(Received for publication, September 23, 1994; and in revised form, October 28, 1994)
From the
A novel Ca-independent phospholipase A
(PLA
) has recently been purified from the murine
macrophage-like cell line P388D
(Ackermann, E. J., Kempner,
E. S., and Dennis, E. A.(1994) J. Biol. Chem. 269,
9227-9233). This enzyme is now shown to be inhibited by palmitoyl
trifluoromethyl ketone (PACOCF
), arachidonyl
trifluoromethyl ketone (AACOCF
), and a bromoenol lactone
(BEL). Both PACOCF
and AACOCF
were found to
inhibit the macrophage PLA
in a concentration-dependent
manner. PACOCF
was found to be
4-fold more potent than
AACOCF
, with IC
values of 3.8 µM (0.0075 mol fraction) and 15 µM (0.028 mol fraction),
respectively. Reaction progress curves in the presence of either
inhibitor were found to be linear, and the
PACOCF
PLA
complex rapidly dissociated
upon dilution.
BEL was also found to inhibit the macrophage
PLA in a concentration-dependent manner, with half-maximal
inhibition observed at 60 nM after a 5-min preincubation at 40
°C. Inhibition was not reversed after extensive dilution of the
enzyme into assay buffer. Treatment of the PLA
with BEL
resulted in a linear, time-dependent inactivation of activity, and the
rate of this inactivation was diminished in the presence of
PACOCF
. In addition, PLA
treated with
[
H]BEL resulted in the covalent labeling of a
major band at M
80,000. Inactivation of the
PLA
by 5,5`-dithiobis(2-nitrobenzoic acid) prior to
treatment with [
H]BEL resulted in the near
complete lack of labeling consistent with covalent irreversible suicide
inhibition of the enzyme. The labeling of a M
80,000 band rather than a M
40,000 band upon
treatment with [
H]BEL distinguishes the
macrophage Ca
-independent PLA
from a
previously identified myocardial Ca
-independent
PLA
and provides strong evidence that the M
80,000 protein is the catalytic subunit.
Phospholipase A (PLA
) (
)has
been the focus of considerable research over the years due to its
potential involvement in the release of arachidonic acid from membrane
phospholipids and the subsequent production of prostaglandins and
leukotrienes (for review, see (1) ). PLA
are also
thought to play key roles in phospholipid metabolism, digestion, and
various disease states. Even though it is now evident that they
represent a very large and diverse family of enzymes (for review, see (2) ), the vast majority of the structural and mechanistic
information available is from the Ca
-dependent Group
I, II, and III secreted PLA
(sPLA
). These
enzymes are characterized by their low molecular weight, high disulfide
bond content, conserved three-dimensional structures, and a requirement
for calcium during hydrolysis (for reviews, see (3) and (4) ).
Recently, a number of unique intracellular cytosolic
PLA have been identified and purified that are distinct
from the sPLA
. Two well studied examples are the 85-kDa
Group IV cytosolic PLA
(cPLA
) (5, 6) and the M
40,000
myocardial Ca
-independent PLA
(iPLA
)(7) . In addition, we have also
reported the purification of an apparent M
80,000
cytosolic Ca
-independent PLA
from the
macrophage-like cell line P388D
(8) . Unlike the
sPLA
, very little is known about the catalytic mechanisms
of these enzymes, their intracellular roles, or their relationships to
one another. This is especially true in the case of the myocardial
iPLA
and the macrophage iPLA
. These two enzymes
are unique among the known PLA
in that they are both
modulated by ATP and they both form high molecular weight complexes of
400,000(8, 9) . Because of these similarities,
there has been some uncertainty as to whether they represent similar
enzymes modulated by the same regulatory protein (10) or
whether they are truly distinct enzymes. Unfortunately, sequences have
not been available for either of these two iPLA
.
One
advantageous method for studying and comparing kinetic and chemical
mechanisms between enzymes is through the use of inhibitors. Recently,
two active site-directed inhibitors have been reported in the
literature, arachidonyl trifluoromethyl ketone (AACOCF),
which reportedly displays specificity for the Group IV cPLA
versus the Group II sPLA
(11) , and
a bromoenol lactone (BEL), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one,
which reportedly displays specificity for the myocardial iPLA
versus both Group I and III
sPLA
(12) . In this study, we have investigated the
action of each of these inhibitors as well as several additional
compounds including a new potent inhibitor, palmitoyl trifluoromethyl
ketone (PACOCF
), toward the purified macrophage
iPLA
, and we have utilized [
H]BEL to
help identify the catalytic subunit of the macrophage iPLA
.
