From the Division of Nephrology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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ABSTRACT |
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A novel pathway for ceramide
metabolism, 1-O-acylceramide formation, was previously
reported (Abe, A., Shayman, J. A., and Radin, N. S. (1996)
J. Biol. Chem. 271, 14383-14389). In this pathway a
fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of
ceramide. An enzyme that catalyzes the esterification of
N-acetylsphingosine was purified from the postmitochondrial
supernatant of calf brain through consecutive steps, including ammonium
sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose,
Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose
chromatography. The molecular mass of the enzyme was determined to be
40 kDa by gel filtration on Sephadex G-75. The enzyme bound to
concanavalin A-agarose column was eluted with the buffer containing 500 mM -methyl-D-mannopyranoside. Further
purification by heparin-Sepharose chromatography resulted in separation
of two peaks of enzyme activity. Coincidence between the transacylase
activity and a stained protein of a molecular mass of 40 kDa was
observed, as determined by SDS-polyacrylamide gel electrophoresis and
recovery after separation over an acidic native gel. The second peak of
activity from the heparin-Sepharose chromatography represented a
purification of 193,000-fold. These results are consistent with the
enzyme being a glycoprotein of a molecular mass of about 40 kDa with a
single polypeptide chain. The purified enzyme had a pH optimum at pH
4.5. The divalent cations Ca2+ and Mg2+
enhanced but were not essential for the transacylase activity. Neither
activation nor inactivation of the enzyme activity was observed in the
presence of 2 mM ATP or 2 mM dithiothreitol.
Preincubation of the enzyme with 1 mM
N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 µM bromoenol lactone, a potent inhibitor
of cytosolic Ca2+-independent phospholipase A2,
had no significant effect on the enzyme activity. The enzyme activity
was completely abolished in the presence of greater than 773 µM Triton X-100. Partial inhibition of the enzyme
activity was observed in the presence of 10-100 µg/ml heparin. In
the absence of N-acetylsphingosine, the enzyme acted as a
phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a
novel phospholipase A2.
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INTRODUCTION |
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Recently ceramide has been identified as a candidate lipid mediator regulating cell proliferation, growth, differentiation, development, and apoptosis (1, 2). In many studies, proof of concept for a biological role of ceramide has relied on the use of N-acetylsphingosine (NAS),1 a truncated and membrane-permeable homologue of endogenous ceramides. Reliance on NAS is problematic since this compound is subject to metabolism when exogenously applied to cultured cells. We observed that MDCK cells and NIH 3T3 cells actively metabolized and converted NAS to other sphingolipids and resulted in a considerable increase in levels of sphingosine, natural (long chain) ceramide, and glucosylceramide (3-5).
Previously reported work on the metabolism of NAS in MDCK cell homogenates revealed the formation of a unique, alkali-labile lipid, 1-O-acylceramide (5). An enzyme activity that catalyzed esterification of a hydroxyl group at C1 position of NAS was described. The enzyme displayed an acidic pH optimum and was mainly recovered in a soluble fraction after centrifugation at 100,000 × g. In the presence of NAS and using liposomes consisting of phosphatidylcholine, [2-14C]arachidonylphosphatidylethanolamine and sulfatide, the enzyme catalyzed both the transacylation of NAS and the release of arachidonic acid from phosphatidylethanolamine. In the absence of NAS, the release of arachidonic acid was still observed. The enzyme predominantly catalyzes the deacylation or transacylation of both phosphatidylethanolamine and phosphatidylcholine. Additionally, Ca2+ was not required for the enzyme activity. This enzyme appeared to be a novel Ca2+-independent PLA2. In the present paper, we report the purification and characterization of this novel acid phospholipase A2 with transacylase activity from calf brain.
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EXPERIMENTAL PROCEDURES |
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Materials
The reagents and their sources were
L--1-acyl-2-[1-14C]arachidonoylphosphatidylethanolamine
(53 mCi/mmol) from Amersham Pharmacia Biotech; [3H]acetic
anhydride (50-100 mCi/mmol) from American Radiolabeled Chemicals;
Ecolume scintillation fluid from ICN; N-ethylmaleimide, Triton X-100, phosphatidylethanolamine (PE) from bovine brain, dicetylphosphate, D-erythro-sphingosine,
heparin, phenylmethylsulfonyl fluoride (PMSF),
-methyl-D-mannopyranoside, and S-Sepharose from Sigma;
dioleoylphosphatidylcholine (DOPC) from Avanti;
DL-dithiothreitol (DTT) and nonadecyltetraenyl
trifluoromethyl ketone (AACOCF3) from Calbiochem;
(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-(2H)-pyran-2-one (BEL) from Cayman Chemical.
