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INTRODUCTION |
Platelet-activating factor
(PAF),1
1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine
(alkylacetyl-GPC), is a potent lipid mediator with a wide variety of
biological activities related to physiological and pathological
phenomena (1, 2). PAF is produced by either de novo or
remodeling pathway (1). In addition, we found the PAF could also be
metabolized by a novel pathway catalyzed by membrane-associated
transacetylase that transfers the acetate group of PAF to
lysoplasmalogen in HL 60 cells (3). This enzyme is CoA-independent and
transfers the acetyl group from PAF to a variety of lysophospholipids
acceptors in the order of radyl-GPC > radyl-glycerophosphoethanolamine (GPE) > acyl-glycerophosphoserine > acyl-glycerophosphoinositol > acyl-glycerophosphate > alkyl-glycerophosphate > fatty alcohol,
whereas alkylglycerol, acylglycerol, or cholesterol are inactive as
acceptors. This PAF-dependent transacetylase is participated in the biosynthesis of acyl analog of PAF (4), which is
the predominant molecular species of PAF in hematopoietic cells
including endothelial cells, mast cells, and basophils, etc.
(5). In endothelial cells, PAF-dependent transacetylase activity is regulated by phosphorylation/dephosphorylation (4).
Recently, we have demonstrated that a similar PAF-dependent
transacetylase transfers the acetyl group of PAF to sphingosine in HL
60 cells (6, 7). This enzyme activity appears to be responsible for the
presence of acetylsphingosine (C2-ceramide) in the
biological systems. For instance, C2-ceramide occurred in
the micromolar range in undifferentiated and differentiated HL-60 cells
(6). This is the concentration range that C2-ceramide has
been shown to exert as a second messenger and a lipid mediator by many
investigators (8, 9). Since C2-ceramide has many biological
activities that differ from PAF and sphingosine, therefore, this enzyme
may serve as a modifier for the functions of PAF and sphingosine (6).
Both enzyme activities are also found in rat tissues (6). Rat kidney
membrane fractions have the highest PAF:sphingosine transacetylase
activity, while both rat kidneys and lung have the highest
PAF:lysoplasmalogen transacetylase activity.
To elucidate the relationship of both enzyme activities, we attempted
to purify the transacetylase from rat kidney membranes, in which the
highest enzyme activity toward both lipid acceptors (6, 7). In the
present report, we have achieved purification of the transacetylase to
homogeneity and shown that this enzyme possesses three catalytic
activities, namely, PAF-acetylhydrolase, PAF:lysophospholipid
transacetylase, and PAF:sphingosine transacetylase.
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EXPERIMENTAL PROCEDURES |
Materials--
l-O-Hexadecyl-2-acetyl-GPC was
obtained from Sigma.
1-O-Hexadecyl-2-N-methylcarbamyl-GPC and
C2-ceramide were purchased from Biomol.
1-O-Alkyl-2-acetyl-GPC was from Avanti. Sphingosine was bought from Matreya, Inc. Alkenyllyso-GPE was a product from Serdary Research Lab. 1-O-hexadecyl-2-[3H]acetyl-GPC
was the product of NEN Life Science Products. DEAE-Sepharose, phenyl-Sepharose, activated CH-Sepharose, PBE 94, and Polybuffer 74 were from Pharmacia Fine Chemicals (Uppsala, Sweden). Hydroxyapatite was obtained from Bio-Rad. Adult male Sprague-Dawley rats were from Taconic.
Enzyme Assays--
PAF:lysoplasmalogen transacetylase and
PAF:sphingosine transacetylase activities were determined according to
our previously described methods (3, 6). The assay system of
PAF:lysoplasmalogen transacetylase consisted of 50 µM
1-O-hexadecyl-2-[3H]acetyl-GPC (0.3 µCi),
300 µM lysoplasmalogen (suspended in 0.1% bovine serum
albumin (BSA)-saline), 100 mM Tris-HCl (pH 7.4), 2 mM sodium acetate, and 10 mM EDTA in a total
volume of 250 µl. Incubations were carried out at 37 °C for 15 min. The lipids were extracted by the method of Bligh and Dyer (10).
