(Received for publication, October 19, 1995)
From the
Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel
membrane-associated enzyme that catalyzes the dephosphorylation of the
phosphate of DGPP to yield phosphatidate and P
. DGPP
phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of
microsomal membranes followed by chromatography with DE53, Affi-Gel
Blue, hydroxylapatite, and Mono Q. The procedure resulted in the
isolation of an apparent homogeneous protein with a subunit molecular
mass of 34 kDa. DGPP phosphatase activity was associated with the
34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and
8.5 and was dependent on Triton X-100 for maximum activity. The enzyme
was inhibited by divalent cations, NaF, and pyrophosphate and was
relatively insensitive to thioreactive agents. The turnover number
(molecular activity) for the enzyme was 5.8
10
min
at pH 6.5 and 30 °C. DGPP phosphatase
exhibited typical saturation kinetics with respect to DGPP (K
= 0.55 mol %). The K
value for DGPP was 3-fold greater than
its cellular concentration (0.18 mol %). DGPP phosphatase also
catalyzed the dephosphorylation of phosphatidate, but this
dephosphorylation was subsequent to the dephosphorylation of the
phosphate of DGPP. The dependence of activity on phosphatidate (K
= 2.2 mol %) was cooperative
(Hill number = 2.0). DGPP was the preferred substrate for the
enzyme with a specificity constant (V
/K
) 10-fold greater than that for
phosphatidate. In addition, DGPP potently inhibited (K
= 0.35 mol %) the
dephosphorylation of phosphatidate by a competitive mechanism whereas
phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was
neither a substrate nor an inhibitor of pure phosphatidate phosphatase
from S. cerevisiae. DGPP was synthesized from phosphatidate
via the phosphatidate kinase reaction.
Diacylglycerol pyrophosphate (DGPP) ()is a novel
phospholipid metabolite recently identified from the plant Catharanthus roseus by Wissing and Behrbohm(1) . DGPP
contains a pyrophosphate group attached to diacylglycerol (Fig. 1). This compound was previously observed by several
workers (2, 3, 4) as the phospholipid product
of a lipid kinase reaction in plants that was not identified
correctly(1) . It is now known that DGPP is synthesized from PA
and ATP through the reaction catalyzed by the novel enzyme PA
kinase(1) . PA kinase is a ubiquitous membrane-associated
enzyme found in the plant kingdom(5) . The enzyme has been
purified from suspension-cultured C. roseus cells (5) and characterized with respect to its enzymological and
kinetic properties(6) .
Figure 1: Structure of DGPP.
Metabolic labeling studies using C. roseus have shown that DGPP is rapidly metabolized by a
membrane-associated phosphatase, which has been named DGPP phosphatase. ()DGPP phosphatase catalyzes the dephosphorylation of DGPP
to form PA and P
.
In addition to being present
in plant cells, DGPP phosphatase activity is also present in membrane
fractions of Escherichia coli, Saccharomyces
cerevisiae, and rat liver.
Whereas it is unclear what
role DGPP plays in phospholipid metabolism and cell growth, PA, the
product of the DGPP phosphatase reaction, plays a major role in lipid
metabolism. PA is the precursor of all phospholipids and
triacylglycerol(7, 8) . In addition, PA regulates the
activity of several lipid-dependent enzymes (9, 10, 11, 12) and has mitogenic
effects in animal cells (13, 14, 15) . Thus,
the discovery of DGPP phosphatase activity in a wide range of organisms
suggests that this enzyme may play an important role in phospholipid
metabolism and cell growth.
A purified preparation of DGPP phosphatase is required for defined studies on the mechanism and regulation of this novel enzyme of phospholipid metabolism. Owing to its amenable molecular genetic system, we are using the yeast S. cerevisiae as a model eucaryote to study DGPP phosphatase. We report in this paper the purification of DGPP phosphatase to apparent homogeneity. The purified enzyme was characterized with respect to its enzymological and kinetic properties. Moreover, we demonstrated that S. cerevisiae synthesized DGPP via the PA kinase reaction.