N-[3H]Acetylsphingosine
([3H]NAS) was prepared by the method of Gaver and Sweeley
(6). High performance thin layer chromatography silica gel plates, 10 × 20 cm, were purchased from Merck. DEAE-Sephacel,
phenyl-Sepharose CL-4B, Sephadex G-75, and heparin-Sepharose were from
Amersham. Concanavalin A (Con A)-agarose was from Seikagaku Inc.,
Japan. Protein molecular mass standards used for Sephadex G-75 gel
filtration and SDS-polyacrylamide gel electrophoresis included the
following: bovine serum albumin, 68 kDa (Amersham); ovalbumin, 43 kDa (Sigma); carbonic anhydrase, 29 kDa (Amersham); and cytochrome
c, 12.5 kDa (Amersham).
Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis in the absence of SDS was performed by the modified method of Reisfeld et al. (7) on 10% acrylamide gels at pH 4.3. Polyacrylamide gel electrophoresis in the presence of SDS was performed by the method of Laemmli and Favre (8) on 12% acrylamide gel. The protein bands were stained with Coomassie Brilliant Blue R-250 or using a silver staining kit (Bio-Rad). The protein content was determined by the bicinchroninic acid protein assay (Pierce) with bovine serum albumin as a standard. The solution with a low concentration of protein was concentrated by the deoxychorate-trichloroacetic acid precipitation method (9) and then assayed.
When the transacylase activity was extracted from a polyacrylamide gel, two gels were loaded with the partially purified enzyme (Step 7, see below) in 0.02% methyl green plus 10% glycerol. Electrophoresis was performed in a cold room. One gel was sliced into 2-mm segments. Each gel was minced by a glass homogenizer with 650 µl of 50 mM sodium citrate (pH 4.5) and vortexed overnight at 4 °C. The samples were centrifuged for 10 min at 2,000 × g to remove the gel particles. The transacylase activity was measured for each sample using 400 µl of supernatant. The other gel was equilibrated for 2 h against 2.3% SDS, 5% 2-mercaptoethanol, 10% glycerol, 62.5 mM Tris-HCl (pH 6.8) and then electrophoresed to a slab polyacrylamide gel (12% separating gel) in the presence of SDS.
Enzyme Assay
The assay conditions are described in the figure and table legends. In general, liposomes consisting of DOPC (70 mol%), PE (30 mol%), and dicetylphosphate (10 mol%) were used as acyl group donors. Constituent lipids in chloroform were mixed and dried under a stream of nitrogen. Fifty mM sodium citrate (pH 4.5) was added to the dried lipids at a volume 1 ml/1.28 µmol of lipid phosphorus. The lipids were dispersed into the buffer for 8 min in an ice water bath using a probe sonicator. The donor liposomes (64 nmol of phospholipid) were incubated with 5 nmol of [3H]NAS (10,000 cpm), 5 µg of bovine serum albumin and the transacylase activity at 37 °C in a total volume of 500 µl of 38-40 mM sodium citrate (pH 4.5). The reaction was terminated by adding 3 ml of chloroform/methanol (2/1). The activity was determined as described in the previous paper (5). In the assay using nonradioactive NAS, the lipids were extracted and chromatographied as in the use of radioactive NAS. The activity was determined by image scan analysis of O-acyl-NAS band obtained by charring.
Purification of Transacylase from Calf Brain
Postmitochondrial Supernatant (Step 1)--
Fresh calf brains
were stored at 80 °C as thin blocks after removal of pia mater.
Frozen brains (600 g) were partly allowed to thaw in the cold room
overnight and then cut in small pieces, after which all manipulations
were performed at 4 °C. A 30% homogenate in 10 mM
Tris-HCl (pH 7.4 at room temperature), 1 mM EDTA, 0.5 mM PMSF (buffer A) was prepared by a Polytron for 1 min at
20,000 rpm. The mixture was centrifuged at 14,000 × g
for 30 min. The pellet was reextracted as described above.