The lipids were separated by thin layer chromatography (TLC)
using a solvent system of
CHCl3/CH3OH/CH3COOH/H2O
(50:25:8:4, v/v/v/v), and the radioactivities of the areas
corresponding to PAF and alk-1-enylacetyl-GPE were counted using liquid
scintillation fluid. The assay system of PAF:sphingosine transacetylase
contained 15 µM
1-O-hexadecyl-2-[3H]acetyl-GPC (1 µCi), 50 µM sphingosine (suspended in equal molar ratio of BSA),
100 mM Tris-HCl (pH 7.4), 2 mM sodium acetate, and 10 mM EDTA in a total volume of 250 µl. Incubations
were carried out at 37 °C for 30 min. The lipids were separated by
TLC using a solvent system of CHCl3/CH3OH
(90:10, v/v). The radioactivities of the areas corresponding to PAF and
C2-ceramide were measured. PAF-AH activity was assayed
according to the method previously described (11). The assay system of
PAF-AH was composed of 20 µM
1-O-hexadecyl-2-[3H]acetyl-GPC (0.1 µCi), 1 mM EDTA, and 100 µM potassium phosphate (pH
8.0) in a total volume of 500 µl. Incubations were carried out at
37 °C for 10 min. The reaction was stopped by sequential additions
of 1 ml of CHCl3, 1 ml of CH3OH, and 0.5 ml of
10% sodium bicarbonate. The upper phase was washed with 1 ml of
CHCl3 three times, and the radioactivities in an aliquot of
0.4 ml were determined.
Preparation of Rat Kidney Membranes--
The kidneys were
dissected out from male rats weighing 150-250 g and homogenized with
four volumes of 0.25 M sucrose, 20 mM Tris-HCl
(pH 7.4), 1 mM DTT, 1 mM EDTA, and 1 µg/ml
leupeptin. The homogenates were centrifuged at 440 × g
for 10 min; the supernatant was collected as postnuclear fraction. The
postnuclear fraction was centrifuged at 15,000 × g for
10 min to isolate the mitochondrial pellets. The postmitochondrial
fractions were centrifuged at 100,000 × g for 1 h
to obtain the microsomal fractions. Both mitochondrial and microsomal
fractions were washed with the same buffer solution twice, and the
washed mitochondria and microsomes were used as a source for enzyme purification.
Solubilization of the Enzyme--
Mitochondrial fractions were
suspended in 20 mM Tris-HCl (pH 7.4) containing 0.04%
Tween 20, 1 µg/ml leupeptin, 1 mM EDTA, and 1 mM DTT at a detergent/protein (w/w) ratio of 0.1. The
mixture was gently stirred at 0 °C for 1 h and then centrifuged
at 100,000 × g for 1 h. The enzyme activity was
twice extracted from microsomal fractions by detergent in the same way
as described for the mitochondria. High salt wash (0.5 M
NaCl) without the presence of detergent (data not shown) could not
further elute the enzyme activity from mitochondria or microsomes.
Column Chromatographies--
We have observed that the
mitochondrial fraction had the highest specific activity and the
microsomal fraction contained the highest total activity of
transacetylase among the subcellular fractions isolated from HL-60
cells (7). Therefore, we decided to combine both solubilized
mitochondria and microsomes as the starting materials for column
chromatography. The solubilized enzyme was applied onto a column of
DEAE-Sepharose (2.5 × 6.11 cm, 30 ml), which was equilibrated
with 20 mM Tris-HCl (pH 7.4), 1 mM DTT, 1 µg/ml leupeptin, and 0.02% Tween 20. The column was washed with the
same buffer solution and was then eluted with 0.15 M NaCl
in 20 mM Tris-HCl (pH 7.4), 1 mM DTT, and
0.02% Tween 20. During initial experiments, the solubilized enzyme
bound to the DEAE-Sepharose column was eluted by a linear gradient of
NaCl. There was only one major eluted peak of enzyme activity that
started to come out at 0.15 M NaCl from the column. Based
on this information, stepwise elution of the enzyme activity with 0.15 M NaCl from the DEAE-Sepharose column was performed
subsequently in order to speed up the purification process.
