure [13]. In the typical experiment, CaCl2 was added first, than the monolayer was formed, l0 rain later a surface pressure was fixed and monitored during 20 min, afterwards (if an area of the monolayer was constant) the phospholipase was added. No changes of the monolayer area was observed during the first 10-40 rain after the phospholipase addition, then a decrease of this area was observed (the enzyme concentration was found to cause the area to decrease at a rate of 0.3-0.6 cm2/min). All the measurements were performed at least 1 h after the phospholipase injection or at least 20 min after

V.M. Mirsky / Chem. Phys. Lipid~ 70 (1994) 75-81

the lipolysis product addition, when a steady rate of lipolysis was observed. In the control experiments (without additions of the reaction products), in 30-60 min after the phospholipase addition, a linear kinetic of the monolayer hydrolysis was observed, and by the moment, when a moving barrier reached the reaction compartment, a deviation from the linear kinetic was non-systematical and did not exceed 30%. The reaction products were added from 2 mg/ml solutions in the ethanol. The same quantity of the pure ethanol did not affect the enzyme activity. Monolayer viscosity was measured by means of monitoring of a monolayer diffusion rate through a long narrow channel (0.5 x 30 ram) in the barrier under the gradient of the surface pressure. All the experiments were performed at room temperature (21-23°C). Deionized water was additionally purified by the system 'Millipore-Milli-Q'. We used the solvents and inorganic compounds from 'Merck', dilauroyllecithin and hydrolysis products (LLL and LA) from 'Sigma' and phospholipase A2 (isolated from Bee Venom) from 'Boeringer Mannheim'. 3. Results An addition of LA at surface pressure 25 mN/m results in lipolysis inhibition. This inhibitory effect was slightly decreased under higher calcium concentration (Fig. la) and did not change at lower (16 mN/m) surface pressure (Fig. l b). Quite different dependence of the lipolysis rate on the LA was observed under high surface pressure of the lipid monolayer - - in such conditions an activation effect of the LA additions was observed (Figs. lb-d). In the LA concentration range, corresponding to the 60-80% inhibition of the initial hydrolysis rate under the low surface pressure 0 6 - 2 5 mN/m), the 50-70% acceleration of this reaction occurred under high (39 raN/m) surface pressure (Fig. lb). A further increase of the LA concentration resulted in an inhibition of the reaction. The activating effect of LA was much higher (20-25 times) under low calcium concentration (Fig. lc), but was suppressed by high ionic strength of the aqueous subphase (Fig. ld) - - the

77

V.M. Mirsky / Chem. Phys. Lipids 70 (1994) 75-81 activity

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23 mN/m (a), 39 mN/m (c,d). CaCI2:5 mM (b), 0.25 mM (d). NaCI: 10 mM (a-c). Buffer: 2 mM imidazole (pH 6.64).

Fig. 1. E f f e c t o f the L A o n the p h o s p h o l i p a s ¢ a c t i v i t y t o w a r d s d i l a u r o y l i c c i t h i n m o n o l a y e r . S u r f a c e p r e s s u r e :

activation was only 3 times in the solutions 0.25 mM CaCI, 1 M NaCI and only 1.5 times in the solution of 5 mM CaCI, 10 mM NaCI. An increase of the LA concentration up to 160 mM resulted in an inhibition of the lipid hydrolysis, followed by monolayer destruction. At each stage of these experiments the lipid hydrolysis could be blocked by means of calcium binding with an excess of EDTA. An increase of the lipid monolayer area after LA addition is presented on the Fig. 2.

Addition of LLL resulted in progressive inhibition of the lipid hydrolysis. The inhibitory effect of LLL was promoted by increase in monolayer surface pressure (Fig. 3). No activation of phospholipase by LLL was observed. A calcium effect on the phospholipase activity (Fig. 4) under our conditions (for phospholipase from Bee venom) was very similar to the effect on pancreatic phospholipase, measured on the same system [13]. This dependence was well fitted by

KM. Mirsky / Chem. Phys. Lipids 70 (1994) 75-81

78

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Fig. 2. Increase of the area of dilauroyllecithin monolayer under fixed surface pressure (39 mN/m) after addition of the LA into the aqueous solution (10 mM NaCI, 0.25 mM CaCl 2, 2 mM imidazole, pH 6.64) under the monolayer.

