(Received for publication, December 26, 1995; and in revised form, February 9, 1996)
From the
We recently identified a cytosolic phospholipase A activity in bovine brain and testis that preferentially
hydrolyzes phosphatidic acid substrates. We also showed that the enzyme
displays sigmoidal kinetics toward phosphatidic acid substrates in a
Triton X-100 mixed micelle assay system (Higgs, H. N., and Glomset, J.
A.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9574-9578).
In the present work we purified the bovine testis enzyme 14,000-fold
and used a combination of size exclusion chromatography, labeling with
the phospholipase A inhibitor, methyl arachidonyl fluorophosphonate,
and SDS-polyacrylamide gel electrophoresis to provide evidence that it
is a homotetramer of 110-kDa subunits. Studies of the molecular basis
of the enzyme reaction in Triton micelles revealed that (a) a
nonhydrolyzable sn-1-alkyl-2-oleoyl analogue of phosphatidic
acid activated the enzyme 30-fold in a sigmoidal fashion (Hill
coefficient 3.2, EC
4 mol %) without substantially
affecting its preference for specific diacyl phosphoglyceride
substrates, (b) the activator promoted tight binding of the
enzyme to micelles, and (c) the enzyme's activity toward
unsaturated phosphatidic acid substrates was affected by the location
and nature of the fatty acyl chain double bonds.
Phospholipases of the A type (PLA
) (
)cleave the sn-1 fatty acyl group from diacyl
glycero-phospholipids, producing a free fatty acid and an sn-2-lysophospholipid. A variety of PLA
activities
have been identified. Membrane-bound PLA
s are present in
platelets, liver, and heart(1, 2, 3) . There
also seem to be a number of soluble lysosomal enzymes(4) , some
of which can also catalyze transacylation reactions (5) . In
addition, cytosolic PLA
activities have been identified in
heart, brain, and
testis(6, 7, 8, 9) . This diversity
of PLA
activities strongly suggests a diversity of cellular
functions. Given the ever expanding roles of phospholipid metabolites
in cellular signaling(10) , it is likely that one role of
PLA
enzymes is to produce or remove such metabolites.
A
detailed enzymatic examination of these enzymes would greatly assist
the elucidation of their cellular roles. Unfortunately, comparison of
the enzymological properties of the PLA enzymes has been
hampered by the fact that different investigators have used
fundamentally different assay systems. Some systems present the
substrate as an undefined aggregate of either individual classes of
phospholipid (8, 11) or phospholipids in combination
with submicellar concentrations of detergent(7, 12) .
In either case, the probability of artefactual results is high, due to
the drastically different structures formed by single phospholipids of
varying headgroup and acyl chain composition(13) .
Interpretation of results obtained with these systems is also hindered
by the inability to alter the surface concentration of phospholipid,
making comparisons of accepted kinetic parameters impossible.
A number of well-defined systems for the study of other lipolytic enzymes have been developed. One of the most convenient is the mixed micelle system, in which the phospholipid is presented as a low percentage component in detergent micelles(14) . This system has clear advantages over those mentioned above: (a) micelle structure is primarily determined by the detergent, reducing the variability of surface architecture between different lipids, (b) the surface concentration of substrate can be varied, allowing the determination of Michaelis-Menten parameters, and (c) other lipids can be added to the system in a controlled fashion, aiding in the determination of cofactor requirements.
We recently used a Triton X-100 mixed micelle
system to study a cytosolic PLA (PA-PLA
) that
displayed interesting enzymatic properties. First, it preferentially
hydrolyzed phosphatidic acid (PA) by 4-fold over phosphatidylinositol
(PI), 5-fold over phosphatidylserine (PS), 7.5-fold over
phosphatidylethanolamine (PE), and 10-fold over phosphatidylcholine
(PC). Second, it hydrolyzed diunsaturated molecular species of PA, such
as diarachidonoyl (478 nmol/min/mg) or dioleoyl PA (208 nmol/min/mg) in
preference to molecular species with an sn-1 saturated fatty
acyl chain, such as sn-1-palmitoyl-2-oleoyl (80 nmol/min/mg)
or sn-1-stearoyl-2-arachidonoyl PA (42 nmol/min/mg). Third, it
appeared to be cooperatively activated by PA, as each PA substrate
displayed sigmoidal kinetics with a Hill coefficient of 3(9) .