Active fractions of DEAE-Sepharose were applied onto a column of
hydroxyapatite (1.5 × 8.49 cm, 15 ml), which was equilibrated with 20 mM Tris-HCl (pH 7.4), 1 mM DTT, 1 µg/ml leupeptin, and 0.02% Tween 20. The column was washed with the
same buffer solution, and the enzyme activity was eluted with 50 mM potassium phosphate (pH 7.0) containing 1 mM
DTT, 1 µg/ml leupeptin, and 0.02% Tween 20. The rationale to use the
stepwise elution from hydroxyapatite with 50 mM potassium
phosphate was similar to that of the experiments with DEAE-Sepharose
column chromatography. We had previously found that the most of the
enzyme activities was eluted from hydroxyapatite column starting at 50 mM potassium phosphate during a linear gradient run of the chromatography.
The active fractions isolated from hydroxyapatite column chromatography
were applied onto a column of phenyl-Sepharose (1.0 × 6.37 cm, 5 ml), which was equilibrated with 5% (w/v) ammonium sulfate in 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM DTT, 1 µg/ml leupeptin, and 0.02% Tween 20. The
column was washed with the same solution, and the enzyme activity was
eluted with 20 mM Tris-HCl (pH 7.4), 1 mM EDTA,
1 mM DTT, 1 µg/ml leupeptin and 0.02% Tween 20.
The active fractions pooled from phenyl-Sepharose were applied onto a
column of PBE94 (1.0 × 6.37 cm, 5 ml), which was equilibrated with 20 mM Tris-HCl (pH 7.4), 1 mM DTT, 1 mM EDTA, 1 µg/ml leupeptin, and 0.02% Tween 20. The
column was washed with 25 mM histidine-HCl (pH 6.2), 1 mM DTT, 1 mM EDTA, 1 µg/ml leupeptin, and
0.02% Tween 20, and the enzyme activity was eluted by decreasing pH
with Polybuffer 74 (pH 4.0) in 1 mM DTT and 0.02% Tween
20. Five-ml fractions were collected into the tube containing 0.7 ml of
1 M Tris-HCl (pH 7.4) to neutralize the pH. The enzyme
activity was unstable at pH 5.0, and 88% of enzyme activity was lost
16 h thereafter. It was necessary to neutralize the pH of the
sample solution soon after the enzyme was eluted from the column. The
active fractions from chromatofocusing were concentrated into 4.5 ml by
using a small size of hydroxyapatite column (1 × 0.27 cm, 1 ml).
Native-PAGE--
The enzyme was further purified by native-PAGE
according to the method of Ornstein and Davis (12). Separating gel (2 ml) consisting of 7.5% polyacrylamide, 0.375 M Tris-HCl
(pH 8.8), and 0.02% Tween 20 was prepared in a glass tube (15 × 0.5 cm). Stacking gel (0.1 ml) composed of 5% polyacrylamide, 0.125 M Tris-H3PO4 (pH 6.8), and 0.02%
Tween 20 were overlaid to the separating gel. The enzyme solution (0.4 ml) was added to each tube. Electrophoresis was carried out at a
constant voltage of 80 V and 4 °C until the dye migrated toward the
end of the gel. The gels were then removed from the glass tubes and
horizontally sliced into fragments (2 mm distance). The individual
pieces were transferred into microtiter plate wells, soaked in 50 µl
of 25 mM Tris-HCl (pH 7.4), 1 mM DTT, 0.02%
Tween 20 for 16 h, and the supernatants with the highest enzyme
activities were collected.
Preparation of Affinity Gel
Matrix--
1-O-Hexadecyl-2-N-methylcarbamyl-GPC
(5 mg, 9.28 µmol) dissolved in 0.5 ml of 0.16 M acetate
(pH 5.6) containing 80 mM CaCl2 was combined
with 1 ml of cabbage phospholipase D and 0.5 ml of 20% ethanolamine.