For the competition
experiments, PACOCF and BEL were added simultaneously to
the enzyme and incubated as described above. Upon dilution into the
assay mixture, the concentration of PACOCF
was
1.1
µM, a concentration that results in
16% inhibition of
the PLA
activity (see Fig. 1). For a control, the
PLA
was routinely preincubated in the presence of
PACOCF
alone, and the resulting activity (measured after
dilution into the assay mixture) was defined as 100% of control for all
assays in which PACOCF
was present.
Figure 1:
Concentration-dependent inhibition of
the P388D Ca
-independent PLA
by PACOCF
and AACOCF
. The P388D
PLA
was assayed in the presence of increasing
concentrations of PACOCF
(
), AACOCF
(
), palmitic acid ([
), or arachidonic acid
([
) utilizing a mixed micelle assay containing Triton X-100
and DPPC. The enzyme activity is plotted as the percentage of the
control enzyme assayed in the absence of inhibitor. Each point
represents the average of duplicates.
For dilution
experiments, the PLA was concentrated
6-fold using a
Centricon 10 apparatus (Amicon, Inc.). The resulting enzyme preparation
was preincubated at 40 °C for 5 min with either Me
SO
alone or 10 µM BEL and Me
SO. After
preincubation, a 2-µl aliquot was removed and diluted 1500-fold
into 3 ml of assay buffer containing 400 µM Triton X-100,
100 µM DPPC (with 200,000 cpm
1-palmitoyl-2-[1-
C]palmitoyl-sn-glycero-3-phosphorylcholine/50
µl of assay buffer), 100 mM Hepes, pH 7.5, 0.8 mM ATP, 1.0 mM dithiothreitol, and 5 mM EDTA. At
the indicated time intervals, a 50-µl aliquot was removed, and the
released radiolabeled fatty acid was measured as described above.
Control experiments were also carried out that
demonstrated that under these conditions, [H]BEL
inhibited
83% of the PLA
activity. For these
experiments, the PLA
activity was assayed utilizing a
double-labeled phospholipid
(1-[1-
C]palmitoyl-2-[1-
C]palmitoyl-sn-glycero-3-phosphorylcholine),
and the activity was measured by following the release of
1-[1-
C]palmitoyl-2-lyso-sn-glycero-3-phosphorylcholine
separated by TLC as described previously(8) . This method was
used because the presence of [
H]BEL would have
interfered with the detection of released radiolabeled fatty acid in
the Dole extraction method.
Because AACOCF has recently been shown to
be a slow, tight-binding inhibitor of the Group IV
cPLA
(11) , we examined the time course and
reversibility of inhibition with the P388D
Ca
-independent PLA
. The P388D
PLA
was assayed in the presence of either 8
µM PACOCF
or 12 µM AACOCF
or in the absence of inhibitor. As shown in Fig. 2, linear
progress curves were observed under all three conditions, with each
curve passing through the origin, indicating that these are not slow
binding inhibitors. In addition, preincubation of the P388D
Ca
-independent PLA
with 300
µM PACOCF
for 5 min at 40 °C followed by a
1500-fold dilution into assay buffer resulted in near complete recovery
of the PLA
activity at all time points measured
(15-240 min). This is in contrast to the slow dissociation rate
observed for the
Ca
AACOCF
cPLA
complex, where under similar conditions, only 14% of the complex
dissociated over a 5-h period(11) . Thus, the
association/dissociation rates observed between these inhibitors and
the P388D
PLA
appear to be much faster than the
rates observed between the Group IV cPLA
and
AACOCF
.
Figure 2:
Reaction progress curves of the
P388D Ca
-independent PLA
in
the presence of trifluoromethyl ketone inhibitors. The P388D
PLA
was assayed under standard assay conditions in
the absence of inhibitor (
) or in the presence of either 12
µM AACOCF
(
) or 8 µM PACOCF
([
). Each point represents the
average of duplicates.
Figure 3:
Concentration-dependent inhibition of the
P388D Ca
-independent PLA
by
BEL. The P388D
PLA
was incubated with the
indicated concentrations of BEL for 5 min at 40 °C in buffer
containing 10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 1 mM Triton X-100, 10% glycerol, and 1
mM ATP. After this preincubation, the enzyme was diluted
10-fold into the assay mixture, and the remaining activity was
measured. Each point represents the average of duplicates and is
plotted as the percent of control (enzyme preincubated in the absence
of inhibitor).
