(Received for publication, April 19, 1995; and in revised form, June 13, 1995)
From the
An N-ethylmaleimide-insensitive phosphatidate phosphohydrolase, which also hydrolyzes lysophosphatidate, was isolated from the plasma membranes of rat liver. The specific activity of an anionic form of the enzyme (53 kDa, pI < 4) was increased 2700-fold. A cationic form of the enzyme (51 kDa, pI = 9) was purified to homogeneity, but the -fold purification was low because the activity of the highly purified enzyme was unstable. Immunoprecipitating antibodies raised against the homogeneous protein confirmed the identity of the cationic protein as the phosphohydrolase and were used to identify the anionic enzyme. Both forms are integral membrane glycoproteins that were converted to 28-kDa proteins upon treatment with N-glycanase F. Treatment of the anionic form with neuraminidase allowed it to be purified in the same manner as the cationic enzyme and yielded an immunoreactive protein with a molecular mass identical to the cationic protein. Thus, the two ionic forms most likely represent different sialated states of the protein. An immunoreactive 51-53-kDa protein was detected in rat liver, heart, kidney, skeletal muscle, testis, and brain. Little immunoreactive 51-53-kDa protein was detected in rat thymus, spleen, adipose, or lung tissue. This work provides the tools for determining the regulation and function of the phosphatidate phosphohydrolase in signal transduction and cell activation.
Phosphatidate phosphohydrolase (PAP) ()catalyzes the
dephosphorylation of PA to DAG. Jamal et al.(1) characterized two distinct PAP activities in rat
liver. One activity requires Mg
and is completely
inhibited by NEM. In contrast, the other activity has no requirement
for Mg
and is NEM-insensitive. The NEM-sensitive PAP
translocates between the cytosol and membrane fractions in response to
insulin, glucagon, cyclic AMP, and fatty acids(2) . Its
regulation and subcellular localization indicate that the NEM-sensitive
PAP is primarily involved in synthesis of triacylglycerol and
phospholipids de novo. The NEM-insensitive PAP is an integral
plasma membrane protein whose role in cell metabolism is not well
defined. Because of its cellular location(1) , it may regulate
the relative concentrations of PA and DAG in the plasma membrane (3, 4) and thus participate in signal
transduction(5) .
The prominent role of DAG as a second messenger became evident when it was identified as an important product formed by agonist-stimulated hydrolysis of phosphatidylinositol bisphosphate. DAG directly activates protein kinase C(6, 7) , which phosphorylates numerous target proteins(8) . Such effects are involved in the induction of DNA synthesis(9) , oocyte maturation(10) , and morphological changes in fibroblasts(11) . After an initial increase in DAG (from hydrolysis of phosphoinositides), a second, larger increase in DAG mass is generated directly by the action of phospholipase C on phosphatidylcholine (12, 13, 14) or indirectly via the sequential actions of phospholipase D and PAP(15, 16, 17, 18) .
It is
likely that the rapid accumulation of PA, which is observed after
agonist stimulation, arises from phospholipase D-mediated hydrolysis of
plasma membrane phosphatidylcholine(18, 19, 20, 21, 22, 23) or via
the sequential actions of phospholipase C and DAG
kinase(5, 24) . In addition to giving rise to DAG
(from PAP activity), PA itself is a potent cellular activator. PA
stimulates the respiratory burst in neutrophils by activating NADPH
oxidase (25) independent of DAG(26, 27) . PA
also directly activates hepatic monoacylglycerol
acyltransferase(28) , phospholipase C-(29) , and
phosphatidylinositol-4-phosphate kinase (30) . PA has potent
mitogenic effects in Swiss 3T3 fibroblasts (31, 32, 33) , rat fibroblasts(9) ,
and human A431 cells(34) . The mechanism of its mitogenic
action is not well defined. PA may control intracellular Ca
levels(35) , activate phosphatidate-dependent protein
kinases(36) , or activate p21
by
inhibiting Ras GTPase activating protein (37, 38) or
Ras guanine nucleotide releasing factor(39) . When added
exogenously to cells, PA decreases adenylate cyclase activity (40) and activates phospholipase D(9, 41) . PA
may be a precursor for lyso-PA. Lyso-PA, which is released from
activated platelets(42) , is a potent mitogen for fibroblasts.
Treatment of cells with lyso-PA activates tyrosine kinase activity and
results in many of the same effects seen with
PA(9, 33, 43, 44, 45) .
