Activation of Phospholipase D by Phosphatidic Acid
ENHANCED VESICLE BINDING, PHOSPHATIDIC ACID-Ca2+ INTERACTION, OR AN ALLOSTERIC EFFECT?*

Dong Geng, Justin Chura, and Mary F. RobertsDagger

From the Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The activity of bacterial phospholipase D (PLD), a Ca2+-dependent enzyme, toward phosphatidylcholine bilayers was enhanced 7-fold by incorporation of 10 mol % phosphatidic acid (PA) in the vesicle bilayer. Addition of other negatively charged lipids such as phosphatidylinositol, phosphatidylmethanol, and oleic acid either inhibited or had no effect on enzyme activity. Only negatively charged lipids with a free phosphate group, phosphatidylinositol 4-phosphate and lyso-PA, had the same effect as PA on enzyme activity. Changes in vesicle curvature and fusion were not the reason for PA activation; rather, a metal ion-induced lateral segregation of PA in the vesicle bilayer correlated with PLD activation. Significant PA activation was also observed with monomer phosphatidylcholine substrate upon the addition of PA vesicles. The PA activation was caused by Ca2+·PA interacting with PLD at an allosteric site other than active site.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Phospholipase D (PLD)1 enzymes cleave the distal phosphodiester bond of phospholipids generating phosphatidic acid (PA) and a free base (1). In addition to hydrolytic activity, PLD enzymes also catalyze a transphosphatidylation reaction in the presence of a high concentration of primary alcohol (2, 3). This reaction, which is consistent with a phosphoryl-enzyme intermediate (4), has been used to monitor the presence of PLD in a variety of cells. PLD activities, observed in both membrane and cytosolic fractions of mammalian cells, play key roles in membrane trafficking and regulation of mitosis as well as signal transduction (5). The lipophilic product of PLD cleavage, PA, and its PLA2 degradation product, lyso-PA, are second messengers and have activation roles in a wide variety of cells (6-9). Lyso-PA appears to be the more potent lipid mediator (10); concentrations in the nM range elicit diverse biological actions, e.g. activation of DNA synthesis. PA can also be converted to a nonsignaling lipid via the PLD transferase activity and DAG to produce bis-PA (11, 12). In mammalian cells, basal PLD activity is low, although it can be activated very rapidly. Given its physiological importance, determining the factors that activate or inhibit PLD is of considerable interest.

PLD enzymes are also present in plants and various microorganisms. Many of these enzymes have been purified and well characterized kinetically. Plant and bacterial PLD enzymes share a number of kinetic characteristics with the recently purified mammalian PLD isozymes and so may be reasonable models for the latter enzymes. PLD from Streptomyces chromofuscus is a water-soluble enzyme purified from the culture supernatant (13). It can mimic the effect of endogenous PLD when added to a variety of mammalian cells. For example, the addition of exogenous S. chromofuscus PLD induces an activity similar to that of endogenous PLD in ovarian granulosa cell culture (14). Addition of bacterial PLD to the medium of vascular smooth muscle cells induces DNA synthesis along with formation of choline and PA (15). With this in mind, we have examined the effect of the lipophilic PLD product PA on PLD from S. chromofuscus. The inclusion of PA in a phosphatidylcholine (PC) bilayer, rather than inhibiting the enzyme (as might be expected for simple product inhibition), activates the enzyme allosterically and enhances activity significantly. This type of interaction may be relevant to signal transduction because phosphatidylinositol monophosphate (PIP) (and presumably phosphatidylinositol bisphosphate (PIP2)) can also activate the enzyme.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals-- Phospholipids including POPC, dimyristoyl-PC, diC4PC, POPA, LPA, PMe, PI, and oleic acid were purchased from Avanti in chloroform solutions and used without further purification. Triton X-100 and beta -octyl glucoside were obtained from Sigma. Pyrene-labeled phospholipids (C16pyr-COOH, C16C6pyr-PC, and C16C6pyr-PMe) were purchased from Molecular Probes.

PLD Purification-- PLD from S. chromofuscus, obtained form Sigma, gave rise to three major bands in SDS-polyacrylamide gel electrophoresis analysis. The enzyme was purified further with the following steps. The commercially available enzyme was dissolved in 1 M (NH4)2SO4 and 50 mM phosphate buffer, pH 7.0, and loaded onto a Hitrap HIC column (Amersham Pharmacia Biotech) preequilibrated with the same buffer. The column was then eluted with a (NH4)2SO4 gradient in phosphate buffer. Two protein fractions with PLD activity were obtained. PLD1 (the first protein eluted) showed one single band with a subunit molecular mass of 57 kDa. This appears to be the same as the PLD enzyme reported previously (13). The second protein fraction containing PLD activity (termed PLD2) showed two bands on SDS-polyacrylamide gel electrophoresis, at 42 and 19.7 kDa. Although both PLD1 and PLD2 showed PLD activities, only PLD1 was used in extensive kinetic studies of the effect of PA because its subunit molecular mass corresponded to that reported previously for PLD from this organism (13). Commercially available PLD from cabbage (obtained from Sigma) was used without further purification. Enzyme concentrations were measured by the Lowry assay using bovine serum albumin as a standard (16).