The reaction was carried out at room temperature for 16 h (similar
to that described in Ref. 13), and the lipid was extracted by the
method of Bligh and Dyer (10). The reaction product,
1-O-hexadecyl-2-N-methylcarbamyl-GPE, was
purified by TLC using a solvent system of
CHCl3/CH3OH/CH3COOH/H2O
(50:25:8:4, v/v/v/v). The purified phospholipid (8.36 µmol) was
dissolved in 2 ml of 50 mM borate (pH 8.0) in
CH3OH and reacted with activated CH-Sepharose (2 ml)
suspended in the same solution for 16 h at 22 °C. The resulting
gel was washed with 50 mM borate (pH 8.0) in
CH3OH thoroughly, and the remaining reactive sites were
blocked with 1 M Tris-HCl (pH 8.0) for 16 h at room
temperature. Based on results from phosphorus determinations (14), 0.98 µmol of ligand was bound to 1 ml of CH-Sepharose
Purification of the Enzyme by Affinity Gel Matrix--
The
active fractions of native-PAGE were combined with 50 µl of affinity
gel matrix in a microcentrifuge tube, which was equilibrated with 20 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl, 1 mM DTT, and 0.02% Tween 20 and mixed gently at 4 °C for
30 min. The gel was washed with the same buffer and followed by the
same solution without NaCl. The washed gel was combined with 5 mM alkylacetyl-GPC dissolved in 20 mM Tris-HCl
(pH 7.4) in 1 mM DTT, and 0.02% Tween 20 and incubated for
1 h at 4 °C. The mixture was centrifuged for 5 min at
10,000 × g, and the supernatant with enzyme activity was transferred into another tube. PAF in this enzyme preparation was
removed by hydroxyapatite (0.5 ml) column chromatography. The resulting
enzyme solution was dialyzed against 20 mM Tris-HCl (pH
7.4) containing 40% glycerol, 1 mM DTT, and 0.02% Tween
20. The purified preparation was stored at
20 °C, and no
significant decrease in enzyme activity was observed at least for 1 month.
SDS-PAGE--
SDS-PAGE was carried out according to the method
of Laemmli (15) using 10% polyacrylamide gel. The proteins were
visualized by silver staining using a silver staining kit for protein
(Pharmacia Fine Chemicals).
Sequencing--
The sequencing of the protein was carried out at
Harvard Microchemistry Facility (Boston, MA) by tandem mass
spectrometry and Edman degradation analysis. The purified protein was
subjected to electrophoresis on 10% SDS-PAGE gel. The protein band on
the gel was visualized by Coomassie Brilliant Blue. The sliced gel was
subjected to digestion with trypsin and microsequencing.
Protein Determination--
In most instances, protein was
measured by a protein assay kit (Bio-Rad) using BSA as the standard.
However, when the protein concentrations in the samples were low
(e.g. Table III, steps 3-7), a series of one to one (1:1)
dilutions of 1 µg/ml BSA (ranging from 31.25 ng to 1 µg per spot)
and the samples in duplicates were spotted on a 3MM filter paper. The
spots were allowed to air-dry and were stained with 0.25% Coomassie
Brilliant Blue R-250 (Bio-Rad), 45% CH3OH, 10%
CH3COOH for 30 min. The paper was destained with 45%
CH3OH, 10% CH3COOH until the background was
nearly white. Then, the protein amount was determined by comparing the
intensities of the spots from samples with those from BSA standards.
The amount of protein in the final enzyme preparation (Table III, step
8) was estimated by comparing the intensity of the protein band with those of standard proteins after SDS-PAGE and silver staining.
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RESULTS |
Purification of PAF-dependent Transacetylase--
An
essential step in purifying the membrane enzyme is to first solubilize
the enzyme from the membrane. Although there are assortments of
detergents (ionic and non-ionic) and chaotropic agents available
commercially, the detergent selected will be the one that does not
inhibit or inactivate enzyme activity, does not present the enzyme in
an aggregated form, and has a high critical micellar concentration.