The balance in the levels of membrane DAG and PA is important for the appropriate cellular response(s) by cells to extracellular signals. PAP appears to be involved in controlling this balance. For example, in ras-transformed fibroblasts, the specific activity of PAP is decreased relative to nontransformed cells, and agonist-stimulated production of PA relative to DAG is increased(3) . Kanoh et al.(46) reported the purification of an 83-kDa NEM-insensitive PAP from porcine thymus tissue. In this paper, we describe the purification to homogeneity of a 51-53-kDa NEM-insensitive PAP from rat liver. This enzyme differs kinetically and immunologically from that described in porcine thymus. Immunoprecipitating antibodies generated against PAP purified from rat liver confirm its identity and have been used to characterize PAP as an integral plasma membrane glycoprotein that is expressed in liver and a number of other rat tissues. This work provides the basis for determining the metabolic functions of this enzyme.
The pelleted
plasma membranes were homogenized in 5 mM sodium phosphate, pH
6.5, containing 1.5 M NaCl and 0.1% Triton X-100, incubated
for 1 h and centrifuged for at least 2 h at 142,700 g
to remove peripheral membrane proteins. After
the supernatant was decanted, the fluffy reddish upper layer of the
pelleted protein was collected and resuspended in 200 ml of 10 mM sodium phosphate, pH 6.5, and 100 mM NaCl. An equal
volume of acetone (-20 °C) was added slowly with constant
agitation to delipidate the sample. The procedure was carried out at
-17 °C in an ice-salt bath, the temperature of the protein
mixture not rising above -4 °C. Acetone extraction did not
decrease PAP activity and resulted in more stable PAP activity during
subsequent chromatography. The mixture was incubated for 30 min at
-20 °C prior to centrifugation at 350
g
and -10 °C for 20 min. Pelleted
proteins were resuspended gently in 300 ml of 5 mM sodium
phosphate, pH 6.5, 200 mM NaCl, and 10% Triton X-100 by
incubating for 3-6 h on a slowly rotating wheel, after which any
undissolved pellet was resuspended by gentle homogenization. The Triton
extract was then dialyzed twice against 4 liters of 5 mM sodium phosphate, pH 6.5, containing 1% Triton X-100 and was
clarified by centrifugation (105,000
g
for 15 min). The sample was applied immediately to a freshly
poured 240-260-ml HAP column (XK-50 column, Pharmacia)
equilibrated in 5 volumes of 5 mM sodium phosphate, pH 6.5,
containing 1% Triton X-100, at a flow rate 600 ml/h. The column was
washed to equilibrium, and PAP was eluted in about 1,100 ml of
equilibration buffer containing 400 mM NaCl. The eluate was
dialyzed twice against 16 liters of 5 mM sodium phosphate, pH
6.5, and 0.1% Triton X-100.
The dialyzed HAP eluate was incubated with 30 ml of swollen QSFF resin for 90 min, and unbound material was separated by filtration. The filtrate was incubated with 30 ml of swollen SSFF resin for 90 min, and any unbound material was separated by filtration. Washed QSFF and SSFF resins were poured into separate XK-16 columns (Pharmacia), and each column was eluted with 5 mM sodium phosphate, pH 6.5, 2 mM EDTA, 0.1% Triton X-100, and 0.5 M NaCl at a flow rate of 60 ml/h. At pH 6.5, PAP activity bound specifically to either QSFF (anionic PAP) or SSFF (cationic PAP). Lack of binding to QSFF was not a result of overloading since no more activity bound during a second incubation with QSFF (prior to SSFF).
For immunoprecipitation, samples were incubated
at 4 °C for 2-3 h with antibody in 100-200 µl of
Buffer B (100 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 0.1% Triton X-100). Aliquots were taken for analysis of
PAP activity, and immunoreactive protein was precipitated with Protein
A-Sepharose. The pellet was washed 4 times with 1 ml of incubation
buffer prior to analysis of PAP activity. For analysis of
immunoprecipitates by SDS-PAGE, samples were denatured immediately
prior to electrophoresis on 5-15% gradient gels (52) by
heating at 95 °C for 3 min in 2 Laemmli sample buffer
containing 5 M urea. All other samples were denatured in the
presence of 8 mg/ml dithiothreitol. Proteins were either visualized by
silver staining (53) or transferred to polyvinylidine
difluoride membranes for Western blot analysis(54) . Blotting
was performed at room temperature in 10 mM HEPES, pH 7.8, 150
mM NaCl, 1 mM EDTA, 0.5% Tween 20, and 0.25% gelatin.