Enzymatic Synthesis of DiC4PA and C16C6pyr-PA-- 100 mM diC4PC dissolved in 50 mM ammonium formate containing 0.5 mM Ca2+, pH 7.5, was incubated overnight at room temperature with 100 µg of PLD from S. chromofuscus. DiC4PA was purified from unreacted PC by elution from a QAE-Sephadex A-25 column using 1 M ammonium formate. C16C6pyr-PA was generated similarly by S. chromofuscus PLD-catalyzed hydrolysis of C16C6pyr-PC (solubilized in an ether/water/borate buffer emulsion with 0.5 mM Ca2+ at pH 7.5). The hydrolysis product, C16C6pyr-PA, was isolated by extraction of the aqueous reaction mixture several times with a chloroform/methanol (100:10) solution. The purity of C16C6pyr-PA and diC4PA was confirmed by 1H and 31P NMR spectroscopy.

Vesicle Preparation-- Lipid solutions in chloroform were dried under argon, lyophilized, and then suspended in 50 mM imidazole-D2O buffer, pH 7.2 (meter reading). For preparation of small unilamellar vesicles (SUVs), the aqueous lipid suspensions were sonicated in 3-min intervals (using a Branson W-350 sonicator) until maximum optical clarity was achieved. LUVs were prepared by extrusion using a LiposoFast Basic extruder with a 100-nm pore filter. The diameter of 6 mM PC LUVs prepared in this fashion was 80 ± 27 nm (17).

1H NMR Assay of PLD Activity-- 1H NMR (500 MHz) spectra, monitoring choline production, were acquired with a Varian Unity 500 spectrometer using an indirect probe and temperature of 30 °C. The following parameters were used in acquiring spectra: 1.2-s acquisition time, 1.0-s relaxation delay, and 6.2-µs pulse width (90°). Chemical shifts were referenced to the residual water resonance (before presaturation) at 4.75 ppm. The total volume of each assay sample was 400 µl. An initial spectrum was acquired before adding the enzyme and calcium solutions; this served as the zero time control. After the addition of PLD, an arrayed experiment was carried out for about 1 h. Initial rates were obtained from the progress curve for 10-20% PC hydrolysis as monitored by the increase in choline N(CH3)3 intensity. Errors in determining rates with this method were typically <= 15%.

pH-stat Assay of PLD Activity-- For kinetic studies involving diC4PC, a radiometer pH-stat model VIT90 was used to monitor generation of the soluble PA. The pKa2 for short chain PA (both monomer and micelle) is 6.8 (18), so that with an end point of 8.0 essentially all of the product can be titrated. PLD activity at each PC concentration was measured in duplicate or triplicate using 4 mM NaOH as the titrant.

Fluorescence Measurements-- Steady-state fluorescence measurements of vesicle mixing were carried out on a Shimadzu RF5000V spectrofluorometer at 30 °C (19). The pyrene-labeled lipids were excited at 350 nm with both excitation and emission slit widths set at 1.5 nm. The emission spectrum was monitored from 360 to 550 nm; almost all of the fluorescence information for both monomer and excimer bands was included in this range. The final concentration of labeled probe was 20 µM, the volume of sample was 400 µl, and total lipid concentration after dilution was 10 mM. These conditions mimic the reaction conditions used in the NMR experiments.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

S. chromofuscus PLD Activity toward Monomer and Micelle PC-- PLD enzymes, including the crude enzyme from S. chromofuscus, have an absolute requirement for Ca2+ because enzyme activity was abolished in the presence of 2 mM EDTA. DiC4PC is a soluble phospholipid with a very high CMC. More critical for kinetics where PA is the product, diC4PA does not form a precipitate with mM Ca2+. With this soluble substrate/soluble product assay system, the KD for Ca2+ was found to be 0.075 mM. There was no PLD activity without Ca2+ (e.g. in the presence of EDTA). Neither Zn2+ nor Ba2+ could substitute for the Ca2+ requirement. However, low PLD activity was observed in the absence of Ca2+ but with 1 mM Mg2+ added. The PLD specific activity with Mg2+ was about half of the activity of PLD without the addition of any metal ions (but without added EDTA). Enzyme activity did not increase with increasing Mg2+, suggesting that the observed activity was from the low level of contaminating Ca2+ in the assay solution and that the Mg2+ did not compete well for the Ca2+ site on the enzyme. PLD activity toward monomeric PC increased with increasing PC acyl chain length: for diC4PC, Vmax = 29.0 ± 1.2 µmol min-1 mg-1 and Km = 0.36 ± 0.06 mM; for monomeric diC6PC, Vmax = 61.5 ± 4.7 µmol min-1 mg-1 and Km = 0.05 ± 0.04 mM. The PLD specific activity showed no dependence on micellization of a monomeric substrate. PLD specific activity was constant, 61.4 ± 1.8 µmol min-1 mg-1, for five concentrations of diC6PC ranging from 2 to 30 mM. This phospholipid has a CMC of 14 mM; therefore, this bacterial PLD exhibits no interfacial activation. The two-dimensional concentration of the substrate in the interface was also not an important parameter for this phospholipase. With Triton X-100/POPC mixed micelles as the substrate, no decrease in activity was observed when the Triton concentration was increased 3-fold at a fixed PC concentration. The lack of a surface dilution effect as well as interfacial activation under these conditions indicated that this bacterial PLD behaved more like an esterase than a typical lipase.