Langmuir adsorption isotherm with total occupation o f the 50% binding sites if calcium concentration was 0.56 mM. To study the possible influence o f lipolysis products on the mechanical properties o f the lipid

Fig. 4. Effect of the CaC12 on the phospholipase activity. Curve i (A): our results (phospholipase from Bee venom, surface pressure 39 mN/m, aqueous solution: l0 mM NaCl, 2 mM imidazole, pH 6.64); curve 2 (O): was recalculated from [13] (pancreatic phospholipase).

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[Lysolauroyllecit hin], juM Fig. 3. Effect of the LLL on the phospholipase activity towards dilauroyllecithin monolayer. Aqueous solution: l0 mM NaCl, 0.25 mM CaCI2, 2 mM imidazole (pH 6.64).

estimates from the data on phospholipase activation (Fig. ld) on the base of a suggested electrostatical mechanism. Curves 2 and 3 (1"1and O) estimate from the data o f LA incorporation into the lipid monolayer under fixed surface pressure (Fig. 4); curve 2 was calculated for total ionization of LA; curve 3 was calculated if pK of the LA was equal to 4.9. Two triangles around the last point of curve 1 were obtained when the phospholipase activation values, varied by a meansquare deviation, were used for calculations of the surface charge.

79

V.M. Mirsky / Chem. Phys. Lipids 70 (1994) 75-81

monolayer, a monolayer viscosity was measured. Only a small effect was observed - - deceleration of the monolayer diffusion rate under maximal (on the Figs. 1 and 2) product concentrations did not exceed 10%. 4. Discu'~ion

The phospholipase inhibition in presence of LLL was observed in the liposome suspension [14]. We have measured the same effect in the lipid monolayer. The inhibition by LLL was more effective under higher surface pressure (Fig. 3), when an incorporation of the LLL into the lipid monolayer was certainly lower. Therefore it is unlikely that the inhibition was caused by the monolayer dilution. Lauric acid addition can lead to the activation as well as the inhibition of the phospholipase A action towards lipid monolayers. The inhibition was a little lower under higher CaCI2. Most probably, the reason of the effect is a shift of local pH near lipid monolayer: optimal pH of this phospholipase is 8.0 [15], negative charge leads to acidic pH shift, therefore an inhibition of this enzyme after fatty acid addition is not surprising. Screening effect of calcium ions suppress this local pH shift and decreases phospholipase inhibition. The fact that the activation by LA could be suppressed by an increased concentration of ions and also that such suppression was more effective by divalent cations than by monovalent, may suggest an electrostatic nature of this phenomenon. Recently we have observed a modification of the electrical field distribution as a result of phospholipase activity [16-18] and proved that fatty acid accumulation in the planar lipid bilayers does not change a dipole potential drop on the membrane/water interface, but changes a surface potential [18]. It is very likely, that such surface potential changes are involved in the phospholipase activation. Three possible mechanisms of this electrostatic activation can be considered: (i) a direct action of the external electric field on the phospholipase turnover; (ii) an increase of the Ca 2+ concentration near the negatively charged lipid-fatty acid monolayer; (iii) an increase of the phospholipase concentration near the monolayer

(an isoelectric point of the phospholipase A is about 10.5 [19], so the enzyme is positively charged at neutral pH). The existence of the first mechanism is not yet generally accepted. Such an effect was probably observed by Thuren et al. [20], but they used a very complicated experimental system, allowing an alternative interpretation. The second and third mechanisms are physical consequences from negatively charged product formation and positive charge of Ca-ion and phospholipase (at neutral pH), therefore these effects nonetheless exist. So we will consider here only a contribution of the second and third mechanisms, i.e. a local increase of the phospholipase and calcium concentrations near the interface. It has been demonstrated by Jain et al. [20], that phospholipase is active in the monomeric form. As is shown in Fig. 4, a dependence of the phospholipase activity on the calcium concentration can be described by Langmuir adsorption isotherm. Therefore: dP = a C c Cca dt Cc, + Cl/2

