HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 24, 21231-21236, June 14, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/24/21231    most recent M106449200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by El Kirat, K. Articles by Roux, B. Search for Related Content PubMed PubMed Citation Articles by El Kirat, K. Articles by Roux, B. Social Bookmarking                      What's this?

Role of Calcium and Membrane Organization on Phospholipase D Localization and Activity

COMPETITION BETWEEN A SOLUBLE AND AN INSOLUBLE SUBSTRATE^*

Karim El Kirat^§, Françoise Besson, Annie-France Prigent^¶, Jean-Paul Chauvet, and Bernard Roux

From  Laboratoire de Physico-Chimie Biologique, Unité Mixte de Recherche (UMR) Centre National de la Recherche Scientifique (CNRS) 5013, Bâtiment Chevreul, 43 Boulevard du 11/11/1918, F-69622 Villeurbanne, Université Claude Bernard-Lyon 1, France, ^¶ Laboratoire de Biochimie et Pharmacologie, Institut National de la Santé et de la Recherche Medical U352, Bat. Pasteur, 20 Avenue Albert Einstein, F-69621 Villeurbanne, Institut National des Sciences Appliquées de Lyon, France, and  Laboratoire d'Ingénierie et de Fonctionnalisation des Surfaces, UMR CNRS 5621, 36 Avenue Collongue, F-69131 Ecully, Ecole Centrale de Lyon, France

Received for publication, July 10, 2001, and in revised form, April 3, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The phospholipase D (PLD) from Streptomyces chromofuscus is a soluble enzyme known to be activated by the phosphatidic acid-calcium complexes. PLD-catalyzed hydrolysis of phospholipids in aqueous medium leads to the formation of phosphatidic acid (PA). Previous studies concluded on an allosteric activation of PLD by the PA-calcium complexes. In this work, the role of PA and calcium was investigated in terms of membrane structure and dynamics. The role of calcium in PLD partitioning between the soluble phase and the water-lipid interface was tested. The monomolecular film technique was used to measure both membrane dynamics and PLD activity. These experiments provided information on PLD activity at a water-lipid interface. Moreover, the ability of PA to enhance PLD activity toward phosphatidylcholine was correlated to the physical properties of PA itself, affecting the rheology of the membrane. The effect of calcium was investigated on PLD binding to lipids and on the catalytic process by competition experiments between a soluble and a vesicular substrate. These experiments confirmed the absolute PLD requirement for calcium and pointed out the importance of calcium for PLD catalytic process and for the enzyme location at the water-lipid interface.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phospholipase D (PLD)¹ catalyzes the hydrolysis of the phosphoester bond between the phosphatidyl moiety and the choline headgroup of phosphatidylcholine (PC) liberating choline and phosphatidic acid (PA). This reaction involves a molecule of water for the nucleophile substitution on the phosphatidyl enzyme intermediate to liberate PA, and if the nucleophile is an alcohol, a phosphatidyl alcohol is produced. This latter activity is called transphosphatidylation and is specific for the PLD (1). The hydrolytic activity catalyzed by PLD occurs with the P-O bond cleavage of PC as demonstrated previously (2).

The phospholipase D from Streptomyces chromofuscus belongs to the phospholipase D superfamily as well as some endonucleases, some helicases, some lipid synthases, and a lot of enzymes capable of catalyzing the hydrolysis and/or the formation of phosphodiester bonds (3).

Stieglitz et al. (4) have demonstrated that bacterial PLD is activated by anionic lipids and also that it exhibits a high affinity for these lipids. Recently, the role of bacterial PLD on the aggregation, leakiness, or fusion of vesicles has been demonstrated (5).

The PLD from Streptomyces species has been widely studied, a crystal structure of this protein has been solved recently (6). Until now, its hydrolase activity has been determined by several means (7): by radioactive assay using radiolabeled PC, by pH-stat ¹H NMR (8), by a choline oxidase electrode (9), and by polarization modulation infrared reflection-absorption spectroscopy on lipid monolayers at the air-water interface (10). All of these studies concluded on an activation of PLD by phosphatidic acid and calcium.

The PLD from S. chromofuscus is a soluble enzyme that catalyzes the hydrolysis of the PC contained into macromolecular insoluble structures (i.e. the lipidic vesicles). Therefore, there must be some factors enhancing PLD interaction with such a macrostructural substrate.