PLD Activity toward POPC SUVs-- The absolute requirement of PLD for calcium complicates kinetics because Ca2+ forms a precipitate with PA, the PLD hydrolysis product. The Ca2+·PA complexes also cause massive particle growth in short chain (except for diC4PC) micelle systems when PA is generated. The relatively large size of vesicles (so that substrate depletion is not a significant problem for <20% hydrolysis) and tolerance of higher Ca2+ concentrations before precipitation or fusion occur make POPC/POPA vesicles ideal for examining the effects of product on PLD catalysis. However, the high pKa2 of PA in vesicles (pKa2 = 7.6 in predominantly PC bilayers (20)) makes pH-stat assays of PLD action problematic because at an end point of 8.0 only part of the PA is titratable. Furthermore, the PA pKa2 increases as more of the bilayer surface is occupied by PA (20). Therefore, 1H NMR spectroscopy was used to monitor PLD activity by measuring the intensity of the water-soluble choline N-methyl resonance. The N-methyl region of the SUVs of POPC exhibits resonances for inner and outer leaflet PC at 3.15 and 3.18 ppm. The water-soluble choline N-methyl resonance is observed as a sharp resonance at 3.09 ppm, upfield of the inner PC (Fig. 1). The integral of the choline resonance as a function of time provides a sensitive measure of PLD activity. Calcium was added with enzyme to avoid any vesicle fusion before PLD generation of PA. There is a small lag in the reaction progress curve (typically around 5 min) under these conditions (Fig. 2A, filled circles). The lag is independent of the POPC concentration. This is reminiscent of the lag phase toward vesicle substrates observed for 14-kDa PLA2 enzymes acting on PC bilayer substrate (21). For PLA2, the binding affinity of enzyme to the PC vesicle surface was low and represented a slow step in the enzyme reaction. Any factor that can facilitate enzyme surface binding abolished the lag and increased the observable enzyme activity (22). The dependence of PLD activity (measured after the lag phase) toward POPC vesicles on calcium concentration was hyperbolic with a KD for Ca2+ of 3.9 mM, considerably higher than the value for short chain PC monomers. Ca2+ induces fusion in SUVs containing negatively charge phospholipids. Therefore, to optimize PLD activity and minimize fusion when PA (or other negatively charged lipids) was included in vesicles, 5 mM calcium was used in the assays unless otherwise noted. As shown in Fig. 3, the dependence of PLD specific activity on POPC concentration was hyperbolic with an apparent Km = 6.5 ± 1.7 mM and Vmax = 13.5 ± 1.5 µmol min-1 mg-1. Although PLD does not exhibit interfacial activation or surface dilution in micelle systems, perturbations of the bilayer could alter the observed specific activity of PLD in a vesicle system. Incorporation of 5-10 mol % DAG, a lipid that destabilizes bilayers to fusion, into POPC SUVs had little effect on the PLD specific activity. Furthermore, the presence of the DAG had no effect on the lag phase (e.g. see Fig. 2B, where the 5-min lag is still observed).


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Fig. 1.   500 MHz 1H spectra (3.27-2.80 ppm) of 10 mM POPC SUVs (in 5 mM Ca2+, 50 mM imidazole, pH 7.2, 30 °C) as a function of incubation time with PLD (1.2 µg) from S. chromofuscus.


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Fig. 2.   Reaction progress curve for hydrolysis of different POPC SUVs. The amount of hydrolysis product choline was calculated from the 1H integrated intensity of the choline resonance compared with an internal standard. SUVs were composed of (panel A) 10 mM POPC (bullet ) and 9 mM POPC with 1 mM LPA (open circle ); (panel B) 9 mM POPC with 1 mM DAG (diamond ) or 1 mM PI (down-triangle); (panel C) 9 mM POPC with 1 mM PMe (black-diamond ) or 1 mM oleic acid (triangle ). Reaction conditions include 50 mM imidazole, pH 7.2, 5 mM Ca2+, and 1.2 µg of PLD.


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Fig. 3.   Specific activity of S. chromofuscus PLD toward different concentrations of POPC SUVs in the absence (open circle ) and presence of (bullet ) 10 mol % POPA. Reaction conditions are the same as in Fig. 2.

POPC/POPA SUVs as Substrates for PLD-- POPA was deliberately incorporated into POPC SUVs to determine if the product of PLD had any effect on the initial rates of reaction. The addition of 0.5-5 mM Ca2+ caused no massive aggregation or precipitation of POPC/POPA vesicles as long as the PA content was <30%. In studies of PLA2 activity toward PC vesicles, it was found that incorporation into the bilayer of a threshold level of product or other anionic lipids diminished the lag phase and increased the enzyme activities (23). With that phospholipase, negatively charged lipids facilitate the binding of enzyme to the bilayer interface (24). Products also alter the polarity in the interface region, and this facilitates PLA2 action (25). If POPA promotes binding of PLD to the vesicle surfaces, there might be an increase in PLD specific activity as well as the disappearance of the lag phase. On the other hand, PA is a PLD product and might give rise to product inhibition.