Figure 4:
Irreversible inhibition of the P388D Ca
-independent PLA
by BEL. The
P388D
PLA
was preincubated at 40 °C for 5
min with either 10 µM BEL in Me
SO (
) or
Me
SO alone (
). After preincubation, a 2-µl
aliquot was removed and diluted 1500-fold into 3 ml of assay buffer. At
the indicated time intervals, a 50-µl aliquot was removed, and the
amount of released radiolabeled fatty acid was determined. Each point
represents the average of duplicates.
Figure 5:
Time-dependent inactivation of the
P388D Ca
-independent PLA
by
BEL. The P388D
PLA
was preincubated with 30
nM BEL (
), 60 nM BEL (
), or 100 nM BEL (
) for the indicated time periods at 40 °C, followed
by dilution into assay buffer and quantification of remaining activity.
Each point represents the average of duplicates and is plotted on a
semilogarithmic plot as the percent of control enzyme incubated in the
absence of inhibitor.
Time-dependent experiments were also carried out in the presence of
the reversible inhibitor PACOCF. The P388D
PLA
was preincubated with BEL alone or with BEL and
10 µM PACOCF
. As shown in Fig. 6, the
rate of inactivation in the presence of PACOCF
was
significantly less than that observed in its absence. This protection
afforded by PACOCF
suggests that the binding sites on the
enzyme for these two inhibitors are at least partially overlapping.
Figure 6:
Protection of the P388D
Ca
-independent PLA
from inhibition by
BEL. The P388D
PLA
was preincubated with 100
nM BEL in the presence (
) or absence (
) of 10
µM PACOCF
for the indicated time periods at 40
°C, followed by dilution into the assay mixture and quantification
of remaining activity. These results are expressed relative to the
control rate measured in the presence of Me
SO (for
)
or Me
SO plus PACOCF
(for
) and are
plotted on a semilogarithmic plot. Each point represents the average of
duplicates.
Figure 7:
Covalent modification of the P388D Ca
-independent PLA
by
[
H]BEL and its attenuation with DTNB-inactivated
enzyme. A,
2-3 µg of the P388D
PLA
was incubated with 2 µM [
H]BEL for 30 min at 40 °C, followed by
separation by SDS-PAGE on a 10% gel and visualization by
autoradiography. B, control(-) or DTNB-inactivated
(+) P388D
PLA
(1-2 µg) was
incubated with 2 µM [
H]BEL for 30
min at 40 °C. Samples were subsequently separated by SDS-PAGE on a
10% gel and visualized by fluorography.
A similar experiment
was also carried out utilizing enzyme that had been treated with DTNB
prior to inhibition with [H]BEL. Treatment of the
P388D
Ca
-independent PLA
with
1 mM DTNB resulted in the complete loss of PLA
activity. As shown in Fig. 7B, utilization of
this DTNB-inactivated enzyme resulted in the near complete lack of
covalent binding of [
H]BEL to the M
80,000 protein, as well as the complete lack of
the label at the top of the gel, suggesting the necessity for a
catalytically competent enzyme for incorporation of label. It should be
noted that visualization for the experiment shown in Fig. 7B was carried out utilizing fluorography and therefore resulted in a
much higher degree of sensitivity than that observed in Fig. 7A. As can be seen, under these more sensitive
conditions, the M
80,000 band appears much darker
and even overloaded, and there is also a previously unobserved very
faint band near the bottom of the gel at M
36,000. Because this band is only a very small fraction of
the total label and was only detected under these more sensitive
conditions, it is most likely the result of a degradation product or
contamination with another protein that reacts with BEL.
Results
reported herein demonstrate that the P388D PLA
is also inhibited by AACOCF
as well as a new analog,
PACOCF
. Inhibition was found to be concentration-dependent,
with IC
values of
0.028 and 0.0075 mol fractions,
respectively. Competition experiments carried out in the presence of
BEL demonstrate that this inhibition is mediated through a direct
binding of the inhibitor to the enzyme, most likely at the active site,
consistent with results obtained with the cPLA
. However, in
contrast to the Group IV cPLA
, kinetic experiments reveal
linear progress curves in the presence of both inhibitors, and the
PACOCF
PLA
complex was found to rapidly
dissociate upon dilution. Taken together, these data are consistent
with a classical mechanism of reversible inhibition.