Additionally, the ratio of protein to detergent concentration is
important. In preliminary experiment, among the several ionic and
non-ionic detergents we tested (such as CHAPSO, octylglucoside, and
Triton X-100), PAF:lysoplasmalogen transacetylase activity was most
effectively solubilized by Tween 20. Enzyme activity was solubilized
from rat kidney membrane fractions (mitochondria plus microsomes,
100,000 × g pellets) by Tween 20 at a concentration of
0.02-0.05%, and the maximal specific activity was observed at a
detergent/protein (w/w) ratio of 0.1 (Table I).
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Table I
Effects of Tween 20 concentrations and Tween 20: protein ratios on the
solubilization of PAF:lysoplasmalogen transacetylase from the membrane
fraction of rat kidneys
Rat kidneys were homogenized in buffer and the membrane fraction was
isolated as described (13). The membrane fraction suspended in 0.25 M sucrose and 10 mM Hepes, (pH 7.0) was mixed
with 0.2% Tween 20 to attain the desired Tween 20 concentration and
Tween 20/protein ratio. The mixture was stirred at 4 °C for 30 min
and then centrifuged at 100,000 × g for 60 min to
obtain the supernatant and pellet fraction. Transacetylase activities
and protein concentrations in both fractions were measured to ensure
proper recovery. Data represent the average of at least duplicate
determinations.
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When the efficiency of the solubilization was tested on mitochondrial
and microsomal fractions separately instead of using total membrane
fraction, it was found that substantial amounts of enzyme activity
still remained in the 100,000 × g pellets, especially
with that of the microsomal fractions (Table
II). Therefore, a second attempt on the
solubilization of the remaining pellets was carried out. Around 8% and
10% of transacetylase activities were obtained from the second
solubilization of the mitochondrial and microsomal pellets,
respectively. However, the second solubilization from the mitochondrial
fraction decreased the specific activity of the enzyme because
relatively large amounts of unspecific proteins were concomitantly
released. Thus, solubilization procedures were performed twice for the
microsomal fraction and once for the mitochondrial fraction hereon.
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Table II
Solubilization of PAF:lysoplasmalogen transacetylase from mitochondrial
and microsomal fractions of rat kidneys
Four groups of mitochondrial and microsomal fractions were
independently prepared from 21 rats as described under "Experimental
Procedures." The solubilization of enzyme was carried out with 0.04%
Tween 20 at a detergent/protein (w/w) ratio of 0.1. Values were
represented as means ± S.E. (n = 4) per kidney.
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Overall, Tween 20 (0.02%) was included in all purification steps
because it maintained the stability of enzyme activity. The apparent
isoelectric point of the enzyme was estimated to be 5.0 by
chromatofocusing (data not shown). Native-PAGE was the crucial step for
the purification of this enzyme. The specific activity was increased
17-fold, as compared with that in the previous step (Table
III). It should be noted that the enzyme
activity was easily extracted from the polyacrylamide gel by soaking
the gel slice in the buffer solution without squeezing the gel or the
aid of electricity, as commonly performed as a method to extract the proteins from the polyacrylamide gel.
1-O-Hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine
(data not shown) and
1-O-hexadecyl-2-N-methylcarbamyl-GPE, which are
structurally related to PAF, competitively inhibit PAF:lysoplasmalogen transacetylase activity in Tween 20 (0.05%) solubilized membrane fraction from rat kidneys with Km = 9.1 µM, and Ki = 10.1 µM for
hexadecylacetyl-GPC and hexadecyl-N-methylcarbamyl-GPE, respectively (Fig. 1). Thus,
hexadecyl-N-methylcarbamyl-GPE is expected to be useful as a
specific ligand for the affinity gel matrix. This lipid has the
advantage of being resistant to enzymatic hydrolysis, while acetyl
ester of PAF is easily degraded by PAF-acetylhydrolase(s) in the cells.