Ammonium Sulfate Precipitation (Step 2)-- Ammonium sulfate was added to the postmitochondrial supernatant to 20% saturation (10.6 g/100 ml). After stirring for 1 h, the precipitate was removed by centrifugation at 14,000 × g for 30 min. The supernatant was adjusted to 70% saturation with ammonium sulfate (31.2 g/100 ml) to precipitate the enzyme. After standing overnight, the clear supernatant above the precipitate was siphoned off. The precipitate was collected by centrifugation at 10,000 × g for 30 min, dissolved in 60 ml of buffer A, and dialyzed for 2 days against 5 times 4 liters of 25 mM Tris-HCl (pH 7.4, at room temperature), 1 mM EDTA (buffer B). The protein was adjusted to less than 20 mg/ml with buffer B and centrifuged at 100,000 × g for 1 h.
DEAE-Sephacel Chromatography (Step 3)-- The supernatant was applied to a column (5 × 22 cm) of DEAE-Sephacel preequilibrated with buffer B at a flow rate of 100 ml/h. Unbound protein was eluted with 2 liters of buffer B. The enzyme activity was recovered in the unbound fraction.
Phenyl-Sepharose Chromatography (Step 4)--
The unbound
fraction was adjusted to 3 M NaCl and then applied to a
column (2.5 × 20 cm) of phenyl-Sepharose preequilibrated with
buffer B containing 3 M NaCl (buffer C) at a flow rate of 80 ml/h. Unbound material was eluted with 1000 ml of buffer C. Additionally the column was rinsed by 450 ml of buffer B containing 1.5 M NaCl (buffer C') and then by 400 ml of buffer B
containing 0.15 M NaCl (buffer D). A small portion of the
enzyme activity was released from the column during rinsing by buffer
D. Most enzyme activity was eluted by 400 ml of buffer D containing
50% (w/v) ethylene glycol (buffer E) and kept at 80 °C.
S-Sepharose Chromatography (Step 5)-- The dialysate was applied to a column (1.4 × 20 cm) of S-Sepharose preequilibrated with buffer F at a flow rate of 40 ml/h. Unbound protein was eluted by buffer F. The bound protein was eluted by a 480-ml linear gradient of NaCl from 0-0.3 M NaCl in buffer F. The enzyme activity was found in fractions between 60 and 130 mM NaCl as two peaks. Each peak was separately collected and named "S-Sepharose fast" and "S-Sepharose slow" by following the order of elution.
Sephadex G-75 Chromatography (Step 6)-- Both fractions, S-Sepharose fast and S-Sepharose slow, were concentrated by Centriprep-10 (Amicon) and then applied separately to a column (2.6 × 90 cm) of Sephadex G-75 preequilibrated with 0.15 M NaCl, 25 mM Tris-HCl (pH 7.4), 1 mM EDTA (buffer D) at a flow rate of 40 ml/h. Protein was eluted with buffer D. For S-Sepharose slow, a peak of the enzyme activity appeared at Ve/Vo = 1.41. For S-Sepharose fast, two small peaks of the enzyme activity were observed at Ve/Vo = 1.12 and 1.41. The active fractions obtained from S-Sepharose slow were collected and used for further purification.
Concanavalin A-Agarose Chromatography (Step 7)--
The fraction
obtained in Step 6 was adjusted to 1 mM CaCl2
and 1 mM MnCl2 and applied to a column
(0.85 × 1.8 cm) of Con A-agarose preequilibrated in 500 mM NaCl, 25 mM Tris-HCl (pH 7.4), 1 mM CaCl2, and 1 mM
MnCl2 (buffer G) at a flow rate 1.5 ml/h. Unbound protein
was eluted by 10 ml of buffer G. The column was rinsed by buffer G
including 10 mM -methyl-D-mannopyranoside.
During the rinse, a small portion of the enzyme activity was released from the column. Most enzyme activity was eluted by 10 ml of buffer G
including 500 mM
-methyl-D-mannopyranoside
(buffer H) and possessed a very high specific activity in the
transacylase assay. The active fraction eluted with buffer H was
collected, adjusted to 77.3 µM of Triton X-100, and then
dialyzed against 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 77.3 µM (w/v) Triton X-100 (buffer
I).