In this work, the role of membrane structure on PLD activity was investigated. To study membrane structure, an assay based on the monomolecular film technique was developed. The role of calcium on PLD activity and interactions was also studied. To distinguish the effect of calcium on the lipidic vesicles from their effect on PLD itself, a spectrophotometric assay based on the use of a soluble substrate has been developed. These two methods gave complementary results on the effect of PA and calcium on PLD activity. These results provided information on the influence of membrane structure on PLD activity as well as some information on the enzyme partitioning between the soluble phase of the medium and the water-lipid interface. Some factors responsible for PLD activation or inhibition previously described on liposomes were also tested.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- L--Dipalmitoyl-[2-palmitoyl-9,10-³H(N)]phosphatidylcholine was purchased from PerkinElmer Life Sciences. Coomassie Brilliant Blue R and TLC aluminum sheets Silica Gel 60F₂₅₄ were from Merck (Darmstadt, Germany). L--Phosphatidylinositol (PI) from bovine brain, L--dimyristoylphosphatidylcholine (DMPC), L--dimyristoylphosphatidic acid (DMPA), L--dipalmitoylphosphatidylcholine (DPPC), L--dipalmitoylphosphatidylserine (DPPS), N-octyl--D-glucopyranoside (-OG), Triton X-100, bis(para-nitrophenyl)phosphate (bis(pNP)P), and phospholipase D from S. chromofuscus were purchased from Sigma and used without further purification. The SDS-PAGE analysis of PLD gave the same three bands as those obtained by Geng et al. (11). These proteins have been previously identified by sequence analysis (11) as the intact PLD and its two proteolytically processed fragments.

Monolayer Technique-- All experiments were performed at constant temperature (21 °C). The film balance was built by R&K (Wiesbaden, Germany) and equipped with a Wilhemy-type surface pressure measuring system. The subphases were aqueous buffer solutions containing different concentrations of CaCl₂, 150 mM NaCl, and 10 mM Tris-HCl, pH 8.0. The maximum PLD activity has been described previously for 20 µM calcium (9). Therefore, PLD activity was assayed between 2 and 65 µM calcium.

Phospholipids were spread at the air-water interface in hexane/ethanol (9:1 (v/v)) at 0.175 mM to reach a final quantity of 8.75 nmol of lipids. After 15 min, the monolayer was compressed to a lateral pressure of 35 mN·m¹ to obtain a control isotherm. The pressure then was fixed at 30 mN·m¹, and the enzyme (15 µg of protein) was injected in the subphase after the monolayer stabilization. The subphase was stirred with one magnetic stirrer spinning at 100 rpm. Surface elasticity moduli were calculated from the pressure-area data obtained from the monolayer compressions using the following equation (12)

(Eq. 1)

where A is the molecular area at the indicated surface pressure  and high K_s values correspond to low interfacial elasticity among packed lipids forming a monolayer (13). This finding suggests that the higher the K_s value of a monolayer is, the more difficult it is to deform it.

Spectrophotometric Assay-- The bis(pNP)P was solubilized in various concentrations of CaCl₂, 150 mM NaCl, and 10 mM Tris-HCl, pH 8.0. The reaction was monitored with an Uvikon spectrophotometer thermostated at a constant temperature (37 °C) at 420 nm. The reaction time was <1 min. Under these conditions, the molar absorption factor of the para-nitrophenol released by PLD activity on bis(pNP)P was 18,500 M¹·cm¹ at 420 nm. The substrate was first incubated at 37 °C to test its stability, and then the reaction was initiated by the addition of PLD.

Vesicle Preparation-- The lipids (i.e. DMPC and DMPA or DPPS or PI) were dissolved in hexane/ethanol (9:1 (v/v)) at 5 mM. The lipid mixture containing varying molar ratio of DMPA was dried under nitrogen for 2 h. The lipidic film then was resuspended by vigorous agitation within 15 min in 65 µM CaCl₂, 150 mM NaCl, and 100 mM Tris-HCl, pH 8.0, to reach a final concentration of total lipid of 5 mM. The lipid suspension was heated at 60 °C in a thermostated bath and submitted to vigorous agitation for 15 min to obtain the multilamellar vesicles. Vesicles solubilization was performed in 10 mM Tris, 150 mM NaCl, and 65 µM CaCl₂ containing 2.5 mM bis(pNP)P) with successive additions of -OG at 37 °C and monitored as a decrease of the turbidity at 450 nm.

Radioactive Assay-- PLD activity on lipidic bilayers was determined with radiolabeled vesicles of DMPC/DMPA (50:50 (mol/mol)) and 20 µCi of [³H]DPPC. The multilamellar vesicles were prepared exactly the same way as described above with the same final concentration.