Fourth, the tissue distribution of PA-PLA
activity was
limited. High levels were present in bovine testis and brain, and the
specific activity of the enzyme was 10-fold higher in mature testis
than in immature testis. However, PA-PLA
was undetectable
in liver, spleen, heart, or blood. Though the enzyme's biological
role has yet to be determined, this distribution of enzyme activity
suggests that PA-PLA
may be involved in cell signaling.
In the current study, we purified the enzyme to the point where a
single major band could be seen by silver-stained SDS-PAGE and then
addressed the following questions. By what mechanism does PA activate
PA-PLA, and how does this activation affect the
enzyme's preference for PA over other lipids? What is the
structural basis for the enzyme's preference for diunsaturated
PAs? Does PA-PLA
behave similarly to a recently purified
brain PLA
(7, 12) if assayed under the
conditions used for this enzyme, and do results in this assay system
agree with those obtained in the Triton micelle system? The results of
these studies are described below.
Characterization of the enzyme, purified through the
Superdex 200 step, was carried out under different assay conditions.
The final buffer concentrations in the 100-µl assay were 50 mM MOPS, pH 7.2, 100 mM KCl, 1 mM EGTA, and 16
mM total micellar lipid, with the bulk detergent being a 1:1
molar mix of Triton X-100 to Triton X-114. Assays included 0.25 mol %
of [P]PA, with the rest of the substrate PA
being nonradiolabeled. All subsequent steps in the assay were the same
as those outlined above.
When PLA activity toward other phospholipid
classes was assayed, conditions were the same as those described above.
Lyso-PS, lyso-PI, and lyso-PE generation by the enzyme were measured by
the same reversed phase TLC technique as described for PA. PC breakdown
was measured by the release of C-oleic acid, which was
separated from PC and lyso-PC by TLC on Silica 60 plates developed in
6:8:2:2 CHCl
:acetone:acetic acid:water.
Under all conditions used, assays were linear for time and protein concentration. All assays were carried out in borosilicate glass tubes siliconized with dichlorodimethylsilane. For each lipid class tested, the fraction of lyso-lipid in the upper phase during extraction was measured and was factored into calculations. Calculation of kinetic parameters was carried out following previously established methods for micellar systems(14) . The program, GraFit (Erithacus Software Ltd., London), was used for making graphs of kinetic data.
For conversion of the
CDP-DAG to PI, the solvent was evaporated under nitrogen. The CDP-DAG
was then resuspended in 4 ml of 50 mM Tris-HCl, pH 8.0, 30
mM Triton X-100, and 50 mM myo-inositol. When the
CDP-DAG was fully resuspended, MgCl was added to 16
mM, and solubilized yeast membranes were added to 0.3 mg
protein/ml. The yeast membranes were a kind gift of G. M. Carman,
supplied as a 3 mg/ml protein stock in 50 mM Tris-HCl, pH 8.0,
30 mM MgCl
, 10 mM
-mercaptoethanol,
20% glycerol (w/v), and 16 mM Triton X-100. The reaction was
incubated at 25 °C for 16 h with agitation. Lipids were extracted
by the method of Folch(15) , and the solvent from the lower
phase was evaporated under nitrogen. The residue was resuspended in 1
ml of CHCl
and passed over a BakerBond PRS column
equilibrated in the same solvent. After washing with 4 ml of
CHCl
, followed by 8 ml 9:1 CHCl
:MeOH, PI was
eluted with 8 ml of 75:25:3 CHCl
:MeOH:water. The solvent
was evaporated under nitrogen, and the pure PI was resuspended in 1 ml
of CHCl
. Purity was judged by TLC in 6:8:2:2:1
CHCl
:acetone:MeOH:acetic acid:water. When
P-labeled dioleoyl PI was synthesized, its radiochemical
purity was >96%.