Additionally, the amino group of hexadecylmethylcarbamyl-GPE can be
linked to the carboxyl-terminal group of CH-Sepharose covalently. All
the enzyme activity was adsorbed to the resulting gel matrix, while
enzyme activity was not adsorbed to the CH-Sepharose without ligand at
all (data not shown). The enzyme activity was not eluted from the gel
matrix by 0.5% CHAPS, 1% Triton X-100, 50% ethylene glycol, or 0.5 M NaCl. In order to elute the enzyme activity from the
column with a reasonable recovery, more than 5 mM PAF was required. The concentration of the ligand in the gel matrix was estimated to be 0.98 mM; therefore, a relatively high
concentration of PAF was needed for replacement. These results also
indicate that the enzyme specifically recognizes the ligand at the
site, which is necessary for the interaction with the substrate.
Starting from rat kidney membrane, 13,700-fold purification of the
enzyme was achieved with a yield of about 4%. A typical result of the purification procedures is summarized in Table III.

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Fig. 1.
Effect of
1-hexadecyl-2-N-methylcarbamyl-GPE on the PAF:
lysoplasmalogen transacetylase activity: a double-reciprocal plot.
The transacetylase activity was determined with various concentrations
of hexadecyl-[3H]acetyl-GPC and 300 µM
lysoplasmalogen in the absence or presence of 20 µM
1-hexadecyl-2-N-methylcarbamyl-GPE. Tween 20 (0.05%)
solubilized membrane fraction from rat kidneys (1 µg) was used as the
enzyme source. Values are average of duplicate determinations with
variations <10%. The calculated Km for PAF and
Ki for 1-hexadecyl-2-N-methylcarbamyl-GPE
are shown on the graph.
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Purity and Molecular Weight of the Enzyme--
Different fractions
obtained during the enzyme purification were examined by SDS-PAGE (Fig.
2). The enzyme obtained after the final
step of purification showed a single homogeneous band. By comparing the
mobility of the enzyme band on SDS-PAGE with molecular size markers, a
molecular mass of 40 kDa was deduced for the enzyme (Fig.
3). In addition, the intensity of the
band of 40 kDa on SDS-PAGE was correlated with enzyme activity in the individual purification steps including chromatofocusing, native-PAGE, and affinity gel matrix (data not shown), suggesting that the band of
40 kDa is PAF-dependent transacetylase. Although we have attempted to estimate the molecular weight of natural enzyme on gel
filtration column of Sephacryl S-200, the enzyme activity was eluted
near the void volume due to the formation of mixed micelles with Tween
20 (data not shown).

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Fig. 2.
SDS-PAGE of protein fractions isolated from
different steps of purification of PAF-dependent
transactylase from rat kidney membranes. SDS-PAGE was
carried out as described under "Experimental Procedures."
Amounts of transacetylase activities loaded onto each lane are listed
in parentheses. Lane 1, mitochondrial fractions
(1 nmol/min); lane 2, microsomal fractions (1 nmol/min); lane 3, solubilized fractions from
mitochondria (1 nmol/min); lane 4, solubilized
fractions from microsomal fractions (1 nmol/min); lane
5, DEAE-Sepharose (1 nmol/min); lane
6, hydroxyapatite (1 nmol/min); lane
7, phenyl-Sepharose (1 nmol/min); lane
8, chromatofocusing (2 nmol/min); lane
9, native-PAGE (4 nmol/min); lane 10,
affinity gel (4 nmol/min). Molecular weight standards: bovine serum
albumin (Mr = 66,000), ovalbumin
(Mr = 45,000), glyceraldehyde-3-phosphate
dehydrogenase (Mr = 36,000), carbonic
anhydrolase (Mr = 29,000), trypsinogen
(Mr = 24,000), and trypsin inhibitor
(Mr = 20, 100).
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Fig. 3.
Determination of molecular weight of
PAF-dependent transacetylase. The log molecular weight
of standard proteins versus mobility on the SDS-PAGE was
plotted. Molecular weight protein markers were the same as described in
Fig. 2. The molecular mass of the purified transacetylase from rat
kidney membranes is shown by the arrow.