Immunoreactive proteins were detected using enhanced chemiluminescence.
Purification of anionic and cationic forms of PAP is summarized in Table 1. Greater than 90% of the activity eluted from HAP was recovered from the QSFF and SSFF columns. Approximately equal amounts of activity bound at pH 6.5 to QSFF (anionic PAP) and SSFF (cationic PAP). Anionic PAP eluted from Mono Q at pH 6.5 in a biphasic manner (Fig. 1). A sharp peak of activity eluted between 80 and 120 mM NaCl (Peak 1), and a second broad peak eluted between 200 and 300 mM NaCl (Peak 2). Overall recovery of activity from Mono Q chromatography at pH 6.5 was typically 90% of the applied activity, 30-50% of which was in Peak 1. Although Peak 1 contained PAP at the highest specific activity (Table 1), the sample contained at least five major protein bands (approximately 78, 67, 62, 53, and 47 kDa) when visualized with silver staining after SDS-PAGE (Fig. 2, lane2). Neuraminidase treatment of this sample followed by chromatography on Mono S at pH 6.5 failed to effect a significant purification of the enzyme, however antibodies (Ab-A92) were raised against this material. Ab-A92 was instrumental in the identification and purification of cationic PAP from the SSFF-binding material. As described below, desialated anionic PAP is identical, or very similar, to cationic PAP (Fig. 2, lane5).
Figure 1:
Fractionation of
anionic PAP using Mono Q chromatography at pH 6.5.
Phosphoagarose-eluted material was fractionated on Mono Q at pH 6.5.
Anionic PAP activity () consistently eluted in a biphasic manner
from the column with a gradient of NaCl. Peak1 contained 30-50% of the eluted activity and represents the
fraction containing the highest specific activity of PAP. Opensymbols (
) represent the elution profile of PAP that
eluted from phosphoagarose and was incubated in 6.4 M urea at
room temperature for 15 min prior to chromatography on Mono Q at pH 6.5
in the presence of 10 mM sodium phosphate, pH 6.5, 2 mM EDTA, and 6.4 M urea. The dashedline represents the NaCl gradient used to elute the
column.
Figure 2: Analysis of purified cationic and anionic PAP. Purified cationic PAP (lanes1 and 3) and anionic PAP (Peak 1 eluted from Mono Q at pH 6.5, lanes2 and 4) were analyzed by SDS-PAGE under reducing conditions. Lane5 is neuraminidase-treated anionic PAP, further purified by sequential chromatography on Mono Q at pH 10, WGA, and HAP concentrating chromatography. Lanes1 and 2 were silver stained. Lanes3-5 were analyzed by Western blot with affinity-purified Ab-D503. A Western blot of the same samples with preimmune antibody showed no immunoreactive bands (results not shown). Migration of molecular mass markers (kDa) is indicated at the sides of the figure.
Cationic PAP activity eluted from Mono Q at pH 10 in a biphasic manner (Fig. 3A). Overall recovery of cationic PAP activity from the Mono Q column at pH 10 was typically 75% of the applied activity, 50% of which was in Peak 1. Analysis of cationic PAP by SDS-PAGE and Western blot with Ab-A92, revealed that Peak 1 contained only one major immunoreactive protein (Fig. 3B) and fewer silver-stained proteins compared with Peak 2 (not shown). Binding of cationic PAP to Mono Q at pH 10 indicated an isoelectric point between pH 6.5 and 10, and PAP activity eluted from a chromatofocusing column around pH 9. Chromatofocusing was not useful in the purification since the activity eluted in a broad peak. Anionic PAP did not elute from the chromatofocusing column between pH 7 and 4 but was recovered in a 1 M salt wash; thus the pI of anionic PAP was less than pH 4.
Figure 3:
Fractionation of cationic PAP using Mono Q
chromatography at pH 10. A, cationic PAP activity ()
consistently eluted in a biphasic manner from the Mono Q column at pH
10 with a gradient of NaCl. These peaks are designated as Peak1 and Peak2, respectively.