To measure PLD activity, the vesicles corresponding to 2 µCi were incubated with PLD at 37 °C in a 200-µl final volume. The reaction was stopped with 2 ml of chloroform/methanol/HCl (0.1 M) (1:1:0.002 (v/v/v)) and 1 ml of HCl (1 M) containing 5 mM EDTA. The tubes were subjected to vigorous agitation and then centrifuged to 400 × g for 5 min at 4 °C. The organic phases containing the lipids were then deposited on TLC plate with DMPC and DMPA standards. After development in ethyl acetate/isooctane/acetic acid (90:50:20 (v/v/v)), the plate was revealed with Coomassie Brilliant Blue R (14). The spots were then scraped off and counted for radioactivity determination (Wallac Winspectral^TM 1414 Liquid Scintillation Counter, Turku, Finland).

Calcium Content Determination-- Plasma emission spectroscopy (Service Central d'Analyze, CNRS, Vernaison, France) was used to determine calcium content in buffers.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Properties of the DMPC/DMPA Monolayers-- The mixtures containing increasing molar ratio of DMPA over DMPC were spread at the air-water interface. The subphase was always the same with 65 µM CaCl₂ and was thermostated at 21 °C. The influence of DMPA content of the monolayer on the apparent molecular area was first tested. The pressure-area isotherms (Fig. 1) show that the increasing DMPA content in the DMPC monolayer decreased the apparent molecular area. These isotherms were useful to estimate the molecular area, at zero surface pressure, of DMPC (80 A²/molecule) (Fig. 1, isotherm A) and of DMPA alone (40 A²/molecule) (Fig. 1, isotherm F). These values are in agreement with those determined in previous studies (15).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Pressure-area isotherms of DMPC monolayers containing increasing molar percentage of DMPA. DMPA molar ratios in DMPC monolayer were 0 (A), 10 (B), 15 (C), 20 (D), 50 (E), and 100% (F). The subphase was 65 µM CaCl2, 150 mM NaCl, and 10 mM Tris, pH 8.0 at 21 °C.

In addition, the surface elasticity modulus was calculated for all the monolayers before injection of PLD at various DMPA molar percentages (Fig. 2, open circles, dashed line). This result indicates that an increasing molar ratio of DMPA in the monomolecular film of DMPC decreases its K_s value. This means that the more the monolayer contains DMPA, the more it can be deformed.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Influence of DMPA molar percentage in the monomolecular film of DMPC on the surface elasticity modulus of the monolayer and on the lag time before the PLD begins the reaction. The surface elasticity modulus of the monolayers was calculated for 30 mN·m1 surface pressure (open circles, dashed line). The lag time was determined for all the monolayers as the time preceding a 5% decrease in the apparent molecular area (closed circles, solid line). The subphase was 65 µM CaCl2, 150 mM NaCl, and 10 mM Tris, pH 8.0, at 21 °C. One unit corresponds to 61 min for the lag time and 100 mN·m1 for the surface elasticity modulus.

Influence of DMPA on PLD Activity toward DMPC Monolayer-- According to the decrease of the molecular area of the monolayer of DMPC with growing molar percentage of DMPA, if the PLD hydrolyzes DMPC into DMPA, it will lead to a decrease of the apparent molecular area at a constant surface pressure. After the stabilization of the monolayer at 30 mN·m¹ (known to be the internal pressure of biological membranes), the PLD was injected in the subphase buffer. For the monomolecular film of DMPC alone, after injection of PLD, the apparent molecular area slowly decreased. The reaction was monitored within 10 h, and then the monolayer was compressed. The molecular area finally reached 40 Å²/molecule, which is the DMPA molecular area. This experiment showed that the activity of PLD can be monitored as the decrease in apparent molecular area with the time at the fixed surface pressure.

