From the Merkert Chemistry Center, Boston College,
Chestnut Hill, Massachusetts 02167
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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
-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.
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RESULTS |
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 ( ) and 9 mM
POPC with 1 mM LPA ( ); (panel B) 9 mM POPC with 1 mM DAG ( ) or 1 mM
PI ( ); (panel C) 9 mM POPC with 1 mM PMe ( ) or 1 mM oleic acid ( ). 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 ( ) and presence of ( ) 10 mol % POPA. Reaction
conditions are the same as in Fig. 2.
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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.
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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.
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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.
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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 ( , 10 mM; ,
5 mM; , 2 mM; , 0.5 mM; ,
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+. , C16C6pyr-PC/PA, 5 mM Ca2+. ,
C16C6pyr-PC/PA, 5 mM
Ba2+. , C16C6pyr-PC/PMe, 5 mM Ca2+.
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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: ( ) POPC/C16C6pyr-PA, 5 mM Ca2+; ( )
POPC/C16C6pyr-PA, 5 mM
Ba2+; ( ) POPC/C16C6pyr-PA, 0.5 mM Ca2+; and ( )
POPC/C16C6pyr-PMe, 5 mM
Ca2+.
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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.
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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 |
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 C
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.
We thank Emily Speelmon, Boston College, for
some of the kinetic experiments with crude PLD.