PLD activity toward POPC/POPA SUVs was examined as a function of calcium concentration and mole fraction POPA. The relative activities of PLD toward POPC and POPC/POPA SUVs with various Ca2+ concentrations are shown in Table I. The incorporation of POPA in PC SUVs increased the enzyme specific activity about 5-8-fold. The presence of POPA also diminished the lag phase, presumably because it facilitated binding of the PLD to the vesicle. The activation of PLD by PA was calcium-dependent. At low Ca2+ (0.5 mM), incorporation of POPA in the vesicles inhibited PLD; at higher Ca2+ concentrations, the same concentration of POPA activated PLD. The inhibition at low calcium concentrations may be caused by the competition of Ca2+ preferential binding to PA instead of PLD. At 5 mM Ca2+, the apparent Vmax of PLD toward POPC vesicles with 10 mol % POPA in the bilayer was 96.0 ± 8.9 µmol min-1 mg-1, and the apparent Km was 4.8 ± 1.4 mM. The 7-fold increase in Vmax could reflect an increase in PLD adsorption to the vesicle surface as well as an increase in kcat. The slight reduction in the apparent Km for substrate (from 6.5 to 4.8 mM) was within the error of Km determination. At a fixed total phospholipid concentration (10 mM) and 5 mM Ca2+, the mol % of POPA incorporated into the vesicles was also varied. As seen in Fig. 4, PLD activity increased with the mol % of POPA to a maximum ~20 mol % PA. At greater than 30 mol % PA, the presence of the Ca2+ caused vesicle aggregation and fusion; PLD activity also decreased somewhat. However, under these conditions (PC + PA = 10 mM), it is also possible that the substrate (PC) concentration decreased to a level near the apparent Km, leading to the decrease in observed activity.

                              
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Table I
Relative activity of S. chromofuscus PLD toward small unilamellar vesicles of POPC and POPC/POPA
Assay conditions included 50 mM imidazole, pH 7.2, 5 mM Ca2+, a total phospholipid (POPC + POPA) concentration of 10 mM, and 1.2 µg of PLD.


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Fig. 4.   Specific activity of S. chromofuscus PLD toward POPC SUVs with different mol % POPA (PC + PA = 10 mM). Reaction conditions are the same as in Fig. 2.

Effect of Other Anionic Lipids on PLD Activity-- PA is dianionic, and the negative charges of PA may play a major role in the binding of enzyme to the vesicle surface and thus activating PLD. Other lipids and amphiphiles with negative charges, PMe, oleic acid, PI, LPA, and PIP, were incorporated into POPC vesicles to see if they could activate PLD. The relative PLD activities toward POPC vesicles with 10 mol % anionic lipids are shown in Table II. Most of the negatively charged lipids did not activate PLD, nor did they reduce the lag phase (Fig. 2, B and C). PMe had almost no effect, whereas the incorporation of oleate and PI inhibited the enzyme activity. Along with the inhibited PLD activity was an increased lag time (compare Fig. 2, B and C, where the presence of 10 mol % PI and oleate leads to an increased lag phase). However, both PIP and lyso-PA activated PLD. The lyso-PA activation was comparable to that of PA, whereas the PIP activation was 3-fold less. Again, when PLD activation was observed, the lag phase was reduced or abolished (e.g. see Fig. 2A, open circles, for the effect LPA). Activating lipids all have phosphate monoester groups, suggesting that the activation was specific for a membrane-localized phosphate moiety.

                              
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Table II
Relative activity of S. chromofuscus PLD toward POPC/X (9 mM:1 mM) vesicles
Assay conditions included 50 mM imidazole, pH 7.2, 5 mM Ca2+, and 1.2 µg of PLD.

M2+-induced Fusion of PA-containing Vesicles-- Vesicle fusion is always a concern in enzymatic reactions involving vesicles containing anionic lipids and Ca2+. Enzyme can be transferred from one vesicle to another through the fusion, potentially complicating kinetics. High concentrations of Ca2+ enhance fusion of vesicles with negatively charged amphiphiles such as PA and fatty acid (26, 27). To investigate whether PA activation of PLD was caused by calcium-related vesicle fusion, the effect of other divalent cations on the PLD hydrolysis of POPC/POPA vesicles was investigated. Table III shows the effect of Mg2+ and Ba2+ on PA activation. Because there is no activity if Ca2+ is absent, 0.5 or 1 mM Ca2+ was used with 4.5 and 4 mM Mg2+ or Ba2+ added. The addition of the other two metal ions had small effects on PLD activity toward POPC vesicles, with Mg2+ more effective in replacing the excess Ca2+ than Ba2+. A more significant activation was seen with PA-containing vesicles. Neither Mg2+ nor Ba2+ led to amounts of activation comparable to Ca2+, although the addition of Mg2+ enhanced PLD activity to roughly half of that produced by Ca2+ at a comparable concentration. The added Mg2+ caused some precipitation in the POPC/POPA vesicle solution (even without the addition of PLD). In contrast, the POPC/POPA vesicle solution was opalescent with 5 mM Ba2+ and 1 mM Ca2+. POPC/POPA (9:1) vesicle solutions appeared opalescent for several hours after the addition of up to 10 mM Ca2+, indicating that they were reasonably stable in the absence of PLD under these conditions. Thus, an excess of divalent ion along with a minimum amount of Ca2+ for catalysis is critical for optimal PLD activity.