As shown in Fig. 3, the increasing concentration of DMPA in the monolayer decreased the observed lag time (defined as the time preceding a 5% decrease in the apparent molecular area). This resulted also in a significant increase of the velocity of DMPC hydrolysis by PLD with the rising DMPA content of the monolayer. This finding is consistent with the findings of Geng et al. (8) who measured PLD activity with ¹H NMR and described a phosphatidic acid-induced enhancement of its hydrolytic activity and a decrease of the lag time. Moreover, the representation of the lag time as a function of DMPA molar percentage in the monolayer (Fig. 2, closed circles, solid line) showed a strong relationship with the surface elasticity modulus. Both curves present the same profile, the increasing molar content of DMPA decreased the lag time and the surface elasticity modulus in the same way. At high DMPA content, the monolayer was more sensitive to deformation and the lag time before the PLD activity was considerably reduced. PLD activity was also tested twice on a DMPC/DPPC (50:50 (mol/mol)) monolayer. At 30 mN·m¹ surface pressure, this monolayer had a surface elasticity modulus of ~55 mN·m¹, and after PLD injection, the observed lag time was 12 ± 2 min (data not shown). This monolayer has the same surface elasticity modulus and the same lag time than the DMPC/DMPA (85:15 (mol/mol)) monolayer at 30 mN·m¹ surface pressure. However, the velocity of PLD reaction for the DPPC-containing monolayer was approximately two times less than that for the 15 molar percent DMPA-containing monolayer. This velocity increased while PA was produced by PLD in the monolayer. This observation indicates that the same low surface elasticity modulus of these two monomolecular films could allow the PLD to create local deformations in the lipidic monolayer. These deformations seemed to be important for the beginning of the PLD-catalyzed hydrolysis of PC but had no effect on the velocity of the reaction itself.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Influence of DMPA molar percentage in the monomolecular film of DMPC on PLD activity. The apparent molecular area has been normalized to compare the different reactions at 30 mN·m1. DMPA molar ratios in DMPC monolayer were 0 (A), 5 (B), 10 (C), 15 (D), 30 (E), and 50% (F). The subphase was 65 µM CaCl2, 150 mM NaCl, and 10 mM Tris, pH 8.0, and the temperature was fixed at 21 °C.

Role of Calcium on PLD Activity toward DPMC/DMPA Monolayers-- The role of calcium on PLD activity was determined with the monomolecular film technique at a constant surface pressure (30 mN·m¹). For this monolayer assay, the molar ratio of DMPA was fixed to 50%, because the reaction velocity was maximal under these conditions. The calcium concentration varied from 2 to 65 µM in the subphase. In the absence of enzyme, the increasing concentration of calcium had no effect on the isotherm of the DMPC/DMPA (50:50 (mol/mol)) monolayer (data not shown). After stabilization of the monolayer at 30 mN·m¹ and injection of the PLD in the subphase, the velocity of the apparent molecular area decrease was calculated. The influence of calcium on this velocity is presented in Fig. 4, open circles, dashed lines. The velocity increased with rising concentrations of calcium. A saturation of this activity was reached at 20 µM calcium. Moreover, the calcium concentration in the subphase did not significantly affect the lag time preceding PLD-catalyzed hydrolysis of DMPC (data not shown). Therefore, it seems that the lag time observed for different DMPC/DMPA ratio (Fig. 2) is only correlated to the decrease of the surface elasticity modulus with rising DMPA monolayer content.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Role of calcium on PLD activity. The influence of calcium was measured on PLD activity toward the bis(pNP)P (closed circles, solid line) and toward the DMPC/DMPA (50:50 (mol/mol)) monolayer (open circles, dashed line). For the monomolecular film assay, PLD activity was expressed as the velocity of the apparent molecular area decrease. For the monolayer assays, the subphase was 150 mM NaCl and 10 mM Tris, pH 8.0, containing various concentrations of CaCl2 at 21 °C. The curves were determined from the Hanes-Woolf linearization of the experimental data.

Role of Other Anionic Lipids on PLD Activity toward DMPC in Monolayers-- To compare the effect of phosphatidic acid with other anionic lipids, monomolecular films containing PI or DPPS mixed with DMPC were tested. At 30 mN·m¹ surface pressure, the mixtures of DMPC/PI (50:50 (mol/mol)) and DMPC/DPPS (70:30 (mol/mol)) revealed a surface elasticity modulus of 80 and 30 mN·m¹, respectively. The results obtained for the PLD-catalyzed hydrolysis of those two monolayers (Fig. 5I, curves E and D) show that the PI-containing monolayer is less affected by PLD activity than the DPPS-containing monolayer. The lag times determined for these two monolayers were 60 min for DMPC/PI (50:50 (mol/mol)) and 20 min for the DMPC/DPPS (70:30 (mol/mol)) monolayer.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Influence of anionic lipids on PLD activity toward a DMPC monolayer. I, PLD activity was assayed at 30 mN·m1 with the anionic lipids either alone or in mixture with DMPC. A, DMPC; B, DPPS; C, PI; D, DMPC/DPPS (70:30 (mol/mol)); and E, DMPC/PI (50:50 (mol/mol)). II, the phospholipids were also tested alone at 10 mN·m1 to allow their PLD-catalyzed hydrolysis. A, DMPC; B, DPPS; and C, PI. The apparent molecular area has been normalized to allow the comparison of the different reactions. The subphase buffer was 65 µM CaCl2, 150 mM NaCl, and 10 mM Tris, pH 8.0, at 21 °C.