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Partial Amino Acid Sequences--
The purified protein was
digested with trypsin, and five of the peptide fragments were sequenced
as described under "Experimental Procedures." Amino acid sequences
of five peptide fragments were: GTLDPYEGQEVMVR, AML*AF*L*QK,
LFSSGTR, IKEGEKEFHVR, and L*PVSWNGPF*K* (* indicate that isobaric
amino acid residues cannot be unambiguously differentiated in mass
spectrometric sequence, but such residues are displayed by alignment
with a known or homology to a known, and with reference to enzyme
specificity). A search using a protein sequence data bank indicated
that these sequences had homology with the sequences present in bovine
PAF-acetylhydrolase II (GenBankTM/EBI Data Bank accession
number D87559; 78.6%, 100%, 71.4%, 81.8%, and 100%, respectively).
Substrate Specificity--
A different type of transacetylase that
transfers the acetate of PAF to sphingosine forming
C2-ceramide was described in HL 60 cells by us (6). The
ratios of specific activity toward lysoplasmalogens/sphingosine were
49.7 and 80.8 in crude mitochondrial and microsomal membranes,
respectively. In the final purified preparation, it also contains
PAF:sphingosine transacetylase activity. Nevertheless, the
sphingosine transacetylase activity was more labile during
purification and storage than that of lysoplasmalogen transacetylase. Thus, we could not accurately assess the ratio of
lysoplasmalogen/sphingosine transacetylase activities.
Notwithstanding, these data indicate that a single enzyme can
catalyze both kinds of transacetylase activities. The higher ratio
toward lysoplasmalogens as the substrate may partly be explained by the
fact that, in order to assay PAF:sphingosine transacetylase activity,
we had to include equal molar ratio of BSA in the incubations, and BSA severely inhibited sphingosine transacetylase reaction (6). However,
incorporation of BSA as the assay medium for sphingosine transacetylase
was necessary in order to maintain zero order kinetics and reduce
nonenzymatic generation of the product (6). Lysoplasmalogen transacetylase did not show the same BSA requirement as that of sphingosine transacetylase and was measured in the presence of 0.1%
BSA-saline (3).
The finding that the enzyme shared the sequence homology with bovine
PAF-acetylhydrolase II led us to ask the question of whether the enzyme
carried the activity of hydrolyzing PAF. We found that the
PAF-dependent transacetylase also contains PAF hydrolyzing
activities in crude membrane fractions and purified preparations; the
ratios of lysoplasmalogen transacetylase/acetylhydrolase were 0.74, 0.84, and 1.04 (average of two experiments with <10% variation) in
the mitochondria, microsomes, and purified enzyme, respectively.
The reason for the slight increase in ratios of lysoplasmalogen
transacetylase/acetylhydrolase activities during purification is not
clear at present. It is possible that some of the contaminating acetylhydrolase activities were removed during purification procedures or the presentation of substrates might differ between the purified enzyme and crude membrane-bound enzymes due to the possible interaction of membrane phospholipids with the lipid substrates.
Kinetic Properties--
The dependence of enzyme activity on
substrate concentration is shown in Fig.
4. PAF:lysoplasmalogen transacetylase
activity displayed typical Michaelis-Menten kinetics, with an apparent Km of 227 µM for lysoplasmalogen and a
Vmax of 81 µmol/min·mg (Fig. 4A).
Interestingly, the Km for lysoplasmalogens was 106.4 µM in the membranes of HL-60 cells (3) and 80 µM in the homogenates of calf pulmonary artery
endothelial cells (data not shown). These results suggest that the
Km values are varied with different tissues and cell
types. Nevertheless, these values are well below the physiological
concentration of lysoplasmalogens in the cells. Calculation from the
data obtaining by Tessner et al. (16) and Nieto et
al. (17) indicated that the lysoplasmalogen concentration could
reach 0.5-0.7 mM when human neutrophils were stimulated
with ionophore A23187 for 5 min.

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Fig. 4.