                              
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Table III
Effect of divalent metal ions on the specific activity of S. chromofuscus PLD toward POPC and POPC/POPA vesicles
Assay conditions included 50 mM imidazole, pH 7.2, and 1.2 µg of PLD.

PA Activation of Cabbage PLD-- The effect of PA on the specific activity of PLD from cabbage was also examined. Vesicles with 1 mM POPA and 9 mM POPC (with 5 mM Ca2+) exhibited a 26-fold enhanced PLD specific activity compared with pure POPC (10 mM) vesicles. Cabbage PLD is different from the bacterial enzyme in that it appears to show some interfacial activation (28) using short chain PCs as substrates. That PA activates the enzyme might suggest that PA activation is a property for many PLD enzymes.

Effect of Vesicle Curvature on PLD Activity-- The incorporation of dianionic POPA into SUVs stabilizes the curvature of the small vesicles. PLD may bind more effectively to a curved surface than to a flat surface. The effect of vesicle curvature on enzyme activity can be assessed by comparing specific activities toward large and small unilamellar POPC and POPC/POPA vesicles (Table II). The specific activity of PLD toward POPC LUVs with 5 mM Ca2+ was 1.6 µmol min-1 mg-1, considerably lower than toward SUVs at the same concentration (8-11 µmol min-1 mg-1). The PLD specific activity increased to 24.7 µmol min-1 mg-1 toward POPC/POPA (9:1) LUVs. Compared with SUVs, the extent of PA activation was higher for the LUV system, although the final specific activity was 2-3-fold lower than for PC/PA (9:1) SUVs. Vesicle curvature clearly affects PLD, but it cannot be the major cause of PA activation.

Fluorescence Studies of Vesicle Stability-- Pyrene-labeled lipids have been used to study lipid lateral distribution and diffusion in membranes (29-31). Use of pyrene as a fluorophore is based on its ability to form excited state dimers or excimers because of the collisional interaction of a ground state pyrene molecule and an excited state pyrene molecule. The pyrene monomer bands (365-425 nm) dominate the emission spectrum at low pyrene concentration, whereas a broad excimer band (~470 nm) dominates at high pyrene concentration. If nonfluorescent phospholipid molecules are added to the pyrene-labeled vesicles, the excimer band will decrease, and the monomer bands will increase as the collisional cross-sectional interaction of one pyrene-PC with another pyrene molecule decreases. This makes the probe very sensitive to any changes in vesicle distribution, notably fusion. Two kinds of probes were used in the experiments, pyrene-labeled PC or pyrene-labeled anionic lipids (PA, PMe, and fatty acid). Experimental conditions used total lipid concentrations comparable to the NMR assay. A vesicle stock solution composed of 1 mM total lipid concentration (900 µM C16C6pyr-PC, 100 µM unlabeled PA) was diluted to make the vesicle solution 40 µM in labeled phospholipid. This was then mixed 1:1 with 20 mM unlabeled vesicles of the same PC/PA (9:1) composition. The final total lipid concentration in the fusion assays was 10 mM; the final labeled pyrene-PC concentration was 20 µM. In a separate set of experiments, C16C6pyr-PA and C16C6pyr-PMe were sonicated with POPC (9:1, PC/anionic lipid), and these vesicles were mixed with 10 mM total lipid concentration of the same vesicle composition to check for the effect of calcium and barium ions on PA and PMe distribution in the bilayers (i.e. to look for partial phase segregation or clustering of anionic lipids). The final concentration of labeled PA or PMe in the mixtures was also 20 µM.

Without the addition of any metal ions, POPC/POPA vesicles doped with C16C6pyr-PC were stable overnight. The addition of Ca2+ and Ba2+ caused an immediate decrease in the excimer band and an increase in the monomer bands (Fig. 5A). The intensities of monomer and excimer bands were relatively stable after the initial changes, indicating that vesicle fusion occurred rapidly. The increases in monomer intensity for Ca2+ concentrations from 0.5 to 5 mM were essentially the same, indicating similar amounts of fusion (Fig. 5B); higher Ca2+ led to much greater fusion (much higher increase in C16C6pyr-PC monomer fluorescence at 370 nm). Incorporation of PMe or oleate instead of PA in the vesicle also led to extensive fusion when Ca2+ (5 mM) was added (Fig. 6). The SUVs with PMe exhibited the greatest amount of fusion as monitored by the increase in C16C6pyr-PC monomer fluorescence. Replacement of Ca2+ by Ba2+ or lowering Ca2+ still led to fusion of the vesicles. Thus, all of the anionic lipids studied led to some amount of vesicle fusion when a divalent metal ion was added. Because PMe did not activate PLD but did lead to fusion, vesicle fusion could not be the sole cause of PA activation.