PA kinase activity was
measured with 40 mM imidazole-HCl buffer (pH 6.1), 10 mM MgCl, 100 mM NaCl, 0.5 mM dithiothreitol, 10 mM NaF, 5 mM
-glycerol
phosphate, 0.1 mM MnCl
, 6.9 mM Triton
X-100, 0.6 mM PA, 1 mM [
-
P]ATP (100,000 cpm/nmol), and 0.3
mg/ml membrane protein(1, 5) . NaF,
-glycerol
phosphate, and MnCl
were included in the reaction mixture
to inhibit phosphatase reactions. The
P-labeled
chloroform-soluble phospholipid product of the reaction, DGPP, was
analyzed with standard DGPP by thin-layer chromatography as described
above for the DGPP phosphatase assay.
PA phosphatase
(3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) was measured
with 50 mM Tris maleate buffer (pH 7.0), 10 mM 2-mercaptoethanol, 2 mM MgCl, 1 mM Triton X-100, 0.1 mM [
P]PA, and
enzyme protein(27) . A unit of enzymatic activity was defined
as the amount of enzyme that catalyzed the formation of 1 µmol of
product/min unless otherwise indicated. Specific activity was defined
as units/mg of protein.
Figure 2:
Elution profiles of DGPP phosphatase
activity after chromatography with hydroxylapatite, Mono Q I, and Mono
Q II and SDS-polyacrylamide gel electrophoresis of purified enzyme.
DGPP phosphatase was subjected to hydroxylapatite chromatography (panel A). Fractions containing DGPP phosphatase activity
under peaks I and II were subjected to chromatography
with Mono Q I (panel B) and Mono Q II (panel C),
respectively. Fractions were collected and assayed for DGPP phosphatase
activity () and protein (-). Phosphate (panel
A) and NaCl (panels B and C) gradient profiles
are indicated by a dashed line. The protein concentration in
the fractions from the Mono Q columns was too low to be detected
spectrophotometrically. Fractions 15 and 16 (lane 1), 17 and
18 (lane 2), 19 and 20 (lane 3), and 21 and 22 (lane 4) from the Mono Q I column (panel B) were
subjected SDS-polyacrylamide gel electrophoresis (right of
figure). Fractions 1 (lane 1), 2 (lane 2), 3 (lane 3), and 4 (lane 4) from the Mono Q II column (panel C) were subjected SDS-polyacrylamide gel
electrophoresis (right of figure). The position of the 34-kDa
subunit of DGPP phosphatase is indicated in the figures shown in panels B and C.
To confirm that the 34-kDa protein in the purified
DGPP phosphatase preparations was indeed DGPP phosphatase, we used
combination of native and SDS-polyacrylamide gel electrophoresis and
measuring activity from gel slices(34) . Purified enzyme was
subjected to native polyacrylamide gel electrophoresis in the presence
of 0.5% Triton X-100 at 5 °C. Following electrophoresis, one lane
from a slab gel was stained with silver and showed a single protein
band with an R of 0.6. The protein from the native
gel was excised, denatured, and subjected to SDS-polyacrylamide gel
electrophoresis. This analysis showed a 34-kDa protein band. A second
lane was cut into 0.5-cm slices, and each slice was minced with a razor
blade in assay buffer and homogenized at 5 °C. The samples were
then assayed for DGPP phosphatase activity using
[
-
P]DGPP as substrate. DGPP phosphatase
activity was found in those polyacrylamide gel slices which contained
the protein migrating with an R
of 0.6. The counts
of water-soluble
P
hydrolyzed from
[
-
P]DGPP were 50,000-60,000 cpm above
the counts derived from gel slices not containing protein. We were
unable to measure DGPP phosphatase activity directly from
SDS-polyacrylamide gel slices. Since the enzyme purified from
hydroxylapatite peak I was essentially pure and had a higher specific
activity than the enzyme purified from peak II, it was used for the
characterization of DGPP phosphatase activity.