Heparin-Sepharose Chromatography (Step 8)-- The dialysate was applied to a column of heparin-Sepharose (HiTrap heparin, 1 ml; Amersham) preequilibrated in buffer I at a flow rate of 5 ml/h. Unbound protein was eluted by 15 ml of buffer I. Then the column was rinsed by 15 ml of buffer B containing 77.3 µM Triton X-100 (buffer B'). The bound protein was eluted by a 60-ml linear gradient of NaCl from 0 to 0.15 M in buffer B'.
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RESULTS |
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Purification of Transacylase from Calf Brain
Table I summarizes the various steps of the protocol developed for transacylase purification. An eight-step procedure was employed, and the transacylase activity was monitored by the acylation of NAS throughout the purification.
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In the present study, calf brain was chosen as a starting material. The post-mitochondrial supernatant was precipitated by 20-70% ammonium sulfate. After dialyzing the suspension of the precipitates, the 100,000 × g supernatant of the dialysate was applied to the DEAE-Sephacel column, and most of the enzyme activity was recovered in the unbound fraction. The recovery rebounded after the DEAE-Sephacel elution. This step gave rise to about 19-fold purification. The unbound fraction was adjusted to 3 M NaCl and applied to the phenyl-Sepharose column. The enzyme activity was recovered by the buffer containing 50% ethylene glycol (Fig. 1). This step gave rise to an additional 7.6-fold purification.
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In the next S-Sepharose chromatography, the enzyme activity was eluted in fractions between 60 and 130 mM NaCl as two peaks (Fig. 2). There was no enrichment in the purification of the S-Sepharose fast fraction. On the other hand the S-Sepharose slow fraction was purified 4.1-fold.
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Before step 6 (Sephadex G-75 gel filtration), both S-Sepharose fast and S-Sepharose slow fractions were concentrated using Centriprep-10 without loss of enzyme activity. Only a small increase in purity was produced by gel filtration. For S-Sepharose slow, a peak of the enzyme activity appeared at Ve/Vo = 1.41 (Fig. 3B). On the other hand, for S-Sepharose fast two small peaks of the enzyme activity were observed at Ve/Vo = 1.12 and 1.41 (Fig. 3A). The values of Ve/Vo, 1.12 and 1.41, corresponded to molecular masses of 77.5 kDa and 38.6 kDa, respectively. The active fractions shown in the Fig. 3B were collected and used for further purification.
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Most proteins passed through the Con A agarose column (Fig.
4). Buffer containing 500 mM
-methyl-D-mannopyranoside was required to elute the
enzyme (Fig. 4). This step gave rise to an additional 55-fold
purification. Further recovery of the enzyme was accomplished by
maintaining the column in buffer H overnight without elution and then
rinsing with the same buffer. Because the protein concentration in the
active fraction was extremely low, the protein solution was stabilized
with 77.3 µM Triton X-100. At this stage, the purified transacylase was separated by acidic native gel electrophoresis. The
extraction of gel slices and assay of transacylase activity corresponded to a protein of a molecular mass 40 kDa (Fig.
5).
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The active fraction obtained from the Con A column was applied to the heparin-Sepharose column and eluted by a linear gradient of 0-150 mM NaCl. No Con A and Con A fragments were contaminated in the sample after heparin-Sepharose chromatography. The enzyme activity appeared as two peaks between 20 and 50 mM NaCl (Fig. 6). In this single experiment the elution profile of the enzyme activity corresponded to that of protein of a molecular mass 40 kDa, estimated by SDS-PAGE. The lack of a protein band in lane 30 was due to the accidental loss of deoxycholate-trichloroacetic acid precipitate during aspiration. The highly purified enzyme found in the second peak was purified 193,000-fold. The protein appeared as a single band by SDS-PAGE and was used for further characterization of the enzyme. The yield from 10 calf brains (2,400 × g) was 4.5 µg with a specific activity of 25 µmol/min/mg of protein. The overall recovery of the activity was 1.6%. Transacylase activity was always accompanied with phospholipase A activity throughout the purification steps as determined by thin layer chromatography and charring (for example, Fig. 5).
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Characterization of Transacylase
pH Optimum-- The purified transacylase was optimally active at pH 4.5 (Fig. 7). Unlike the homogenate obtained from MDCK cells, the purified enzyme still retained the activity at pH 5-6.5 (5). The enzyme activity was totally abolished at pH greater than 7.6.