PLD activity was also measured at 30 mN·m¹ on monolayers containing only one phospholipid (DMPC, DPPS, or PI) (Fig. 5I, curves A, B, and C, respectively). DPPS seemed to be easily hydrolyzed by PLD in comparison with DMPC and PI. Their K_s were slightly different at 30 mN·m¹ surface pressure, 100 mN·m¹ for DMPC, 70 mN·m¹ for DPPS, and 80 mN·m¹ for PI. These results could explain why DPPS was rapidly hydrolyzed by PLD at 30 mN·m¹. However, without information on the affinity of PLD toward each of these lipids, it is difficult to compare their hydrolyses.

PLD activity was also measured toward DMPC, DPPS, and PI alone at low surface pressure (10 mN·m¹) to test their hydrolyses (Fig. 5II, curves A, B, and C, respectively). Under these conditions, PI was found to be a substrate for PLD. At 10 mN·m¹ surface pressure, both DMPC and PI monolayers have a surface elasticity modulus of ~45 mN·m¹, and the DPPS monolayer has a K_s of 30 mN·m¹. The lag times were low: 10 min for DMPC, 7 min for DPPS, and 34 min for PI. Moreover, PI alone was still hydrolyzed at 30 mN·m¹ but at a lower extent than for 10 mN·m¹ surface pressure (Fig. 5I, curve C). PLD activity was also tested on vesicles containing either PC or PI alone at 5 mM (calcium concentration was 1 mM). After 30-min incubation at 37 °C, TLC analysis of lipids revealed that PLD activity on both types of vesicles generates PA. This finding demonstrates that PI hydrolysis in monolayers was because of PLD activity. These results show that PI, DPPS, and DMPC are substrates for PLD (as seen at 10 mN·m¹ surface pressure), whereas DMPA is not. Therefore, it is difficult to investigate the role of each lipid on the PLD-catalyzed hydrolysis of DMPC in mixed monolayers.

Determination of the Kinetic Parameters for the PLD-catalyzed Hydrolysis of bis(pNP)P-- To measure PLD activity in an homogeneous medium, a spectrophotometric-based assay using bis(pNP)P was developed. The kinetic parameters of PLD toward this soluble substrate were determined at pH 8.0 with 65 µM calcium. These experiments were repeated four times with bis(pNP)P concentrations used from 0.01 to 5 mM. The linearizations of these results gave a V_max of ~131.5 ± 0.3 µmol·min¹·mg¹ for the PLD-catalyzed hydrolysis of bis(pNP)P. The Michaelis-Menten constant for S. chromofuscus PLD toward the bis(pNP)P was also determined, K_m of 0.6 ± 0.1 mM. This K_m is identical to that determined for S. chromofuscus PLD activity toward liposoluble PC (16) and toward soluble dibutyrylphosphatidylcholine (11).

Role of Calcium on PLD Activity toward the bis(pNP)P-- In this case, the system is totally free from liposomes and macrostructural substrate, and the calcium could interact with the phosphate group of the bis(pNP)P. The effect of calcium was measured from 2 to 65 µM on PLD activity toward bis(pNP)P (Fig. 4, closed circles, solid line). Compared with the measurement of calcium effect on monolayer, an identical profile with the same saturation of PLD activity at 20 µM calcium was obtained. PLD dependence upon Ca²⁺ under other conditions (with small unilamellar PC vesicles) with the choline oxidase electrode has been determined previously (9), and their results are consistent with ours. Consequently, calcium is important for PLD activity independent of the nature or the concentration of the substrate. This finding suggests that PLD activity dependence upon calcium is strongly related to the enzyme itself, and it should be attributed to an involvement of this divalent cation in the catalytic process. The apparent dissociation constant, K_D, for the calcium was found to be 10 ± 1.5 µM.