The combination of ammonium sulfate and PEG
3350 precipitations greatly increased the capacity of the subsequent
SP-Sepharose column for the extract. Interestingly, although the high
speed supernatant bound to the SP column in the presence of 20 mM KCl, solubilized PEG precipitates flowed through even in the
absence of KCl. However, these PEG precipitates did bind in the
presence of 3 M urea and 2 mM Thesit, while the
enzyme retained the majority of its activity (>80% after 2 h at 4
°C). Treatment of the PEG precipitate with urea and Thesit probably
served to release other proteins that had become tightly bound to the
PLA during the precipitations.
The inclusion of the
nonionic detergent, Thesit, at concentrations around its critical
micelle concentration (0.1 mM) enhanced the stability of
PA-PLA during the purification. For example, incubation of
the Mono Q-purified fraction for 20 h at 4 °C resulted in a
recovery of 25% without detergent, whereas 80% was recovered if 0.2
mM Thesit was included. Other nonionic detergents, such as
Triton X-100, Triton X-114, and octyl glucoside, behaved similarly.
Thesit was chosen over the others because its low UV absorption did not
interfere with the monitoring of chromatographic steps at 280 nm.
The concentration of KCl present during loading of the Mono Q column (Fig. 1A) affected the overall purification obtained by
that column. Loading at 200 mM KCl gave a 2-fold increase in
enrichment over loading at 100 mM followed by washing with 200
mM. PA-PLA activity eluted at 280 mM KCl.
Thus, it appeared that a number of proteins that otherwise would have
bound to the column and eluted at the same KCl concentration as
PA-PLA
were unable to bind to the column at 200 mM KCl.
Figure 1:
Chromatographic elution profiles and
SDS-PAGE evaluation of PA-PLA purification. Elution
profiles from Mono Q (A) and Superdex 200 (B)
chromatography are shown. Elution volumes for markers on Superdex 200
(molecular mass in kDa in parentheses) were as follows: blue dextran
2000 (2,000,000), 43 ml; thyroglobulin (669), 47 ml; ferritin (440), 55
ml; catalase (232), 65 ml; aldolase (158), 67 ml; phosphorylase B (97),
68 ml; bovine serum albumin (67), 75 ml; ovalbumin (43), 83 ml;
chymotrypsinogen (25), 91 ml. C, silver-stained gradient gel
SDS-PAGE of the Superdex 200 fractions (2 µl each). D,
silver-stained gradient gel SDS-PAGE of the pools from each of the
stages of purification. In lanes 1-7, 1 µg of
protein was loaded. In lanes 8 and 9, 50 ng of
protein was loaded. Lanes 8 and 9 were stained
separately in order to bring out minor bands. Lane 1, ammonium
sulfate precipitate; lane 2, PEG precipitate; lane 3,
SP pool; lane 4, concentrated SP pool; lane 5, Mono Q
pool; lane 6, concentrated Mono Q pool; lane 7,
Superdex 200 pool; lane 8, phenyl-Superose pool; lane
9, phenyl-CL-4B pool.
PA-PLA activity eluted as a peak centered at
440 kDa on Superdex 200 size exclusion chromatography (SEC) (Fig. 1B). The active fractions from this column were
pooled and passed over two successive phenyl-based hydrophobic resins:
phenyl-Superose and phenyl-CL-4B. On each of these columns, the enzyme
eluted over a broad range of NaCl when gradients were applied, even
though the concentrations of salt needed for elution were very
different (900 mM NaCl for phenyl-Superose and <100 mM for phenyl-CL-4B). For this reason, step elutions were applied,
with no loss of enrichment by these columns.