Neuraminidase treated-anionic PAP (
) in the unbound fraction after
Mono Q chromatography at pH 6.5 was dialyzed to pH 10 against 10 mM 2-amino-2-methyl-1-propanol, pH 10, and 0.1% Triton X-100 and then
reapplied to a Mono Q column equilibrated at pH 10. After washing, the
sample was eluted with a gradient of NaCl (dashedline). B, a sample of material from Peak 1
eluted from Mono Q chromatography at pH 10 was separated by SDS-PAGE
under reducing conditions and analyzed by Western blot using Ab-A92.
Migration of molecular mass markers (kDa) is indicated at the side of
the figure.
Recoveries of PAP activity from the WGA agarose column were 20-40% of that applied, when assayed immediately after chromatography. Routine analysis for protein in these column fractions could not be made because of the relatively high concentrations of detergent and reducing sugar; therefore, this chromatographic step is not included in Table 1. To generate a quantifiable sample, the WGA-agarose-eluted material was concentrated, and the deoxycholate and N-acetylglucosamine were removed using HAP. After HAP-concentrating chromatography, no protein was detected (by silver staining of SDS-PAGE), and no PAP activity was present in the unbound material. PAP activity was recovered in the phosphate-eluted fraction, and only one major protein band was detected by silver staining after SDS-PAGE (Fig. 2, lane1), indicating that the eluted PAP was essentially homogeneous. Purified cationic PAP migrated on SDS-PAGE with an apparent molecular mass of 51 kDa, independent of the presence or absence of 100 mM dithiothreitol in the solubilization buffer, indicating that the enzyme is a single polypeptide containing no sulfhydryl-linked subunits. Overall recovery of enzyme activity from the last two chromatographic steps was only 4% (partly due to intrinsic instability); however, this procedure yielded purified PAP. Loss of enzyme activity may also be attributed to the use of WGA-agarose(55, 56) . When the last steps in the purification (WGA/HAP) were performed with radiolabeled protein, only one radiolabeled protein of 51 kDa was detected by autoradiography of the purified fraction after SDS-PAGE (results not shown).
The
ability of anionic PAP and homogeneous cationic PAP (Table 1) to
hydrolyze lyso-PA was evaluated. Optimum reaction rates of lyso-PA
hydrolysis were obtained between 20 and 100 µM lyso-PA
with V values for anionic and cationic PAP that
were 34 and 29% of that for PA, respectively.
Figure 4:
Dose-dependent immunoprecipitation of PAP
activity. A, samples of cationic PAP; B, samples of
anionic PAP (upperpanel) or desialated (neuraminidase-treated) anionic PAP (lowerpanel) were immunoprecipitated with preimmune antibody
() or Ab-D503 (
). PAP activity of the pelleted material is
expressed as a percentage of the sum of the immunoprecipitated and
nonimmunoprecipitated activity.
Figure 5:
Western blot analysis and
immunoprecipitation of radiolabeled protein from partially purified
cationic and anionic PAP. A, samples from chromatographic
fractions were separated by SDS-PAGE under reducing conditions and
analyzed by Western blot with Ab-D503. Lane1, Peak 1
from Mono Q at pH 6.5; lane2, Peak 2 from Mono Q at
pH 6.5; lane3, Peak 2 from Mono Q at pH 10. B, cationic PAP (Peak 1 eluted from Mono Q at pH 10),
radiolabeled with [2,3-H]propionate (lane1) and radioiodinated anionic PAP (Peak 1 eluted from
Mono Q at pH 6.5 (lane3) were immunoprecipitated
with Ab-A92 (lane2), or Ab-D503 (lane4), separated by SDS-PAGE under nonreducing conditions
and analyzed by autoradiography. Migration of molecular mass markers
(kDa) is indicated at the sides of the
figure.
All measurable PAP activity was lost when a cationic sample of PAP was incubated with urea, consequently it was not possible to assess cationic PAP in the same manner. However, Peak 2 material from Mono Q chromatography at pH 10 contained a protein that cross-reacted with Ab-D503 (Fig. 5A, lane3) and migrated on SDS-PAGE with a molecular mass identical to the major immunoreactive protein band in Peak 1 (Fig. 3B).
To test whether PAP exists in a complex with other proteins, partially purified samples of anionic and cationic PAP were radiolabeled using two different methods and then incubated with Ab-D503 or Ab-A92. Only one protein band was immunoprecipitated from either sample (Fig. 5B). The immunoprecipitated radiolabeled proteins migrated on SDS-PAGE with an apparent molecular mass identical to the corresponding protein bands detected by Western blot analysis or silver staining (Fig. 2, 3, and 5A). Less than 10% of the radioactivity was immunoprecipitated with Ab-A92 from the crude radiolabeled cationic sample.