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Fig. 5.   Ca2+-induced fusion of SUVs composed of C16C6pyr-PC (40 µM) with 10 mol % POPA after dilution 1:1 with 20 mM unlabeled POPC/POPA (9:1) SUVs in 50 mM Tris, pH 7.5. Panel A, C16C6pyr-PC monomer fluorescence intensity (plotted as (I - I0)/I0, where I0 is the monomer fluorescence at 370 nm after dilution in the absence of unlabeled SUVs or metal ions, and I is the monomer fluorescence at a given time after the addition of unlabeled vesicles and metal ions) after mixing with different Ca2+ concentrations (bullet , 10 mM; square , 5 mM; open circle , 2 mM; triangle , 0.5 mM; black-diamond , no added Ca2+). Panel B, limiting change in C16C6pyr-PC monomer intensity as a function of Ca2+ concentration.


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Fig. 6.   Fusion (as monitored by changes in the C16C6pyr-PC monomer intensity at 370 nm and quantified as (I - I0)/I0) of SUVs composed of 40 µM C16C6pyr-PC with 10 mol % negatively charged unlabeled lipids diluted 1:1 with 20 mM POPC/anionic lipid (9:1) SUVs in 50 mM Tris, pH 7.5. ×, C16C6pyr-PC/oleate, 5 mM Ca2+. bullet , C16C6pyr-PC/PA, 5 mM Ca2+. triangle , C16C6pyr-PC/PA, 5 mM Ba2+. open circle , C16C6pyr-PC/PMe, 5 mM Ca2+.

It is possible that divalent ions cause aggregation of PA within the bilayer, and this is related to the activation phenomenon. If so, there should be differential effects for PMe, oleate, and PA. This can be probed using pyrene-labeled PA, PMe, or fatty acid. If there is Ca2+-induced aggregation of PA in the plane of bilayer, the monomer band should decrease in intensity; if vesicle fusion and randomization of the lipids occur, then an increase in the pyrene-probe monomer band should be observed. As shown in Fig. 7A, the pyrene-PA monomer intensity did not change dramatically for PC/PA vesicles with the addition of both barium and calcium, whereas the pyrene-PMe monomer intensity increased for PC/PMe vesicles with the addition of calcium. From the fluorescence experiment using pyrene-PC, it is clear that fusion occurred upon the addition of metal ions to both PC/PA and PC/PMe vesicles. When the anionic lipid is labeled with pyrene, we should observe an increase in pyrene monomer band intensity if only fusion occurred, as is the case for PC/PMe vesicles. If both lateral aggregation of anionic lipid and fusion occurred, the increase in monomer band could be balanced by a decrease in that band, giving rise to only small changes in the monomer intensity; the excimer band may also increase slightly under this scenario. This appears to be the case for PC/PA vesicles with the addition of either Ca2+ or Ba2+. As shown in Fig. 7B, the excimer band at 470 nm increased initially with the addition of Ca2+ or Ba2+ for pyrene-labeled PA but not for pyrene-labeled PMe. After 30 min, the excimer band of PA decreased, consistent with overall vesicle fusion occurring as well as lateral aggregation of the PA molecules. As a control, light scattering at 400 nm was monitored for all of the vesicle systems. The addition of calcium and barium ions increased A400 slightly; adding Ca2+ had a more pronounced effect for PC/PMe vesicles compared with PC/PA vesicles. The greater light scattering will reduce the intensity of the pyrene fluorescence. However, because we observed higher intensity changes for the pyrene probe in PC/PMe vesicles, light scattering resulting from particle fusion does not affect the results.


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Fig. 7.   Fusion of SUVs composed of 40 µM pyrene-labeled negatively charged lipids and 360 µM POPC diluted 1:1 with 20 mM unlabeled POPC/anionic lipid (9:1) SUVs. Panel A, change in pyrene monomer fluorescence (I - I0)/I0 at 370 nm, as a function of time after vesicle mixing. Panel B, change in the pyrene excimer (I - I0)/I0 at 470 nm, as a function of time after vesicle mixing. The different POPC/anionic lipid systems include: (bullet ) POPC/C16C6pyr-PA, 5 mM Ca2+; (triangle ) POPC/C16C6pyr-PA, 5 mM Ba2+; (black-diamond ) POPC/C16C6pyr-PA, 0.5 mM Ca2+; and (open circle ) POPC/C16C6pyr-PMe, 5 mM Ca2+.

PA Vesicle Activation of PLD toward DiC4PC-- The fluorescence experiments indicate that the addition of barium and calcium causes the lateral aggregation of PA (but not PMe) in the bilayer surface. Hence, PA activation of PLD appears to be caused by a specific interaction between calcium (or to some extent another divalent metal ion such as Ba2+ or Mg2+), PA, and enzyme. Can such an interaction occur in a monomer substrate system? PLD specific activity toward diC4PC, with a CMC > 280 mM (32), was investigated by pH-stat. Although the KD for Ca2+ in this system was 0.075 mM, higher Ca2+ was used in the assay system to measure kinetic parameters of PLD acting toward the dibutyroylphospholipids because excess Ca2+ is also needed for examining the PA activation. The Km and Vmax values for diC4PC at 5 mM Ca2+ were 0.36 ± 0.06 mM and 29.0 ± 1.2 µmol min-1 mg-1, respectively. DiC4PA was added to diC4PC reaction mixtures to determine whether there was any PA activation in a monomer assay system. There was a consistent 20% increase in PLD specific activity with 5 mM diC4PA; the Km for diC4PC was 0.46 mM with 5 mM diC4PA in the assay system. However, this increase in Vmax was much smaller than that observed with PC/PA vesicles. Therefore, it appears that an interface is necessary for the full activation of PLD by PA.