(1)

where dP/dt is a rate of the products formation, Ce and Cca are the phospholipase concentration on the membrane and calcium concentration near the membrane, CI/2 is a calcium concentration, corresponding to the occupation of 50% Cabinding sites (from Fig. 4 C1/2 = 0.56 raM) and a is a special activity of the enzyme. From the Boltzmann equation for the phospholipase and calcium distribution between aqueous solution and interface it follows that: Ce = fiG,S, exp(-z~I') Cca = fCca.s exp(-2~I')

(2)

where f is the phospholipase adsorption coefficient, Ce. s and CCa,S are the phospholipase and calcium concentrations in the bulk of solution, ~I, is a membrane surface potential in e/kT units (e is the elementary charge, k is the Bolzmann constant and T is absolute temperature) and z is a charge of

V.M. Mirsky / Chem. Phys. Lipids 70 (1994) 75-81

80

the phospholipase in elementary charge units. Let us define an activation 3' as a ratio of the reaction rate to the initial one, then:

3' -

Cca,s + CI/2 Cca,sexp(-2~I') +

Cl/2

sidered as an argument for the suggested 'electrostatical' mechanism of phospholipase activation.

(3)

exp(-z~I, + 2)

According to the Gouy-Chapman equation q = -BX/Cca,s(exp(-21 ') - 1) + C(exp(- ~) - 1) + C(exp(t') - 1)

where q is a surface charge of the lipid monolayer, C is a concentration of the electrolyte and B = 5.861.10 -2 Cul m 2. The numerical solution of the 2 pairs of equations (3) and (4) for 2 values of activation at the different ionic strengths (3' = 23 in 10 mM NaCI and 3' = 3.15 in 1 M NaCI) were found to be: q = - 4 0 mCul/cm, z = +0.6. These two values with data from the Fig. ld and the equation (3) can be used to calculate the dependence of the surface charge on the fatty acid concentration; the result is presented in Fig. 5 (curve 1). This dependence means the surface charge, which should have lipid monolayer to show a phospholipase activity as shown in Fig. ld. The dependence of the monolayer surface charge on the LA concentration can also be calculated from the independent data of LA incorporation into the monolayer under fixed surface pressure (Fig. 2) according to AA e q_ - A + AA A0

(5)

were AA is increase of the monolayer area A after fatty acid addition and A0 is the area of the fatty acid molecule in the monolayer (A0 = 22 A). This dependence is presented in the Fig. 5: curve 2 was calculated for total ionization of LA, curve 3 was calculated if pK of the LA is equal 4.9. The curves 1 and 2 (or 3) were obtained from independent experiments and without free parameters. A qualitative similarity of these curves can be con-

(4)

The phospholipase activation was observed under high surface pressure only. Under the high surface pressure a free energy of the molecule in the monolayer is higher and only a small part of the total phospholipase amount is incorporated into the monolayer. Such dependence was measured for pancreatic phospholipase [22]. Lack of the activation under the low surface pressure can be probably explained by a strong increase of the phospholipase adsorption in such conditions (for the pancreatic phospholipase this shift is about six times for an increase of the surface pressure from 2 mN/cm to 18 mN/cm [22]), so a considerable amount of the enzyme which was adsorbed on the neutral monolayer before accumulation of products and negative surface charge formation can not change essentially this distribution. One can not exclude, however, that fatty acid accumulation leads to an increase of a number of monolayer defects followed by additional phospholipase adsorption on these defects [5-8]. Since only small changes of monolayer viscosity were observed after LA addition, one can consider any essential changes of the monolayer structure as unlikely. Also, if such defects were formed, the same question remains - - how could these defects induce a phospholipase adsorption? An inhibition of phospholipase at high LA concentration at high surface pressure was observed only when the LA concentration was comparable with the calcium one, therefore, this inhibition can be simply caused by calcium laureate formation and subsequent decrease of free calcium ions.