When fractions from
each step were analyzed by silver-stained SDS-PAGE, a band of 110 kDa
was the major band remaining at the end of the purification (Fig. 1D). Furthermore, the elution of this band from
SEC paralleled the elution of PA-PLA activity (Fig. 1, B and C). These results suggest that
the 110-kDa protein contains the PA-PLA
activity. In
purifications where less care was taken to inhibit endogenous protease
activity, additional major bands of 97 and 85 kDa were also observed,
suggesting that these bands were proteolytic products of the 110-kDa
band.
Figure 2:
MAFP inhibition and labeling of
PA-PLA. PA-PLA
(0.3 µg of the Superdex 200
pool) was incubated with 50 µM Triton X-100 or with 4
µM
C-labeled MAFP in 50 µM Triton X-100. At various times, aliquots were removed, diluted in
buffer containing 10 mM DTT, and tested for PA-PLA activity
while other aliquots were removed, boiled in SDS-PAGE loading buffer
containing 10 mM DTT, and run on 2.7-14% gradient
SDS-PAGE. The radioactive bands on the gel were observed by
autoradiography. A, inhibition curve for
C-MAFP. B, silver-stained SDS-PAGE of samples incubated for the
indicated times with
C-MAFP. This gel was heavily
overstained in order to show the minor bands present in the fractions. C, autoradiogram of SDS-PAGE showing time-dependent labeling
of the 110-kDa band with
C-MAFP. The 110-kDa band was
labeled with
C-MAFP on two separate
occasions.
Although PA-PLA migrated
as a 110-kDa band on SDS-PAGE (Fig. 1D), it eluted from
Superdex 200 SEC with an apparent molecular mass of 440 kDa (Fig. 1B). The enzyme also migrated at 440 kDa on a
silica-based, Toso Haas 3000 column (data not shown). One possible
explanation for this difference is that the enzyme exists as a
homotetramer in solution.
Figure 3:
Activation of PA-PLA by
alkyl-oleoyl-phospholipids. A, hydrolysis of varying
concentrations of dioleoyl-[
P]PA by PA-PLA
(0.6 ng) in micelles of 1:1 (mol:mol) Triton X-100:Triton X-114
in the absence of AO-PA (open circles), presence of 10 mol %
AO-PA (closed circles), and presence of variable AO-PA such
that the concentration of total PA (dioleoyl PA + AO-PA) remained
at 10 mol % (open squares). There was 16 mM total
micellar lipid. B, effect of varying concentrations of
AO-phospholipids on PA-PLA
hydrolysis of 0.5 mol %
dioleoyl-[
P]PA in Triton micelles (same
conditions as above). Closed circles, AO-PA; open
circles, AO-PS; closed triangles,
AO-phosphatidylmethanol; closed squares, AO-PC. Each
experiment was conducted in duplicate on two separate occasions with
similar results.
When PA-PLA activity
toward increasing mole percentages of dioleoyl PA was retested in the
presence of 10 mol % AO-PA, the kinetics were hyperbolic (Fig. 3A). Under these conditions, the enzyme displayed
an apparent K
(K
) of
5.22 mol % and a V
of 392 µmol/min/mg (Table 2). When the total mole percentage of PA (dioleoyl PA
+ AO-PA) was kept at 10 while the ratio of AO-PA to dioleoyl PA
was varied from 19 to 0, the results mimicked those observed at 10 mol
% AO-PA. Thus, it appeared that AO-PA did not compete significantly
with hydrolyzable PA for binding to the enzyme's active site.
Other anionic lipids also could stimulate PA-PLA.
AO-phosphatidylmethanol stimulated dioleoyl PA hydrolysis to an extent
similar to that stimulated by AO-PA (Hill coefficient of 2.7) but with
an EC
of 9 mol % (Fig. 3B). Similarly,
AO-PS cooperatively activated the enzyme but at higher concentrations
than for AO-PA. At 20 mol % AO-PS, dioleoyl PA hydrolysis had not fully
plateaued. Higher concentrations of AO-PS were not tried for fear of
severely altering the state of the micelles. In contrast to the
stimulatory effect of these anionic lipids, AO-PC had no effect on the
hydrolysis of PA (Fig. 3B).