Cationic PAP was shown to be a glycoprotein, containing complex polysaccharide by virtue of its ability to bind specifically to WGA agarose (Table 1). The anionic form of PAP had similar lectin-binding characteristics (results not shown). Although anionic and cationic PAP were not immunologically distinguishable, anionic PAP consistently migrated on SDS-PAGE with an apparent molecular mass approximately 2 kDa larger than cationic protein (Fig. 2). Incubation of anionic PAP with neuraminidase did not alter enzyme activity significantly but caused PAP to bind to Mono S rather than Mono Q at pH 6.5. Also, when neuraminidase-treated anionic PAP was chromatographed on Mono Q at pH 10, PAP activity eluted from the column in a manner similar to the cationic form of the protein (Fig. 3A). Neuraminidase treatment of cationic PAP does not affect its chromatographic properties on Mono Q at pH 10 (results not shown). Subsequent purification of the neuraminidase-treated anionic PAP with WGA agarose and HAP after Mono Q chromatography at pH 10 yielded an immunologically cross-reactive protein (with Ab-D503) that co-migrated on SDS-PAGE with the purified cationic PAP (Fig. 2, lane5).
To determine whether PAP, as purified, was a phosphoenzyme, samples of cationic and anionic PAP were incubated with acid phosphatase and/or neuraminidase and then examined by SDS-PAGE and Western blot analysis (Fig. 6). Anionic PAP migrated slightly above cationic PAP. The band shifts in the phosphatase-treated samples indicated that anionic PAP could be a phosphoprotein. The smaller change in the migration of the phosphatase-treated cationic PAP makes the same conclusion equivocal for that form of the protein. The band shift of anionic PAP seen with neuraminidase treatment was intermediate to that seen with phosphatase treatment alone or with neuraminidase and phosphatase (Fig. 6). Neuraminidase had no effect on the migration of cationic PAP. The calculated molecular mass of dephosphorylated and desialated anionic PAP was identical to the phosphatase-treated cationic PAP. The phosphorylation state of anionic PAP did not affect its elution profile on Mono Q at pH 10 (results not shown). The results in Fig. 2and Fig. 6indicate that the primary difference between the two forms of PAP is the degree of sialation of the polysaccharide side chain(s).
Figure 6: Treatment of PAP with acid phosphatase and neuraminidase. Anionic and cationic PAP samples containing 1 µg of protein were incubated for 2 h at 37 °C with or without 0.2 units of neuraminidase in a volume of 22 µl with 10 mM sodium phosphate, pH 6.5, and 0.1% Triton X-100. Citrate-MES buffer (pH 4.8, final concentration of 50 mM) and 2 units of potato acid phosphatase (if indicated) were added, and the samples were incubated for an additional 4 h. The samples were then denatured and analyzed by SDS-PAGE under reducing conditions and Western blot with affinity-purified Ab-D503. Migration of known molecular mass markers (kDa) is indicated at the side of the figure.
Cationic PAP probably contains only N-linked polysaccharides since neither O-glycosidase (Fig. 7) nor neuraminidase (Fig. 6) altered its migration
on SDS-PAGE. The apparent molecular mass of anionic PAP may be
decreased slightly by O-glycosidase. Treatment with N-glycanase F caused both cationic and anionic PAP to shift to
an apparent molecular mass of 28 kDa (Fig. 7). Analogous results
were found when samples of anionic and cationic PAP were labeled with I, treated with N-glycanase F,
immunoprecipitated, and analyzed by autoradiography after SDS-PAGE
(results not shown). To determine whether a fully glycosylated protein
was required for expression of PAP activity, nondenatured anionic PAP
was incubated with N-glycanase F at 37 °C. Although the
specific activity of PAP declined, there was no difference between
control and glycanase-treated PAP activity at any time (Fig. 8A), despite the fact that by 48 h the enzyme had
been fully deglycosylated (Fig. 8B). From this
experiment, it appears that PAP does not have to be fully glycosylated
to remain catalytically active in vitro; however, activity in
both the control and N-glycanase-treated samples was not
stable to extended incubation at 37 °C.