These data
represent the first report of inhibition of a
Ca-independent PLA
by trifluoromethyl
ketone inhibitors. It is difficult to directly compare the exact
potency of inhibition between the macrophage iPLA
and the
cPLA
due to the different types of inhibition observed. In
addition, it should be kept in mind that the IC
values
reported for the macrophage iPLA
are a function of the
assay conditions utilized and should only be taken as an upper limit
estimate of the true K
value, which may be much
lower. In any case, the ability of these compounds to inhibit multiple
intracellular PLA
in the low micromolar range indicates
that their use as specific inhibitors for in vivo studies
should be carried out with some caution. For example, the P388D
macrophages contain a Group IV cPLA
, a Group II
sPLA
, and the Ca
-independent
iPLA
(24) , and therefore, these inhibitors cannot
be used indiscriminately in this cell type to inhibit specifically
either the Group IV cPLA
or the
Ca
-independent iPLA
. On the other hand,
AACOCF
has been used recently in calcium ionophore- and
thrombin-stimulated platelets to implicate the involvement of the
cPLA
rather than the sPLA
in arachidonic acid
release(25, 26) . Structure/function studies were also
carried out with several different compounds besides AACOCF
to help eliminate nonspecific inhibition
effects(25, 26) .
Recently, BEL has been shown to be a
potent suicide inhibitor of the myocardial
PLA(12, 27) . This inhibition was found to
be at least 1000-fold more potent toward the myocardial PLA
than toward the Group I or III sPLA
(12) .
Because of the similarities between the myocardial PLA
and
the macrophage PLA
, we examined the ability of BEL to
inhibit the P388D
PLA
in the hope of gaining
insight into the relationship between these two enzymes. We have found
that (a) BEL is a potent inhibitor of the P388D
iPLA
, with half-maximal activity found at 60 nM after a 5-min preincubation at 40 °C; (b) inhibition
is irreversible when subjected to a 1500-fold dilution and covalent as
demonstrated by the incorporation of [
H]BEL,
which persisted through SDS-PAGE treatment; (c) inhibition is
time-dependent and shows pseudo first-order kinetics; (d) this
time-dependent inactivation is slowed in the presence of the reversible
inhibitor PACOCF
; and (e) a catalytically active
enzyme is necessary for covalent modification as DTNB-inactivated
PLA
had a greatly diminished capacity for incorporation of
label.
Taken together, these data indicate that BEL is a covalent
irreversible inhibitor of the P388D PLA
, with
inactivation proceeding through an enzyme-mediated process. These
results are consistent with the documented cases of suicide inhibition
utilizing BEL with both the myocardial PLA
(12) and
chymotrypsin (17) . In addition, we have found that the
hydrolyzed form of BEL (bromomethyl ketone) is not inhibitory. This
indicates that the bromomethyl ketone (which is proposed to be the
reactive species responsible for irreversible modification of both the
myocardial PLA
and chymotrypsin) is not released from the
enzyme prior to irreversible inactivation, i.e. ruling out a
metabolically activated mechanism(22) . Furthermore, the
sensitivities and characterizations of BEL inhibition observed with the
P388D
PLA
were strikingly similar to those
observed with the myocardial PLA
. Both enzymes were
inhibited by BEL in the nanomolar range, both showed an initial burst
of inhibition followed by a first-order time-dependent inactivation,
and both enzymes lost their ability to incorporate
[
H]BEL label after inactivation with DTNB. These
data are intriguing in that they suggest that these two enzymes may
share very similar active-site environments.
However, despite these
similar sensitivities to BEL, treatment of the macrophage PLA with [
H]BEL resulted in the labeling of a
single major M
80,000 protein, as opposed to the M
40,000 band documented with the myocardial
PLA
. These data distinguish the macrophage PLA
from the myocardial PLA
and provide strong evidence
that the M
80,000 protein is the catalytic
subunit, and not phosphofructokinase. Thus, the ATP activation and
oligomerization observed with both of these enzymes appear to be a
function of distinct regulatory mechanisms.
In conclusion, we have
demonstrated that PACOCF, AACOCF
, and BEL
inhibit the macrophage Ca
-independent PLA
activity. Based on the data presented herein and in analogy with
results obtained with the cPLA
and the myocardial
iPLA
, we propose that the trifluoromethyl ketones are
classical reversible inhibitors of the macrophage iPLA
and
that BEL is a suicide inhibitor. More important, these data demonstrate
that the myocardial iPLA
and the macrophage iPLA
are distinct enzymes and provide new evidence that the M
80,000 protein is the macrophage iPLA
catalytic subunit.