Figure 4:
Influence of AO-PA on micelle binding by
PA-PLA. The enzyme (6 ng) was incubated in 100 µl of
assay buffer containing Triton X-114 micelles with 0.5 mol % dioleoyl
PA and varying amounts of AO-PA (16 mM total micellar lipid)
for 5 min at 25 °C, followed by a 5-min centrifugation at 16,000
g at the same temperature. 80 µl of supernatant
were removed, and the pellet was resuspended in 80 µl of assay
buffer. Both pellet and supernatant were assayed in Triton X-100:Triton
X-114 micelles containing 0.5 mol %
dioleoyl-[
P]PA and 10 mol % AO-PA (16 mM total micellar lipid). In separate experiments, the increase in
PA-PLA
(0.6 ng) activity in response to increasing AO-PA in
Triton X-100:Triton X-114 micelles containing 0.5 mol %
dioleoyl-[
P]PA was tested. Similar results were
obtained in two separate experiments conducted in
duplicate.
The effect of calcium ions on both PA-PLA activity and
surface binding in response to activator further suggested that the
activator functions by enhancing the interaction of PA-PLA
with the surface. With no AO-PA in the assay, CaCl
had a slightly stimulatory effect on PA-PLA
activity (Fig. 5; see legend). In the presence of 10 mol % AO-PA,
however, CaCl
inhibited activity, with an IC50 of 0.6
mM. This inhibition was paralleled by a decrease in
PA-PLA
binding to Triton X-114 micelles (Fig. 5).
These results suggest that calcium ions compete with the enzyme for
binding to the activator, causing less surface binding and, as a
consequence, less PA-PLA
activity. Magnesium ions also
stimulated the enzyme in the absence of activator (Fig. 5; see
legend) but inhibited when 10 mol % AO-PA was included (Fig. 5).
However, activator-containing micelles precipitated in the presence of
magnesium ions, making interpretation of this ion's effect
difficult.
Figure 5:
Effect of divalent cations on PA-PLA activity/binding. Both binding (6 ng enzyme) and activity (0.6 ng
enzyme) measurements used micelles of 16 mM total micellar
lipid containing 10 mol % AO-PA and 0.5 mol % dioleoyl PA. Binding
measurements were conducted in Triton X-114, whereas activity
measurements were conducted in 1:1 (mol:mol) Triton X-100:Triton X-114.
Free calcium ion concentrations were varied in the presence of 2 mM nitrilotriacetic acid, using established binding
constants(32) . The binding or activity in the absence of
CaCl
was set at 100%. In the absence of AO-PA, PA-PLA
activity in the presence of calcium was as follows: 130% at 0.1
mM, 150% at 0.5 mM, and 206% at 2 mM. For
the MgCl
study, conditions were the same as above.
Precipitates were observed at all concentrations of magnesium but grew
more pronounced at higher concentrations. In the absence of AO-PA,
PA-PLA
activity in the presence of magnesium was 108% at
0.5 mM, 121% at 2 mM, 148% at 5 mM, and 177%
at 10 mM. Similar results were obtained in two separate
experiments conducted in duplicate.
The importance of surface binding to PA-PLA activity was further tested by varying the concentration of
micelles in the assay. If the sole effect of AO-PA were to increase the
fraction of enzyme bound to micelles, then in the absence of AO-PA the
activity of PA-PLA
should increase as the total
concentration of micelles increases, but in the presence of AO-PA the
enzyme's activity should be fairly independent of this parameter.
However, we found that the enzyme's activity was independent of
surface concentration under both experimental conditions when the
concentration of total micellar lipid exceeded 12 mM (Fig. 6). These results seem to contradict the micelle
binding experiments, which were conducted at 16 mM total
micellar lipid. A possible explanation is that the enzyme can bind to
surfaces with different degrees of affinity (see
``Discussion'').