Competition between Insoluble and Soluble Substrate for PLD-- In these experiments, the PLD activity in the soluble phase of the medium was assayed with the soluble substrate bis(pNP)P in the presence of vesicles (Fig. 6, closed circles, solid line). To identify the factors that enhance PLD location at the lipid-water interface (i.e. vesicles or monolayers), the competition between vesicles and bis(pNP)P was tested under different concentrations of calcium. As a consequence, when the PLD interacts with the vesicles to catalyze the hydrolysis of DMPC into DMPA, its activity toward the bis(pNP)P will be reduced.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Role of anionic lipids and calcium on the behavior of PLD in a heterogeneous medium. The vesicles were made of DMPC mixed with DMPA (circles), DPPS (triangles), or PI (crosses) at different molar percentages. The concentration for all of the assays was 0.025 mM lipids. Two concentrations of calcium were tested, 65 µM (closed symbols, solid lines) and 3 mM (open symbols, dashed lines). Buffer composition was 150 mM NaCl, 10 mM Tris, pH 8.0, the concentration of bis(pNP)P was kept constant in all experiments (2.5 mM), and temperature was fixed at 37 °C.

To determine the amount of DMPA generated after a 1-min incubation with 0.3 mM DMPC/DMPA (50:50 (mol/mol)), PLD activity was assayed on vesicles with the radiolabeled PC at 37 °C. Under these conditions, 15% DMPC was converted into DMPA. If the vesicles are ten times less concentrated (0.025 mM) according to the Michaelis-Menten equation, the percentage of hydrolyzed DMPC should be negligible.

In these competition experiments, the vesicles of DMPC mixed with DMPA, PI, or DPPS were first incubated at 37 °C with bis(pNP)P at various concentrations of calcium. The PLD then was added to the mixture, and its activity toward the bis(pNP)P was measured for <1 min. For the two calcium concentrations tested (65 µM and 3 mM), PI had no effect on PLD activity toward bis(pNP)P (Fig. 6, crosses, solid line for 65 µM calcium and dashed line for 3 mM). At low calcium concentration, the activity of PLD on bis(pNP)P decreased with increasing DMPA and DPPS content (Fig. 6, closed circles for DMPA and closed triangles for DPPS, solid lines), and this decrease was maximal for 50% DMPA and 50% DPPS vesicles, a 50% loss of activity toward bis(pNP)P. This important decrease of PLD activity toward the bis(pNP)P to the benefit of vesicles is surprising. Considering the concentration of the soluble substrate (2.5 mM), which is one hundred times higher than the lipid concentration (0.025 mM), the probability for the PLD to meet the bis(pNP)P is greater than the probability for the vesicles. This finding indicates that there must be a factor that enhances PLD interaction with the vesicles.

The role of calcium in this inhibition has been tested (Fig. 6, open circles for DMPA and open triangles for DPPS, dashed lines). The vesicles of DMPC mixed with DMPA or DPPS were incubated with 3 mM calcium at 37 °C. Under these conditions, there was no decrease in PLD activity on bis(pNP)P in the presence of vesicles at any concentration from 0.025 to 0.3 mM lipids) (data not shown). This finding indicates that a large part of the enzyme interacts with the soluble substrate but not with liposomes. It seems that for 65 µM calcium, the divalent cation interacts with the vesicles, and this interaction is more efficient when the DMPA (or DPPS) ratio is increased up to 50%. This observation suggests that DMPC/DMPA and DMPC/DPPS vesicles could remove the calcium from the soluble fraction of the medium, reducing its quantity available for PLD-catalyzed hydrolysis of bis(pNP)P. These results are consistent with those obtained on vesicle binding studies of PLD reported previously (4).

Role of Detergents on PLD Activity Measured with bis(pNP)P-- The influence of a detergent, the -OG, was also tested on the competition between vesicles and bis(pNP)P for PLD activity. In this assay, 0.3 mM vesicles of DMPC/DMPA (50:50 (mol/mol)) and DMPC/DPPS (50:50 (mol/mol)) were first incubated with bis(pNP)P (2.5 mM) at 37 °C in the presence of low calcium concentration (65 µM) to allow competition. The solubilization (Fig. 7, open circles, dashed line) of these vesicles by -OG occurred at low concentrations of detergent and was totally accomplished at 6 mM. In parallel, the PLD activity on bis(pNP)P was measured (Fig. 7, closed circles for DMPA and closed triangles for DPPS, solid lines). A total inhibition of PLD activity toward the bis(pNP)P was observed in the absence of detergent for DMPA-containing vesicles, whereas only a 80% inhibition was found for the DPPS-containing vesicles. However, for the two types of vesicles, an enhancement of PLD activity occurred with increasing -OG concentrations. This finding indicates that the progressive solubilization of the vesicles (DMPA- and DPPS-containing ones) (Fig. 7, open circles and triangles, respectively, dashed lines) seems to liberate the enzyme that was first associated to the lipids. The same experiment was performed on vesicles containing only DMPC (data not shown), but in this case, the PLD was not sequestrated by the lipids, and the solubilization did not modify its activity toward the soluble substrate bis(pNP)P. This observation is consistent with previous findings where millimolar concentrations of divalent cations are needed to drive the PLD at the PC vesicle surface (4).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of -OG on the interaction of PLD with DMPC vesicles containing DMPA or DPPS in 50:50 molar ratio at low calcium concentration. Two parameters were measured on DMPA (circles) and DPPS (triangles) containing vesicles. The solubilization of the vesicles was measured by turbidity at 450 nm (open symbols, dashed lines), and PLD activity on bis(pNP)P was measured at 420 nm (closed symbols, solid lines). The measurements were done at 37 °C with 0.3 mM vesicles in 65 µM CaCl2, 150 mM NaCl, 10 mM Tris, pH 8.0, and 2.5 mM bis(pNP)P.