As long as diC4PC does not partition into a bilayer, the effect of PA in a vesicle surface can be examined for its effect on monomeric PC hydrolysis. A POPA/cholesterol (20 mol %) vesicle along with 0.5 mM Ca2+ was used for these studies. The cholesterol was incorporated into the PA vesicles to prevent precipitation of the vesicles (pure PA vesicles will fuse and precipitate with 0.5 mM or higher Ca2+). Low concentrations of these POPA vesicles caused a 2-fold activation of PLD toward 5 mM diC4PC; at higher concentrations, the POPA vesicles inhibited the enzyme activity (Fig. 8A). The addition of PIP vesicles (2 mM) also generated a 1.8-fold activation of PLD under the same conditions. This is not likely to be the maximum activation obtainable because the Ca2+ was kept low to prevent SUV precipitation and fusion. The maximum specific activity at 0.25 mM PA occurs where Ca2+·PA ~ 2. As the PA is increased, there will be competition between PLD and PA for Ca2+. If not enough Ca2+ is available to bind to PLD, enzyme activity will decrease.


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Fig. 8.   Panel A, relative activity of S. chromofuscus PLD toward diC4PC with different concentrations of POPA/cholesterol vesicles. The pH-stat assays included 0.5 mM Ca2+ and 100 mM NaCl. Panel B, profile for 1 mM POPA and 10 mM diC4PC elution on a Sephadex G-25 column equilibrated with 0.5 mM Ca2+, 50 mM Tris, pH 7.5.

Sephadex G-25 gel filtration was used to check for diC4PC partitioning into the POPA vesicles. The sizing column was equilibrated with 1 mM diC4PC and 50 mM Tris, pH 7.5. A sample of 10 mM diC4PC, 1 mM POPA/cholesterol SUVs, and 0.5 mM Ca2+ was applied to the column. Phospholipid concentrations in each fraction were analyzed by 1H-decoupled 31P NMR spectroscopy; Triton X-100 was added to each fraction to micellize the PA for a more accurate measurement of intensity. The resonances for POPA (3.9 ppm) in Triton X-100 mixed micelles and diC4PC (~0 ppm) were well separated in the 31P spectrum, allowing for easy quantitation of each species. As shown in Fig. 8B, these two lipids were also well separated in the elution profile. Because no significant diC4PC was incorporated into the PA vesicles, the PA activation of PLD must involve interfacial PA binding to an allosteric site on the enzyme.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

PLD from S. chromofuscus is a water-soluble enzyme whose substrate forms aggregates in aqueous solution. Although this PLD does not exhibit monomer-micelle interfacial activation like other phospholipases, the transfer of PLD from aqueous solution to the lipid-water interface is necessary for the catalytic reaction to occur with vesicle substrates. PA, the product of PLD cleavage of phospholipids, enhances Vmax when present in the PC bilayer. This is an intriguing observation, and understanding the mechanism for this response could shed light on the rapid activation of mammalian PLD in signal transduction. PLD activation could be caused by the anionic PA enhancing PLD binding to the bilayer surface, by a direct effect of this ligand on the enzyme, or by a combination of both effects. The results in the present work indicate that the charge of the lipid components, vesicle curvature, and vesicle fusion are not controlling factors for PA activation of PLD. Because almost all substrate molecules in these vesicles can be hydrolyzed, either the enzyme can hop from vesicle to vesicle, or vesicle fusion prevents substrate depletion and makes all substrate accessible to the enzyme. Of more relevance to the PA activation of PLD is the observation that there is some divalent metal ion-induced lateral segregation of the PA in PC vesicles. However, both Ba2+ and Ca2+ induce PA clustering, but only Ca2+ leads to maximal PLD activation. Nonspecific perturbations of PC bilayers have only minor effects on PLD activity. Previously, Yamamoto et al. (26) incorporated 1,2-diacylglycerol and cholesterol into egg yolk PC SUVs and checked for the effect of these bilayer-perturbing lipids on the Vmax of PLD from S. chromofuscus. 5-10 mol % of both DAG and cholesterol slightly enhanced the PLD activity, consistent with our observations. Increasing the concentration of cholesterol in the bilayer did not affect Vmax further; however, if the concentration of DAG in the bilayer was increased to 18.3 mol %, the Vmax was increased 5-fold. This concentration of DAG would destabilize vesicles and produce some microemulsions as well as large vesicles.