Figure 6:
Surface dilution properties of unactivated
and fully activated PA-PLA. Micelles of 1:1 (mol:mol)
Triton X-100:Triton X-114 containing either 0.5 mol %
dioleoyl-[
P]PA or 0.5 mol %
dioleoyl-[
P]PA, 10 mol % AO-PA were assayed for
PA-PLA
(0.6 ng) activity at various total micelle
concentrations for 20 min at 25 °C. Similar results were obtained
in two separate experiments conducted in
duplicate.
Figure 7:
Headgroup preference of
PA-PLA. Dioleoyl molecular species of phospholipids were
compared as substrates for PA-PLA
(0.6 ng) in the presence
of 10 mol % AO-PA in micelles of 1:1 (mol:mol) Triton X-100:Triton
X-114 (16 mM total micellar lipid). Closed circles,
PA; open circles, PI; closed squares, PE; open
squares, PC; closed triangles, PS. Similar results were
obtained in two separate experiments conducted in
duplicate.
Figure 8:
Acyl chain preference of
PA-PLA. A, dioleoyl PA (closed circles)
is compared with diarachidonoyl PA (closed squares),
dilinolenoyl PA (open squares), dilinoleoyl PA (crosses), stearoyl-arachidonoyl PA (closed
triangles), and palmitoyl-oleoyl PA (open triangles) in
micelles of 1:1 (mol:mol) Triton X-100:Triton X-114 containing 10 mol %
AO-PA (16 mM total micellar lipid) as substrates for
PA-PLA
. B, dioleoyl PA (closed circles)
is compared with dielaidoyl PA (closed squares) and
dipetroselinoyl PA (open squares) as substrates for
PA-PLA
under the same conditions as in A. Similar
results were obtained for each compound in two separate experiments
conducted in duplicate.
To test the
possibility that the testis PA-PLA might be related to the
brain PLA
, we assayed the activity of PA-PLA
under conditions similar to those used for the brain enzyme.
Under these conditions, PA-PLA
behaved similarly to the
brain enzyme in all properties tested (Table 3). We then asked
whether PA was a preferred substrate in this system. Under the exact
conditions of the brain assay system, dioleoyl PA was 8-fold less well
utilized than was sn-1-palmitoyl-2-arachidonoyl PE. However,
the buffer in this system contained a superphysiological concentration
of MgCl
, which we had previously shown to be inhibitory to
AO-PA-stimulated PA hydrolysis by PA-PLA
in the Triton
assay system (Fig. 5). When MgCl
was removed from
the brain system, PA-PLA
activity toward PA increased over
8-fold, making PA a slightly better substrate than PE (Fig. 9).
Thus, the catalytic properties of PA-PLA
in the brain
PLA
assay system are very similar to those of the brain
enzyme and are in conflict with those obtained in the Triton micelle
system. However, when the brain assay system was made more similar to
our system by removal of magnesium ions, PA was again found to be the
preferred substrate.
Figure 9:
Effect of MgCl on PA-PLA
activity in the brain PLA
assay system. PA-PLA
(0.4 ng) was assayed in 100 mM Tris-HCl, pH 7.5, 1
mM EDTA, 70% glycerol, and 325 µM CHAPS, in the
absence or presence of 3 mM MgCl
, for the
hydrolysis of 10 µM dioleoyl [
P]PA, sn-1-palmitoyl-2-[
C]arachidonoyl PE, or sn-1-palmitoyl-2-[
C]arachidonoyl PC.
For details of the assay procedure, see ``Experimental
Procedures.'' Assays were conducted in duplicate on two separate
occasions.
We purified bovine testis PA-PLA 14,000-fold and
used a Triton mixed micelle assay system to examine its properties. The
purified enzyme had an apparent molecular mass of 440 kDa as determined
by SEC but corresponded to a major band of 110 kDa as determined by
SDS-PAGE and labeling with the phospholipase inhibitor, MAFP. This
strongly suggests that PA-PLA
exists as a homotetramer of
110-kDa subunits in solution. However, definitive proof of the
enzyme's identity will have to await cloning of the cDNA for the
110-kDa protein and evidence that the cellular expression of this cDNA
is associated with a parallel increase in PA-PLA
activity.