V.M. Mirsky / Chem. Phys. Lipida 70 (1994) 75-81

The calculations, presented in this paper are only simplified estimations. The main simplification is to ignore pH-dependence of phospholipase. Optimal pH for phospholipase A2 from bee venom is 8.0 [15], therefore this effect is opposite to the electrostatical activation. As a result we could underestimate surface charge. Also, for a qualitative description one has to take into account an entropy contribution into the enzyme adsorption [23], specific calcium adsorption, partial solubilization of the lipid monolayer under high LA concentration, lateral LA distribution and an influence of monolayer structure on the phospholipase adsorption energy. But, as we have demonstrated here, even simple electrostatical estimations based on the Gouy-Chapman model allow explanation of many features of the phospholipase behaviour. 5. Acknowledgements Financial support by the Alexander-vonHumboldt Foundation is gratefully acknowledged. I am indebted to Professor Dr. Klaus D. Heckmann for his hospitality and continuing support, to Dr. Valerij Sokolov and Dr. Edmumd Ziomek for fruitful discussions and to Dr. Ortwin Lossen for his help in development and adjustment of experimental devices.

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4 5 6 7 8 9 10 11 12 13 14 15 16

17 18 19

20

6. References 21 1 H. Brockerhoff and R.G. Jensen (1974) Lypolitic Enzymes, Academic Press, New York. 2 R. Verger (1980) Enzyme kinetics of lipolysis. Methods Enzymol. 64, 340-392. 3 M.K. Jain, M.R. Egmond, H.M. Verhej, R. Apitz-Castro,

22 23

R. Dijkmann and G.H. deHaas (1984) Biochim. Biophys. Acta 688, 341-348. M.K. lain and D.V. Jahagirdar (1985) Bichim. Biophys. Acta 814, 313-318. F. Ramirez and M.K. Jain (1991) Proteins: Strnct. Funct. Genet. 9, 229-239. T. Wieioch, B. Borgstrom, G. Pieroni, F. Pattus and R. Verger (1982) J. Biol. Chem. 257, 11523-11528. R. Apitz-Castro, M.K. Jain and G.H. deHaas (1982) Bichim. Biophys. Acta 688, 349-356. D. Lichtenberg, G. Romero, M. Menashe and R.L. Biitonen (1986) J. Biol. Chem. 261, 5334-5340. D.A. Pink, K. Farrell, G. MacNeil and E. Sackmann (1991) Biochim. Biophys. Acta, 1065, 167-176. D.W. Grainger, A. Reichert, H. Ringsdorf and C. Salesse (1989) FEBS Lett. 252, 73-82. D.W. Grainger, A. Reichert, H. Ringsdorf and C. Saiesse (1990) Biochim. Biophys. Acta 1023, 365-379. R. Verger and G.H. deHaas (1973) Chem. Phys. Lipids 10, 127-136. G. Zografi, R. Verger and G.H. deHaas (1971) Chem. Phys. Lipids 7, 185-206. K.M. Conricode and R.S. Ochs (1989) Biochim. Biophys. Acta 1003, 36-43. R.C. Cottrell (1981) Methods Enzymol. 71,698-702. V.M. Mirsky, V.V. Cherny, V.S. Sokolov and V.S. Markin (1991) J. Biochem. Biophys. Methods 21, 277-284. V.V. Cherny, V.M. Mirsky, V.S. Sokolov and V.S. Markin (1989) Bioelectrochem. Bioenerg. 21,373-378. V.V. Cherny, M.G. Sihuralidze, V.M. Mirsky and V.S. Sokolov (1992) Biol. Membr. 9, 733-740. R.A. Shipolini, G.L. Callewaert, R.C. Cotrell, S. Doonan, C.A. Vernon and B.E.C. Banks (1971) Eur. J. Biochem. 20, 9459-9465. T. Thuren, A.-P. Tulkki, J.A. Virtanen and P.K.J. Kinnnnen (1987) Biochemistry 26, 4907-4910. M.K. Jain, G. Ranadive, B.-Z. Yu and H.M Verhej (1991) Biochemistry 30, 7330-7340. J. Rietsch, F. Pattus, P. Desnuelle and R. Verger (1977) J. Biol. Chem. 252, 4313-4318. M. Mosior and S. McLaughlin (1992) Biochim. Biophys. Acta 1105, 185-187.