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Effect of Ca2+-- The enzyme activity was slightly inhibited in the presence of 5 mM EDTA. One and 10 mM Ca2+ enhanced the activity by 48 and 27%, respectively (Table II). A similar effect was observed in the presence of Mg2+, in which 1 and 10 mM Mg2+ enhanced the activity by 32% and 43%, respectively.
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Effect of Other Agents-- Nucleotides, in particular ATP, have been reported to stabilize and activate a cytosolic Ca2+-independent PLA2 (10, 11). The transacylase activity was slightly inhibited in the presence of 2 mM ATP (Table II). Neither the thiol compound, DTT, nor the thiol reagent, NEM, had significant effects on the enzyme activity (Table II). The transacylase activity was slightly inhibited by the pretreatment of the enzyme with 1 mM PMSF (Table II). Both BEL, a potent inhibitor of cytosolic Ca2+-independent PLA2 (iPLA2), and AACOCF3, a potent inhibitor of cytosolic PLA2 (cPLA2), slightly inhibited the enzyme activity (Table II and Fig. 8).
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Dual Enzyme Activity, Transacylase and Phospholipase
A2--
As noted above, the transacylase was always
accompanied with phospholipase A activity. To confirm the existence of
both transacylase and phospholipase A activities,
L--1-acyl-2-[1-14C]arachidonoyl-PE was
employed as an acyl donor. The enzyme acted as not only a transacylase
but also as a PLA2 in the presence of NAS and
sn-2-radiolabeled PE (Fig.
10). On the other hand, the enzyme
showed only PLA2 activity in the absence of NAS as an
acceptor. Interestingly, the total activity of the transacylase
and PLA2 in the presence of NAS was exactly equal to
PLA2 activity in the absence of NAS. Only 7% of total
radiolabeled products was
2-[1-14C]arachidonoyllysophosphatidylethanolamine after
30 min incubation in the presence or absence of NAS. These data are
consistent with a high degree of specificity for the enzyme in using
the sn-2 fatty acid of
PE.2
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DISCUSSION |
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Our previous study demonstrated that the transacylase activity of brain is higher than that of other tissues (5). Therefore brain was chosen as a starting material. In a preliminary study, it was confirmed that the transacylase activity is efficiently released by sonication from MDCK cell homogenate prepared in isotonic buffer, consistent with its localization in an intracellular compartment. In the present study, fresh calf brains were frozen once before use. The thawed tissues were homogenized with a hypotonic buffer using a Polytron. This procedure presumably lysed intracellular compartments efficiently and resulted in an increased release of the enzyme. However, a very low recovery (20-30%) of enzyme activity was initially obtained after ammonium sulfate fractionation. Recovery was markedly improved by diluting the dialysate before the 100,000 × g centrifugation. A high protein concentration might have caused a nonspecific coprecipitation of the enzyme with insoluble materials during ultracentrifugation. The observed rebound of enzyme activity after DEAE-Sephacel chromatography might have been due to separation of the enzyme from an endogenous inhibitor.
The concanavalin A-agarose chromatography step was one of the most
successful purification steps. The enzyme was tightly bound to
concanavalin A-agarose resin and specifically released from the resin
with 500 mM -methyl-D-mannopyranoside (Fig.
4). At this stage, a major band of molecular mass about 40 kDa and some minor bands of that between 43 and 60 kDa were observed in the active
fractions from the concanavalin A column by SDS-PAGE (Fig. 5). Further
purification by the heparin-Sepharose column showed an exact
coincidence between the transacylase activity and a stained protein of
a molecular mass of about 40 kDa as determined by SDS-PAGE (Fig. 6),
consistent with the molecular mass assignment by Sephadex G-75
chromatography (Fig. 3B). These data are consistent with the
purified transacylase being a 40-kDa oligomannose-containing glycoprotein with a single polypeptide chain. This enzyme has a pH
optimum of 4.5. Although suggestive of a lysosomal origin for the
transacylase, the enzyme activity was still observed at pH 5-6.5 (Fig.
6). Thus the enzyme may function outside the acidic environment of
lysosomes.