Proteins of lower molecular weight (97 and sometimes 85 kDa),
observed in earlier purification trials, appeared to be proteolytic
products of the 110-kDa polypeptide. They were not observed in later
purifications, where proteolysis was reduced by freezing fresh cubes of
tissue in liquid nitrogen immediately after removing testes from bulls,
rapid thawing of the tissue cubes during homogenization, and adding an
extensive protease inhibitor mixture to all purification buffers.
Furthermore, when an earlier enzyme preparation containing the 110-,
97-, and 85-kDa bands was treated with radiolabeled MAFP and analyzed
by SDS-PAGE, all three bands incorporated the label. ()
Pete et al. recently purified a soluble
PLA from bovine brain that displayed bands of 112 and 95
kDa on SDS-PAGE (7) . Though further studies were not done to
determine whether either of these polypeptides corresponded to the
enzyme, analysis of the native protein by SEC revealed active material
of 365 kDa, suggesting that this protein might also be a tetramer. The
protease inhibitors included in buffers during this purification were
similar to those we used when three bands appeared on SDS-PAGE,
suggesting that the 95-kDa band might have been a proteolytic product
of the 112-kDa band. A phospholipase A
from macrophages
also appeared to be a homotetramer of 80-kDa subunits in
solution(25, 26) . These similarities are intriguing,
but their functional significance is unknown.
It would be
interesting to know whether the tetrameric nature of these enzymes
provides a means for their regulation. We observed sigmoidal kinetics
in our enzyme assay experiments with mixed micelles of Triton
containing either increasing concentrations of PA substrates or a fixed
concentration of PA substrate and increasing concentrations of AO-PA (Fig. 3, A and B). In addition, our studies of
PA-PLA binding to mixed micelles of Triton X-114 revealed
an activator-dependent, sigmoidal increase in surface binding that
paralleled the activator-dependent increase in enzyme activity (Fig. 4). These sigmoidal effects may reflect cooperative
interactions between the subunits of the tetramer, but could also be
due to interactions involving multiple sites within subunits. A number
of monomeric proteins display sigmoidal binding to lipid surfaces. For
example, small basic peptides, protein kinase C, and annexins bind with
apparent cooperativity to negatively charged
surfaces(27, 28, 29) . Therefore, mechanisms
other than those involving the tetrameric state of PA-PLA
could be responsible for the observed sigmoidal effects.
The parallel sigmoidal effects of AO-PA on the enzyme's binding and activity do, however, suggest that an increase in some type of binding may be responsible for enzyme activation. In our current working model, each enzyme subunit contains two different types of binding site, a single catalytic site that preferentially binds diacyl PA and at least one activator-binding site that binds diacyl PA, AO-PA, or another anionic lipid. In support of this model, AO-PA activated the enzyme without noticeably inhibiting the hydrolysis of diacyl PA (Fig. 3, A and B). In addition, AO-PS effectively activated the enzyme even though diacyl PS was a very poor substrate (Fig. 3B and Fig. 7). Furthermore, the fact that AO-PA increased the enzyme's ability to hydrolyze PE and PC, both poor substrates compared with diacyl PA, suggests that the enzyme first binds to AO-PA via noncatalytic sites and then interacts with diacyl phosphoglyceride substrates via its catalytic site.
The
model might also explain the apparently contradictory results of our
surface dilution studies, which revealed that the enzyme's
activity was not affected by changes in micelle concentration in excess
of 12 mM, even in the absence of AO-PA (Fig. 6). This
suggested that all of the enzyme was bound to the surface even in the
absence of the activator. However, several states of enzyme activity
and binding might exist if the PA-PLA contains multiple
activator-binding sites and if the enzyme's activity and strength
of binding to micelles increase in parallel with increasing occupancy
of these sites, as illustrated in .
where E is unbound enzyme, M is micelle surface, E-M is the enzyme-micelle complex, PA,
PA
, and PA
are sequentially binding activators,
and E*
-M, E*
-M, and E*
-M are the increasing affinities of E-M
binding due to increasing activator concentration. Following this
model, the enzyme might bind weakly to the micelle surface in the
activator's absence and dissociate during the centrifugation step
of the Triton X-114 binding assay. Conversely, in the presence of
activator, PA-PLA
might bind more strongly to the surface
and pellet with the micelles upon centrifugation. Stronger binding
might also bring all four catalytic sites into proximity with
substrates, leading to maximal enzyme activity.