The influence of -OG and Triton X-100 on PLD activity assayed with bis(pNP)P alone has also been investigated (from 0 to 60 mM for -OG and from 0 to 10 mM Triton X-100). Under these conditions, the data on the effect of detergents on the PLD itself were obtained. None of the two tested detergents had any effect on the hydrolysis of the bis(pNP)P by the enzyme (data not shown), whereas PLD activity toward monomeric dibutyrylphosphatidylcholine has been shown to be increased by ~3.75-fold with 40 mM -OG and by 2-fold with 10 mM Triton X-100 (11, 17). The explanation for the enhancement described on dibutyrylphosphatidylcholine could be the interaction of the monomeric PC with detergents that could not occur with the soluble substrate bis(pNP)P.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Role of the Membrane Structure on PLD Activity-- The monolayer technique is a powerful method for assaying the PLD-catalyzed hydrolysis of various lipids. This method requires only small amounts of substrate and provides information on the structure of the macromolecular substrate.

The measurements of PLD activity on monomolecular films of DMPC/DMPA at various molar ratios suggested that PLD is activated by DMPA, inducing a decrease of the lag time that occurs before DMPC hydrolysis and leading to an increase of the velocity of this hydrolysis. This latter effect might be attributed to the lateral segregation of DMPA induced by calcium (18). The surface elasticity modulus of the monolayers at 30 mN·m¹ decreased with increasing DMPA content. The representation of the lag time of the reaction as a function of DMPA molar ratio in the monolayer gave the same profile as the one for the surface elasticity modulus. This finding indicates that there is a relationship between the membrane dynamic and the lag time before the onset of PLD activity. If the monolayer is deformed, the PLD will have no difficulty in reaching the phosphate site of DMPC and will catalyze its conversion into DMPA. The increasing DMPA content in the monolayer attributed to PLD hydrolysis of DMPC will enhance this deformity, and as a consequence, PLD activity also will be enhanced. PLD activity was also tested on a DMPC/DPPC (50:50 (mol/mol)) monolayer. This monolayer had a K_s similar to that of the DMPC/DMPA (85:15 (mol/mol)) monolayer (55 mN·m¹). The PLD-catalyzed hydrolysis of those two monomolecular films was identical in terms of lag time. However, the 15% DMPA-containing monolayer was hydrolyzed two times faster than the 50% DPPC-containing monolayer. Therefore, the lag time seems to be independent of the presence of anionic lipids in the monolayer. Recently, Stieglitz et al. (5) have described the insertion of PLD into lipidic vesicles leading to leakiness. This insertion of PLD could be favored by the high deformity induced by phosphatidic acid.

Role of Calcium on PLD Location in a Heterogeneous Medium-- The PLD from S. chromofuscus is water-soluble, whereas its natural substrate is not and forms macromolecular associations. Hence, the PLD activity takes place in a heterogeneous medium, and some factors could enhance the enzyme removal from the soluble part of the medium (i.e. aqueous solution) to the particular part (i.e. the lipidic vesicles or monolayer).