A more likely explanation for the kinetic activation of PLD is that Ca2+·PA binds to the enzyme at an allosteric site (possibly one specific for anchoring the enzyme to the interface) and in so doing enhances PLD activity. Studies of bacterial PI-PLC indicate that an allosteric binding site for a PC interface increases the PI-PLC activity toward the water-soluble substrate myo-inositol 1,2-(cyclic)-phosphate dramatically (33, 34). Iwasaki et al. (35) have cloned the PLD enzyme from Streptomyces antibioticus in Escherichia coli. The sequence was compared with PLD sequences from Streptomyces acidomyceticus, S. chromofuscus, and other Streptomyces species as well as other phospholipases including PI-PLC from Bacillus cereus, and various rat PI-PLC isozymes. There was a high similarity in the primary sequence for PLD enzymes. The comparison also revealed a region of homology between PI-PLCs and PLD. In this region, 22% of the amino acids were identical between S. antibioticus PLD and B. cereus PI-PLC. There was 41% homology if conservative changes were considered. Moreover, this region involves a part of the X region, which is highly conserved among bacterial and eukaryotic PI-PLCs (36). The X region is supposedly involved in substrate binding. If so, PLD might have the same binding mode as PI-PLCs along with a related allosteric activator site. For the monomeric substrate diC4PC, the addition of diC4PA had only a small effect on activity. However, PLD could be activated toward this monomer substrate with the addition of POPA or PIP vesicles. Because the substrate diC4PC has no detectable affinity for the PA vesicles, enzyme activation must arise from PLD binding to the PA surface but at a site distinct from the active site.

Mammalian PLD is activated rapidly in cells. Exogenous PLD from S. chromofuscus can mimic the reaction of endogenous PLD in the cell. Several examples show that PLD can also be stimulated in vitro by PIP2 (38). An ARF-responsive human PLD in Sf9 insect cells was activated by PIP2 and inhibited by oleate (37). Decreasing PIP and PIP2 levels in the cell led to a decrease in the PLD activity in vivo (38). The expressed human PLD was shown to occur in two alternatively spliced forms. Both forms were strongly activated by PIP2, PIP3, and ARF and to a lesser extent by Rho proteins (37)). Protein kinase Calpha also activated both isoforms in the absence of ATP. These data indicate that regulators (ARF, Rho proteins, PIP2, and protein kinase C) of PLD directly activated both PLD enzymes by interacting at different sites on the enzymes. The PA activation of bacterial PLD may be related to PIP2 activation. In the present work, lipids with a phosphate group, PA, LPA, and PIP, stimulated the activity of bacterial PLD toward both monomer and vesicle substrates. Although PIP2 was not examined, it is likely that the effect it will have on PLD will be the same as that on PIP. Because PLD from cabbage was also activated by PA, the phenomenon may be universal for members of the PLD family.

PLD activities need Ca2+, and evidence shows that cytosolic Ca2+ can stimulate PLD activities (39). Depletion of cellular Ca2+ by EGTA results in inhibition of the stimulation of the enzyme by agonists linked to the heterotrimeric G proteins. However, no evidence indicates physiological control of PLD by changes in cytosolic Ca2+. From our observations, Ca2+ is not only required for PLD basal activities but also needed for PA activation. The function of Ca2+ may rely on its ability to induce a local lipid segregation of PA for specific binding to PLD and subsequent activation.

    ACKNOWLEDGEMENT

We thank Emily Speelmon, Boston College, for some of the kinetic experiments with crude PLD.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 26762 (to M. F. R.).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.

Dagger To whom correspondence should be addressed: Merkert Chemistry Center, Boston College, 2609 Beacon St., Chestnut Hill, MA 02167. Tel.: 617-552-3616; Fax: 617-552-2705; E-mail: mary.roberts{at}bc.edu.

1 The abbreviations used are: PLD, phospholipase D; PA, phosphatidic acid; PLA2, phospholipase A2; lyso-PA, 1-acyl-2-hydroxyglycero-3-phosphate; DAG, diacylglycerol; PC, phosphatidylcholine; PIP, phosphatidylinositol monophosphate; PIP2, phosphatidylinositol bisphosphate; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; diC4PC, dibutyroylphosphatidylcholine; LPC, 2-lauroyl-2-hydroxyglycero-3-phosphocholine; POPA, 1-palmitoyl-2-oleoylphosphatidic acid; PMe, phosphatidylmethanol; PI, phosphatidylinositol; C16pyr-COOH, 1-pyrenehexadecanoic acid; C16C6pyr-PC, 1-hexadecanoyl-2(1-pyrenehexanoyl)-sn-glycero-3-phosphocholine; C16C6pyr-PMe, 1-hexadecanoyl-2(1-pyrene-hexanoyl)-sn-glycero-3-phosphomethanol; C16C6pyr-PA, 1-hexadecanoyl-2(1-pyrenehexanoyl)-sn-glycero-3-phosphate; diC4PA, dibutyroylphosphatidic acid; C16C6pyr-PA, 1-hexadecanoyl-2(1-pyrenehexanoyl)-sn-glycero-3-phosphate; SUV, small unilamellar vesicle; LUV, large unilamellar vesicle; LPA, 2-lauroyl-2-hydroxyglycero-3-phosphate; PLC, phospholipase C; CMC, critical micelle concentration; ARF, ADP-ribosylation factor.

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