Secreted PLA2s, group I, II, and III, contain 5-8 disulfide bonds essential for the enzyme activity (13, 14). Reducing these disulfide bonds by thiol reagents results in inhibition of the lipase activity. The calf brain transacylase activity was insensitive to DTT when preincubated at pH 7.4 in Tris buffer or when added directly to the assay mixture (Table II). Using a partially purified enzyme (step 7), it was found that the enzyme was tightly bound to organomercurial-agarose resin (Affi-Gel 501; Bio-Rad) and eluted by 200 mM 2-mercaptoethanol (data not shown). This indicates that the enzyme has free thiol group(s) exposed on its molecular surface. Pretreatment of the purified enzyme with NEM had no effect on the enzyme activity (Table II). These results suggest that the free thiol group(s) of the enzyme that is thiol reagent-sensitive is neither functionally important nor found in the catalytic site.
Recently human cPLA2 was found to have a transacylase activity that is CoA-independent, raising the possibility that cPLA2 forms an acyl-enzyme intermediate (15). Site-directed mutagenesis of cPLA2 demonstrated that the serine 228 essential for the catalytic activity of the 85-kDa cPLA2 may be the active site nucleophile. (17). Additionally, it was reported that AACOCF3 is both a slow and tight binding inhibitor of cPLA2 that may form a hemiketal enzyme-inhibitor complex (16, 18). The reaction mechanism of cPLA2 thus might be similar to that of serine protease, esterase, or lipase (15-18). In general, these types of enzymes are also sensitive to PMSF and diisopropylfluoride. In the present study, neither 1 mM PMSF nor 28 µM AACOCF3 significantly inhibited the activity of the purified transacylase (Table II and Fig. 8). The action of these inhibitors on the enzyme in 100,000 × g supernatant from MDCK cells homogenate was also not significant (5). These results suggest that neither the calf brain transacylase nor MDCK cell transacylase has an essential serine residue for the transacylase reaction.
Although the purified transacylase did not require Ca2+ or Mg2+ for activity, these cations enhanced the activity (Table II). In the previous study, it was observed that 1 mM Ca2+ slightly enhanced the transacylase activity in the 100,000 × g supernatant obtained from MDCK cells homogenate (5). It is known that divalent cations affect the physical state of lipid assembly (12). The enhancement of the enzyme activity is thought to be due to formation of ionic bridges by these divalent cations on liposomes, thereby modulating molecular packing and availability of substrate.
Recently, it has been reported that myocardial cytosolic iPLA2 and macrophage cytosolic iPLA2 interact with ATP, acting on the enzyme as an activator or a stabilizer (10, 11). The purified calf brain transacylase was not activated in the presence of ATP (Table II). It has also been reported that BEL is a potent covalently bound inhibitor of these iPLA2s but not other PLA2s (secreted Ca2+-dependent PLA2 or cytosolic Ca2+-dependent PLA2) (19, 20). Myocardial iPLA2 activity was completely abolished by pre-incubation of the iPLA2 with 1 µM BEL (19), and half-maximal inhibition of macrophage iPLA2 activity was observed at 60 nM after a 5 min preincubation (20). Preincubation of the calf brain transacylase with 3 µM BEL had no significant effect on the enzyme activity (Table II). These results suggest that a functional group sensitive to BEL inhibitors does not exist around the active site and/or lipid recognition site of the calf brain transacylase.
Bartolf and Franson (22) reported that a lysosomal iPLA2 from bovine adrenal medulla was stabilized by adding Triton X-100, although the lipase activity was reduced in a dose-dependent manner at higher Triton X-100 concentrations (22). A similar effect was observed on the purified calf brain transacylase, where activity was completely abolished in the presence of more than 773 µM Triton X-100 (Fig. 9). On the contrary, iPLA2 (39 kDa) partially purified from bovine brain has the optimal concentration of Triton X-100 at 773-1546 µM, suggesting that the transacylase is distinct from the 39-kDa iPLA2 (27).