Figure 7:
Treatment of PAP with endo- and
exoglycosidases. Purified cationic and anionic PAP were denatured by
heating for 3 min at 95 °C in the presence of 12% methanol and then
cooled, buffered with 50 mM sodium phosphate, pH 6.0,
containing 0.5% Triton X-100, 0.1% bovine serum albumin, and incubated
without glycosidase (lanes1), with 0.1 unit of N-glycanase F (lanes2), 2 milliunits of O-glycosidase (lanes3) overnight at 37
°C. Samples were then denatured in 2 Laemmli buffer,
separated by SDS-PAGE under reducing conditions, and analyzed by
Western blot with Ab-A92 (cationic PAP) or Ab-D503 (anionic PAP).
Neither Ab-A92 nor Ab-D503 showed immunoreactivity with N-glycanase F or O-glycosidase. Migration of
molecular weight markers (kDa) is indicated at the side of the
figure.
Figure 8:
Time
course incubation of anionic PAP with N-glycanase F. A, anionic PAP, which had not been denatured, was incubated at
37 °C in the presence () or absence (
) of N-glycanase F for up to 96 h. At the indicated times, aliquots
were removed from the incubation and were assayed for PAP activity.
Values represent the average of two independent experiments. B, aliquots from the same incubation of anionic PAP were also
analyzed by SDS-PAGE under reducing conditions and Western blot with
affinity-purified Ab-D503. Migration of molecular mass markers (kDa) is
indicated at the side of the figure.
Figure 9: Tissue distribution of PAP activity and immunologically cross-reactive protein. A, homogenates of rat tissues obtained by freeze-clamping dissected organs from an anesthetized animal were screened for PAP activity. The PAP assay included a preincubation in the presence of NEM. B, samples from tissue homogenates containing 50 µg of protein were analyzed using SDS-PAGE under reducing conditions and by Western blot with affinity-purified Ab-D503 as described under ``Experimental Procedures.'' Migration of molecular mass markers (kDa) is indicated at the side of the figure.
During the purification of PAP, two ionically distinct forms of the protein were identified and characterized. The specific activity of anionic PAP (53 kDa, pI < 4) was increased 2,700-fold. This degree of purification is an underestimate since the total PAP activity was distributed between cationic and anionic forms, each of which was further separated into two peaks of activity. The specific activity of the 51-kDa cationic PAP (pI = 9) was lower than that of anionic PAP (at least in part due to instability); however, cationic PAP was purified to homogeneity. In addition to appearing essentially homogeneous by silver staining, cationic PAP elicited antibody production against only one protein, attesting to the homogeneity of the purified sample. Only one protein from anionic PAP and one protein from cationic PAP was immunoprecipitated using Ab-D503, and PAP activity co-purified with the immunoreactive proteins. Antibodies generated against either form of PAP immunoprecipitated PAP activity from any PAP sample and cross-reacted on a Western blot with a 51-53-kDa protein. Neuraminidase treatment of anionic PAP decreased the apparent molecular mass of the protein by about 2 kDa and produced an enzyme having essentially the same characteristics as cationic PAP. It is concluded that cationic PAP is probably a desialated form of anionic PAP. In support of this conclusion, the molecular masses of both forms of PAP are decreased to 28 kDa by treatment with N-glycanase F, and both deglycosylated proteins cross-react with either Ab-A92 or Ab-D503.
PAP in the partially purified samples did not associate with other cellular proteins (Fig. 5B), therefore this is not an explanation for its biphasic elution profile from Mono Q. It is possible that self-association or aggregation of PAP may alter its chromatographic behavior. The presence of an approximately 112-kDa immunoreactive protein on Western analysis in Peak 2 from Mono Q at pH 6.5 (Fig. 5A, lane2) may represent a dimer of PAP(65) . Self-association or aggregation may also explain our lack of ability to employ nondenaturing PAGE or size exclusion chromatography in the analysis or purification of PAP.
N-linked polysaccharide constitutes about 46% of the total
mass of PAP. Several other mammalian glycoproteins have been purified
that are as much as half polysaccharide by weight. For example,
polysaccharide contributes 54% of the mass of the -subunit of the
porcine gastric
H
/K
-ATPase(66) , 47-50%
of an angiotensin receptor from human myometrium(67) , 52% of a
major lysosome-associated protein(68, 69) , and 37% of
contactinhibin(70) . The polysaccharide portion of a
glycoprotein may be required for proper folding during synthesis,
appropriate cellular localization, stability, or biological
function(71) . Complete deglycosylation of native anionic PAP
does not affect its activity toward PA, but the activity of desialated
PAP appears to be inherently less stable than that of the anionic form
of the enzyme.