The AO-PA-activated
enzyme catalyzed the hydrolysis of diacyl PA at a much greater rate
than it did the other diacyl phosphoglycerides tested. This strongly
suggests that the catalytic site preferentially interacts with the
phosphate headgroup over those containing phosphodiesters. In addition,
the fully activated enzyme catalyzed the hydrolysis of diunsaturated
molecular species of diacyl PA at greater rates than it did sn-1-saturated-2-unsaturated molecular species. The molecular
basis of this preference has yet to be determined, but both dielaidoyl
(di18:19trans) and dipetroselinoyl PA (di18:1
6cis) were
appreciably poorer substrates than was dioleoyl-PA (di18:1
9cis).
The former two species would be predicted to be more ordered than
dioleoyl PA, based on comparisons of the phase transition temperatures
of the corresponding molecular species of PC and
PE(30, 31) . Moreover, diunsaturated molecular species
of PC and PE species are less ordered than sn-1-saturated-2-unsaturated species, based on similar
comparisons. The degree of substrate acyl chain order could conceivably
affect the enzyme's ability to bind substrate or transfer
substrate from the surface of a micelle into its substrate-binding
pocket.
Whereas our experiments with mixed micelles seemed to yield
important new information about the enzyme's properties, our
experiments with the assay system used in the study of a brain
PLA yielded very different results, as PA-PLA
mimicked the brain enzyme in a number of characteristics (Table 3). These results raise issues concerning both the
enzyme's identity and its properties. First, the similarity of
enzymatic properties between the two enzymes, coupled with similarities
in molecular mass (see ``Results''), suggests that they may
be closely related. Studies of the molecular biology of bovine testis
PA-PLA
are in progress in our laboratory and may help to
clarify this point.
The second issue concerns the validity of the
enzymatic data collected with these different assay systems. Whereas
our studies with Triton micelle assay systems identified PA as the
major substrate, PE was a better substrate in the brain enzyme assay
system. One reason for this was that the superphysiological levels of
MgCl present in the buffer specifically inhibited PA
hydrolysis (Fig. 9). When the brain PLA
assay system
was modified by removing magnesium ions, PA was slightly preferred over
PE as a substrate. The reason why the enzyme showed a higher relative
activity toward PE in this assay than it did in the Triton micelle
assay remains to be explained. However, PE is known to have special
physical properties. For example, liposomes can be made from pure
suspensions of PC but not from pure suspensions of PE. Furthermore,
some molecular species of PE readily form nonbilayer structures and
adopt inverted micellar conformations(13, 31) . Such
conformations cause a greater exposure of acyl chains than do
continuous bilayer structures, and the low dielectric constant of the
brain PLA
assay buffer (70% glycerol, no NaCl or KCl) might
further favor acyl chain exposure. PA-PLA
might interact
well with the exposed acyl chains of PE because it binds strongly to
hydrophobic chromatographic resins. In contrast, PE acyl chains are
probably not significantly exposed in the Triton micelle assay system.
Thus, the difference in relative activity toward PE in the two assay
systems might be due to the fundamentally different conformations
adopted by this lipid in the respective systems.
What is needed at
this point is an assay system that presents substrates in well defined
lipid bilayers. Such a system could be used to investigate in more
detail the properties elucidated in this study as well as to reveal
other as yet undetected properties. Experiments with unilamellar
liposomes are planned and would seem a logical next step in our efforts
to learn about how testis PA-PLA functions in intact cells.