Using iPLA2 (39 kDa) partially purified from bovine brain, Yang et al. (21) reported that heparin at either 100 or 200 µg/ml produced 25% inhibition of the iPLA2 activity. The calf brain transacylase was bound to heparin-Sepharose resin under a low ionic condition and eluted by a low concentration of NaCl consistent with weak ionic binding (Fig. 6). In the present study, 50% inhibition of the transacylase activity occurred at 10-100 µg/ml heparin (Table II). The interaction between the enzyme and heparin might be very weak in the assay system because of the ionic strength. Alternatively, heparin is thought to interact with liposomes and to change the surface properties and the molecular packing of liposomes. As a result, an apparent activity of the enzyme might be partially suppressed.
iPLA2s have been found in many mammalian tissues and cells (23), and some successful purification of iPLA2s has been reported, including canine myocardium (10), P388D1 macrophages (11), and rat lung (24), brain (25), and kidney (26). Some biochemical properties of the calf brain transacylase, including insensitivity to DTT and Ca2+ independence for enzyme activity, are found among iPLA2s. However, many of iPLA2s have a neutral pH optimum. For example, although two kinds of cytosolic iPLA2, 110 kDa and 39 kDa, were partially purified from bovine brain, the optimal pH values of 110-kDa and 39-kDa enzymes were 7.4 and 8.0, respectively (27). The purified rat kidney soluble iPLA2 has a molecular mass of 28 kDa and a neutral pH optimum (26). Although a soluble acidic pH-optimum iPLA2 was purified from rat brain by a protocol similar to ours, the enzyme displayed a molecular mass of 58 kDa and was highly phosphatidic acid-selective (25). Recently Wang et al. (24) purified an acidic-pH optimum iPLA2 from rat lung. This enzyme displayed some properties similar to the calf brain transacylase, including insensitivity to 2-mercaptoethanol, ATP, AACOCF3, and the binding ability to heparin-Sepharose resin. However, the molecular mass of the lung iPLA2 was 15 kDa (24). A soluble, lysosomal iPLA2 partially purified from bovine adrenal medulla showed a basic pI, Triton X-100 inhibition, and binding to concanavalin A-agarose resin (22). These profiles are common to the calf brain transacylase. This enzyme, however, gave a molecular mass of 30.6 kDa.
More recently, a calcium-independent lysosomal PLA2 has been cloned. This enzyme displayed a similar pharmacologic profile to the 15-kDa protein but has a molecular mass of 25.8 kDa (28).
In the previous study, it was strongly suggested that MDCK cell
transacylase has dual enzyme activity, i.e. transacylase and PLA2 (5). In the present study, both transacylase and
phospholipase A activities were observed in the purified protein. The
assay in which
DOPC/L--1-acyl-2-[1-14C]arachidonoyl-PE/dicetylphosphate
liposomes was used as an acyl donor demonstrated that the calf brain
transacylase possesses dual enzyme functions, viz.
transacylase and phospholipase A activities (Fig. 10). The enzyme also
produced 2-[14C]arachidonyllyso-PE but only as a small
fraction of the total radioactive products. This supports the view that
the phospholipase activity of the transacylase has sn-2
fatty acid specificity. These data are consistent with the
interpretation that 1-O-acylceramide synthase is a novel
Ca2+-independent PLA2.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the advice and comments of Norm Radin and Philip Majerus.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1 DK41487 and RO139255 and a Merit Review Research Award from the Veterans Administration (to J. A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
An Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Nephrology Div., Dept. of
Internal Medicine, University of Michigan, Box 0676, Rm. 1560 MSRBII, 1150 West Medical Center Dr., Ann Arbor, MI
48109-0676. Tel.: 313-763-0992; Fax: 313-763-0982; E-mail:
jshayman{at}umich.edu.
1 The abbreviations used are: NAS, N-acetylsphingosine; AACOCF3, nonadecyltetraenyl trifluoromethyl ketone; BEL, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one; PLA2, phospholipase A2; cPLA2 and iPLA2, cytosolic and calcium-independent PLA2, respectively; DOPC, dioleoylphosphatidylcholine; DTT, DL-dithiothreitol; MDCK, Madin-Darby canine kidney; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; PE, phosphatidylethanolamine; Con A, concanavalin A; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.
2
The reaction was carried out for 30 min with
purified enzyme and
DOPC/L--1-acyl-2-[1-14C]arachidonoyl-PE/dicetylphosphate
liposomes as an acyl donor both in the presence and absence of NAS. In
the presence of NAS the product profile in cpm was arachidonic acid
14356, 1-O-acyl-NAS 7050, and sn2-arachidonoyl-PE
1652. The lyso-arachidonoyl-PE represented 7.2% of the reaction
products. In the absence of NAS the products were arachidonic acid
21,734 and lyso-arachidonoyl-PE 1657. The lyso-arachidonoyl-PE
represented 7.1% of the reaction products.
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REFERENCES |
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