It is unclear whether the purification of two ionic forms of PAP from liver homogenate is artifactual or if it reflects the presence of distinct ionic forms in vivo. The absence of a doublet of immunoreactive proteins at 51-53 kDa in the Western blot analysis of tissue homogenates could indicate that only one form of the protein exists in the intact liver. However, the appearance of a single tight band in the homogenate, compared with a relatively diffuse one in purified fractions, indicates that PAP migrates differently in a complex mixture of proteins. Thus, two forms of a glycoprotein differing in mass by 2 kDa may not be resolved under these conditions. Two-dimensional gel electrophoresis could not be used to address this issue because PAP precipitated when we attempted isoelectric focusing. If two ionic forms of PAP are present in vivo, the difference may be functionally significant. Different states of sialation affect the clearance of circulating glycoproteins from the blood(72) , and increased sialation of contactinhibin reduces its ability to mediate contact inhibition of cell proliferation(70) .
Band
shift experiments indicate that PAP is a phosphoprotein. Further
evidence for the phosphorylation of PAP was obtained by labeling
cultured rat hepatocytes with P
and
immunoprecipitating a radiolabeled 51-53 kDa band with
affinity-purified Ab-D503. (
)Phosphorylation does occur on
amino acid residues,
but it could also occur on residues of
the polysaccharide(73, 74) . Phosphorylation-dependent
regulation of PAP has been implicated in previous work (3, 5) and could explain, at least in part, the
difference in specific activity or stability of the two ionic forms of
PAP. Phosphorylation may also explain the lack of correlation between
the specific activity of PAP and the intensity of the immunoreactive
51-53-kDa proteins in the various tissues (Fig. 9).
However, the relative abundance of PAP may be tissue-specific, or
tissues may express different isoforms of PAP, for which our antibodies
have different affinity. The 86-kDa protein in brain and the 17- and
36-kDa proteins in thymus and spleen may be tissue-specific isoforms of
PAP. The existence of tissue-specific isoforms is supported by the
observation that lyso-PA does not inhibit PAP purified from pig thymus (46) and that anti-thymus 83-kDa PAP antiserum failed to
immunoprecipitate PAP activity from rat liver samples, nor did it
cross-react with any protein bands on a Western blot of rat liver
homogenate (results not shown). In contrast, purified rat liver PAP
hydrolyzed lyso-PA with a V
of about 30% that
for PA, and the two substrates are mutually competitive. (
)There also may be other forms of PAP that do not
cross-react well with our antibodies (for example, in lung or adipose
tissue). Furthermore, differential regulation of PAP in adipose tissue,
compared with heart and liver, is indicated in insulin-resistant
rats(59) . The reasons for the lack of correlation between the
activity and band intensity (Fig. 9) will be clarified when
sequence information is known and physical modifications of PAP in the
various tissues have been characterized.
PAP has also been purified
from yeast(75, 76) , but this enzyme resembles the
NEM-sensitive and Mg-requiring PAP in liver. Neither
Ab-A91 nor Ab-D503 immunoprecipitates NEM-sensitive PAP activity from
liver microsomes or cytosol (results not shown), confirming that the
two enzymes are distinct proteins(1, 58) . Purified
hepatic PAP is not alkaline phosphatase; since the pH optimum for PAP
is 6.5, it does not require Mg
, and antibodies to
alkaline phosphatase do not interact with PAP (results not shown).
An NEM-insensitive ``ecto-PAP'' in neutrophils (63) may be the same protein we have purified from rat liver. If the active site of PAP in the plasma membrane is oriented externally, it could degrade exogenous PA and lyso-PA, thus mitigating the effects of these lipids on cell signaling and cell division(5, 9, 33, 34) . Alternatively, PAP activity oriented internally could degrade PA present in the inner leaflet of the plasma membrane, thus altering the balance of PA and DAG and their actions on intracellular target proteins(3, 5) .
In this paper we describe the purification and characterization of a novel 51-53-kDa NEM-insensitive PAP from rat liver. This enzyme is an integral plasma membrane glycoprotein that is expressed in rat liver, brain, kidney, testis, heart, and skeletal muscle. Immunoprecipitating antibodies directed against purified PAP protein will be useful tools for further characterization and investigation into the regulation of PAP and of its proposed role in signal transduction.