A number of protocols have been developed to measure PLD activity (7, 19), but there are no assays based on the hydrolysis of a soluble substrate. The bis(pNP)P was useful in characterizing some of the factors responsible for the location of PLD at the surface of the vesicle. PLD-catalyzed hydrolysis of this soluble substrate showed the same dependence on calcium as PC (9). The K_m values of PLD for PC and bis(pNP)P also were similar (11, 16). The results obtained for competition between the lipidic vesicles and the soluble substrate indicate that calcium plays a key role in PLD partitioning between the monomeric and the macromolecular substrate. The PI-containing vesicles had no effect on PLD activity toward the bis(pNP)P. When the vesicles contained DMPA or DPPS, there was a competition between the activity on lipids and the activity on bis(pNP)P. Only a small amount of DMPA was necessary to obtain inhibition, whereas the molar percentage of DPPS needed to obtain the competition was in a higher range (of >20 molar percent). This competition was dependent on calcium concentration, which is consistent with the findings of Stieglitz et al. (4). For the lowest concentration of calcium (65 µM) and the highest concentration of vesicles (0.3 mM), the divalent cation content was not sufficient to interact with both the lipids and the soluble substrate. As a consequence, all of the calcium seemed to interact with the DMPA or DPPS-enriched vesicles, leading to a loss of PLD activity on bis(pNP)P. The excess of calcium in the medium in regard to the DMPA concentration induced no inhibition of the bis(pNP)P hydrolysis, indicating that PLD was located where it could find calcium ions. Thus, this protein should be activated by the substrate-calcium complex. This means that it is not the calcium-protein interaction that provokes the translocation of S. chromofuscus PLD from the soluble phase of the medium to the lipid-water interface. Furthermore, the gradual solubilization of DMPC/DMPA (or DPPS) (50:50 (mol/mol)) vesicles with -OG induced a gradual recovery of PLD activity on the soluble substrate. This finding indicates that the solubilization of anionic lipid-calcium domains liberates the PLD from the vesicles. Therefore, the anionic lipid (i.e. DMPA or DPPS) interaction with calcium is necessary to enhance PLD activity toward the vesicles.

Role of Calcium on PLD Activity-- It is possible to consider the calcium as an assistant of the nucleophile substrate for PLD in phosphodiester-bound hydrolysis. The dependence of PLD activity upon calcium concentration can be described by the Michaelis-Menten equation. The same saturation was observed for different assays involving different substrates (bis(pNP)P and PC) at different concentrations (for review on PC vesicles see, Ref. 9). Therefore, the saturation attributed to the divalent cation is independent of the amount and the nature of the substrate. As a consequence, to reach the 20 µM calcium saturation of the activity, there must be a limiting step in the catalytic process that could be the formation of the enzyme-substrate covalent intermediate (20). This result shows that calcium should play a key role in the phosphatidyl enzyme geometry for the nucleophilic attack of water on the P-O bond.

As a conclusion, the effect of various lipids on PLD activity should be investigated in terms of affinity for these lipids, interaction between ions and lipids, and membrane dynamic and structure induced by these lipids (21, 22). Our results show the great importance of the physical state of membranes on PLD activity. Moreover, the study of membrane rheology should lead to a better understanding of PLD catalytic mechanism and affinities. Another important point is the possibility for PLD location and activity to be modulated by physiological (i.e. micromolar) concentrations of calcium. This divalent cation is known to be cytotoxic at submicromolar concentrations, which suggest that PLD requirements for calcium should be in the same range.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel: 33-4-72431542; Fax: 33-4-72431543; E-mail: elkirat@univ-lyon1.fr.

Published, JBC Papers in Press, April 8, 2002, DOI 10.1074/jbc.M106449200

	ABBREVIATIONS

The abbreviations used are: PLD, phospholipase D; PC, phosphatidylcholine; PA, phosphatidic acid; PI, phosphatidylinositol; DMPC, L--dimyristoylphosphatidylcholine; DMPA, L--dimyristoylphosphatidic acid; DPPC, L--dipalmitoylphosphatidylcholine; DPPS, L--dipalmitoylphosphatidylserine; -OG, N-octyl--D-glucopyranoside; bis(pNP)P, bis(para-nitrophenyl)phosphate; K_s, surface elasticity modulus; mN, millinewton.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				K. Wagner and G. Brezesinski Phospholipase D Activity Is Regulated by Product Segregation and the Structure Formation of Phosphatidic Acid within Model Membranes Biophys. J., October 1, 2007; 93(7): 2373 - 2383. [Abstract] [Full Text] [PDF]

				H. Yang and M. F. Roberts Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: Covalent versus noncovalent catalysis Protein Sci., September 1, 2003; 12(9): 2087 - 2098. [Abstract] [Full Text] [PDF]

				C. Zambonelli and M. F. Roberts An Iron-dependent Bacterial Phospholipase D Reminiscent of Purple Acid Phosphatases J. Biol. Chem., April 11, 2003; 278(16): 13706 - 13711. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/24/21231    most recent M106449200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by El Kirat, K. Articles by Roux, B. Search for Related Content PubMed PubMed Citation Articles by El Kirat, K. Articles by Roux, B. Social Bookmarking                      What's this?