Figure 3:
Time dependence of DGPP phosphatase
activity and identification of reaction products. Panel A,
DGPP phosphatase activity was measured with 10 nmol of
[-
P]DGPP as substrate for the indicated
time intervals. Following the incubations, the water-soluble
P
hydrolyzed from
[
-
P]DGPP was analyzed by scintillation
counting. Panel B, DGPP phosphatase activity was measured with
10 nmol of [
-
P]DGPP as substrate for the
indicated time intervals. Following the incubations, the
chloroform-soluble
P-labeled substrate and product of the
reaction were analyzed by thin-layer chromatography. Duplicate lanes
are shown for each time point in the figure. The positions of DGPP and
PA standards are indicated. Panel C, DGPP phosphatase activity
was measured with 10 nmol of [
-
P]DGPP as
substrate for the indicated time intervals. Following the incubations,
the chloroform-soluble
P-labeled product PA was separated
from [
-
P]DGPP by thin-layer chromatography
and then analyzed by scintillation counting. The water-soluble
P
hydrolyzed from
[
-
P]DGPP was analyzed by scintillation
counting.
Figure 4:
Effect of pH, divalent cations, and Triton
X-100 on DGPP phosphatase activity. DGPP phosphatase activity was
measured at the indicated pH values with 50 mM Tris-maleate-glycine buffer (panel A); the indicated
concentrations of MgCl, CaCl
, and MnCl
(panel B); and the indicated concentrations of Triton
X-100 (panel C).
The effect of the major S. cerevisiae phospholipids and diacylglycerol on DGPP phosphatase activity was examined. In these experiments, the surface concentration of lipids was 20-fold greater than that of DGPP. None of these lipids affected DGPP phosphatase activity by more than 50%. PA, the product of the reaction, did not affect activity.
Figure 5:
Dependence of DGPP phosphatase activity on
the surface concentration of DGPP. DGPP phosphatase activity was
measured as a function of the surface concentration (mol %) of
[-
P]DGPP. The molar concentration of DGPP
was held constant at 0.1 mM (
), 0.15 mM (
), and 0.2 mM (
) while the Triton X-100
concentration was varied. The inset is a reciprocal plot of
the data using DGPP at a molar concentration of 0.1 mM. The line drawn is the result of a least-squares analysis of the
data.
Figure 6:
Kinetics of the dephosphorylation of PA by
DGPP phosphatase. Activity was measured as a function of the surface
concentration (mol %) of [P]PA. The molar
concentration of PA was held constant at 0.1 mM (
) and
0.2 mM (
) while the Triton X-100 concentration was
varied.
We next
examined the effect of DGPP on the ability of DGPP phosphatase to
dephosphorylate PA. DGPP inhibited the dephosphorylation of PA in a
dose-dependent manner (Fig. 7). A kinetic analysis was performed
to examine the mechanism of DGPP inhibition on the activity using PA as
the substrate. The dependence of activity on the surface concentration
of PA was measured in the absence and presence of 0.5 mol % DGPP (Fig. 7, inset). The kinetic patterns of the enzyme
reaction were cooperative (Hill number of 2.0) in the absence and
presence of DGPP. The data were transformed to a double-reciprocal plot
where the PA surface concentration was raised to the Hill number of
2(36) . DGPP did not affect the V value
for the enzyme but did cause an increase in the K
for PA (Fig. 7, inset). These results were
consistent with DGPP being a competitive inhibitor with respect to PA.
A K
value for DGPP was calculated to be 0.35 mol
%.
Figure 7:
Effect of DGPP on the dephosphorylation of
PA by DGPP phosphatase. Activity was measured with 3 mol %
[P]PA (molar concentration of 0.1 mM)
as substrate in the presence of the indicated surface concentrations of
DGPP. Inset, activity was measured as a function of the
surface concentration (mol %) of [
P]PA in the
absence (
) and presence (
) of 0.5 mol % DGPP (molar
concentration of 0.1 mM). The data are plotted as 1/V (units/mg) versus the reciprocal of the PA surface
concentration raised to the Hill number of 2. The lines drawn
are the result of a least-squares analysis of the
data.
Figure 8:
Effect
of DGPP on PA phosphatase activity. The activity of pure PA phosphatase
was measured with 3 mol % [P]PA (molar
concentration of 0.1 mM) in the presence of the indicated
surface concentrations of DGPP.
Figure 9:
Identification of DGPP as the reaction
product of PA kinase. PA kinase activity was measured for 5 min with
[-
P]ATP and PA as substrates using the
total membrane fraction as the source of enzyme. Following the
incubation, the chloroform-soluble
P-labeled product of
the reaction was analyzed by thin-layer chromatography. The position of
standard DGPP is indicated in the figure. The radiolabeled spots near
the bottom (right of figure) of the chromatogram were
polyphosphoinositides.
Figure 10: High performance liquid chromatography of DGPP. Phospholipids were extracted from S. cerevisiae cells and analyzed by high performance liquid chromatography as described under ``Experimental Procedures.'' The elution position (23.3 min) of DGPP is indicated in the figure. The elution positions of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and PA were between 15 and 21 min. The peaks in the chromatogram eluting before min 15 and after DGPP were not identified.
We undertook the purification of DGPP phosphatase from S. cerevisiae to facilitate well defined studies on the biochemical regulation of this novel enzyme of phospholipid metabolism. Purification of DGPP phosphatase required the solubilization of the enzyme from microsomal membranes with Triton X-100 followed by conventional chromatography steps performed in the presence of Triton X-100. The presence of Triton X-100 in the buffers used for enzyme purification was required to prevent aggregation of this integral membrane protein. The hydroxylapatite chromatography step resulted in two distinct peaks of DGPP phosphatase activity. Each peak of activity was purified separately by Mono Q chromatography. The seven-step purification procedure reported for the enzyme from hydroxylapatite peak I resulted in a DGPP phosphatase preparation that was essentially homogeneous as evidenced by native and SDS-polyacrylamide gel electrophoresis. DGPP phosphatase was identified in this preparation as a 34-kDa protein. Overall, DGPP phosphatase was purified 33,333-fold relative to the activity in the cell extract to a final specific activity of 150 µmol/min/mg. The enzyme from hydroxylapatite peak II was purified 24,666-fold over the activity in the cell extract to a final specific activity of 111 µmol/min/mg. This near homogeneous enzyme preparation also contained the 34-kDa protein, which was associated with DGPP phosphatase activity. The reason for the different chromatographic properties of the two DGPP phosphatase preparations is unclear. Additional studies are required to address these differences. The molecular mass of native DGPP phosphatase in Triton X-100 micelles cannot be determined until the interaction of the enzyme with detergent has been quantitated(40) .
The fold purifications and final specific activities of the DGPP phosphatase preparations described here were higher than any other phospholipid-dependent enzyme purified from S. cerevisiae (reviewed in (8) and (39) ). That such a high degree of purification was required to obtain purified DGPP phosphatase indicated that the abundance of this enzyme in S. cerevisiae was relatively low when compared with other phospholipid-dependent enzymes.
Pure DGPP phosphatase derived from
hydroxylapatite peak I was used for the biochemical characterization of
the enzyme. The enzyme had a broad pH optimum and required Triton X-100
for maximum activity. The enzyme did not have a divalent cation
requirement, and activity was insensitive to NaEDTA.
Divalent cations, especially Mn
ions, potently
inhibited DGPP phosphatase activity. DGPP phosphatase was also
inhibited by NaF but was relatively insensitive to thioreactive
compounds. Pyrophosphate and, to a lesser extent, ADP (which contains a
pyrophosphate group) inhibited DGPP phosphatase activity. We did not
test whether these compounds were substrates for DGPP phosphatase.
Pyrophosphate is the specific substrate for inorganic pyrophosphatase
in S. cerevisiae(41, 42) . DGPP phosphatase
differed from inorganic pyrophosphatase in that inorganic
pyrophosphatase is a cytosolic enzyme, which is dependent on
Mg
ions for activity(41, 42) .
DGPP phosphatase catalyzed the dephosphorylation of the
phosphate of DGPP yielding PA and P
. DGPP phosphatase
activity was not inhibited by its product PA. This lack of product
inhibition was consistent with the length of time in which the reaction
was linear and the enzyme catalyzing the near quantitative conversion
of DGPP to PA. Whereas the enzyme was not inhibited by PA, DGPP
phosphatase catalyzed the dephosphorylation of PA. The
dephosphorylation of PA was subsequent to the dephosphorylation of the
phosphate of DGPP. Moreover, the dephosphorylation of PA by DGPP
phosphatase was potently inhibited by DGPP. DGPP was a competitive
inhibitor with respect to PA and the K
value for
DGPP was similar to the K
value for DGPP (Table 3). Taken together, these results suggested that the DGPP
and PA binding sites on the enzyme were the same.
DGPP phosphatase activity followed surface dilution kinetics (35) using Triton X-100/phospholipid-mixed micelles. The role of Triton X-100 in the assay of phospholipid-dependent enzymes is to form a mixed micelle with the phospholipid substrate and, therefore, provide a surface for catalysis(35) . Indeed, DGPP phosphatase activity was dependent on the surface concentration of DGPP. The reaction of the dephosphorylation of PA also followed surface dilution kinetics. However, in contrast to the typical kinetic behavior (saturation kinetics) the enzyme exhibited toward DGPP, the dependence of activity on the surface concentration of PA was cooperative.
DGPP was clearly
the preferred substrate for pure DGPP phosphatase based on the relative
values for V and K
(Table 3). Moreover, the specificity constant (V
/K
) for DGPP was 10-fold
higher than that for PA (Table 3). Although the cellular
concentration of DGPP was 16-fold lower than that of PA (Table 3), an argument can be made for DGPP being the the
preferred substrate for the enzyme in vivo. PA, at a
concentration 20-fold higher than that of DGPP, did not inhibit DGPP
phosphatase activity, and DGPP potently inhibited the enzyme's
ability to catalyze the dephosphorylation of PA. Moreover, the K
value for DGPP was very close to its cellular
concentration (Table 3). Our studies, however, do not rule out
the possibility that the enzyme could use PA as a substrate in
vivo.
The enzyme in S. cerevisiae which is responsible
for the dephosphorylation of PA is PA phosphatase(22) . PA
phosphatase has been purified and extensively characterized from S.
cerevisiae(22, 33, 38, 43, 44, 45) .
DGPP phosphatase differed from PA phosphatase with respect to molecular
mass, substrate specificity, cofactor requirement, and sensitivity to
thioreactive agents. For example, PA phosphatase requires
Mg ions for activity and is inhibited by N-ethylmaleimide(22, 43) .
If DGPP
phosphatase was to play an important role in phospholipid metabolism,
it was important for us to demonstrate that DGPP was synthesized in S. cerevisiae. Indeed, we demonstrated that S. cerevisiae possessed PA kinase activity and DGPP was identified in growing
cells. PA kinase activity (7 pmol/min/mg) in S. cerevisiae was
very low when compared with the activity (3.3 nmol/min/mg) in membranes
from C. roseus(5) . This low level of activity may be
a reflection of a low level of PA kinase expression, the
product's rapidly hydrolyzation by DGPP phosphatase, and/or not
knowing the correct assay conditions for the enzyme. Additional studies
are needed to characterize PA kinase activity in S.
cerevisiae. DGPP was previously not identified in S.
cerevisiae. In previous studies (reviewed in (39) ), a
relatively large percentage of unidentified phospholipids have been
labeled as ``others.'' Perhaps DGPP was a minor phospholipid
among the unidentified phospholipids described in previous studies.
DGPP accounted for only 0.18 mol % of the major phospholipids in S.
cerevisiae. The identification of DGPP was dependent on the
procedure used to extract phospholipids. Method IIIB of Hanson and
Lester (32) was the best method to extract DGPP. This method is
commonly used for the extraction of polar lipids such as
polyphosphoinositides and sphingolipids(32) . This method also
minimizes creation of artifacts such as lysolipids(32) . The
cellular concentration of DGPP was 3-fold lower than the K value (0.55 mol %) for DGPP. Thus, DGPP
phosphatase activity would be expected to be very sensitive to changes
in the cellular concentration of DGPP.
It is unclear what roles DGPP and DGPP phosphatase play in phospholipid metabolism. One could speculate that DGPP is the precursor of the PA used for phospholipid synthesis or neutral lipid synthesis, DGPP is the precursor of the PA which acts as a signaling molecule, and/or DGPP itself is a signaling molecule. The activity of DGPP phosphatase could regulate the levels of DGPP and PA in the cell. Since DGPP phosphatase also dephosphorylated PA, the enzyme may play a role in regulating diacylglycerol levels. The studies reported here provide the foundation for future molecular genetic studies directed toward understanding the roles DGPP and DGPP phosphatase play in phospholipid metabolism and cell growth.