Kinetics of PAF-dependent
transacetylase. A purified preparation (0.86 ng) of
PAF-dependent transacetylase was incubated with varying
amounts of lysoplasmalogen (A) or sphingosine
(B). Double-reciprocal plot according to Lineweaver and Burk
is shown in the inset. Each value represented the average of
triplicate determinations. Correlation coefficient was >0.98 when
lysoplasmalogen was used as the substrate and >0.95 when sphingosine
was the substrate. Data were expressed as means ± S.D.
(n = 3).
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In contrast, inhibition of sphingosine transacetylase activity (Fig.
4B) was resulted when sphingosine concentrations were higher
than 50 µM. If the points during the ascending portion of
the curve were used, the calculated Km for
sphingosine was 4.1 µM. This value is similar to the
Km reported for sphingosine of the purified rat
kidney sphingosine kinase (5 µM) (18).
We have shown that, using mixed substrate experiments and crude
membrane fractions from HL-60 cells (6), either CoA-independent transacetylase has a higher substrate affinity for sphingosine than any
of the other substrate analogs, or the possibility of two isoforms of
the transacetylase might be involved in the transfer of acetate from
PAF to sphingosine and lysophospholipids. When mixed substrate
experiments were performed in the present studies using purified
enzyme, similar results to those using crude membranes from HL-60 cells
were obtained (6). Sphingosine at 15 and 50 µM inhibited
the lysoplasmalogen transacetylase activity by 32% and 80%,
respectively. On the other hand, lysoplasmalogens at 15 and 50 µM had no effect on the sphingosine transacetylase
activity. Thus, we further confirmed that transacetylase has a higher
substrate affinity for sphingosine than lysophopholipids.
Effect of Inhibitors--
PAF:lysoplasmalogen transacetylase
activity was inhibited by diisopropyl fluorophosphate in a
dose-dependent manner (Table IV and data not shown). These data
suggest that serine residue is involved in the active site of the
enzyme. The specific inhibitor of plasma PAF-AH and serine esterase
(19), p-aminoethyl benzenesulfonyl fluoride (Pefabloc),
completely inhibited PAF:lysoplasmalogen transacetylase at a
concentration of 0.1 mM. These results are in good
agreement with the fact that the transacetylase partially shared
structural homology with PAF-AH II, and human PAF-AH II exhibited 43%
predicted amino acid identity to human plasma PAF-AH (20). Both
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and
N-ethylmaleimide (NEM), which react with the sulfhydryl
group of cysteine, partially block the enzyme activity, indicating that
sulfhydryl group is important for enzyme activity. Diethyl
pyrocarbonate, a modifier of the histidine, at 1 mM
partially inhibited the PAF:lysoplasmalogen transacetylase activity. In
parallel, the PAF:sphingosine transacetylase activities were also
affected by these inhibitors except with different sensitivities
especially with the sulfhydryl reagents (Table IV).
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Table IV
Effects of inhibitors on PAF:lysoplasmalogen and PAF:sphingosine
transacetylase activities purified from rat kidney membranes
Each inhibitor was incubated with the purified enzyme for 30 min at
37 °C, and then the enzyme reaction was started by adding the
substrate.
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DISCUSSION |
PAF-dependent transacetylase was purified from rat
kidney membrane 13,700-fold. The final preparation, which was judged to be nearly homogeneous by SDS-APGE, yielded a single protein band of 40 kDa. DTT was included throughout the purification because enzyme
activity was rapidly decreased in the absence of DTT. DTT is thought to
be necessary for protecting the sulfhydryl group of cysteine from
oxidation because DTNB and NEM partially abolish the enzyme activity
(Table IV). NEM and DTNB inhibit PAF:lysoplasmalogen and
PAF:sphingosine transacetylases with different inhibitory potencies.
For example, PAF:sphingosine transacetylase activity (22.6% of enzyme
activity remained after 30 min of 0.1 mM NEM treatment) was
more susceptible to NEM than PAF:lysoplasmalogen transacetylase (which
retained 90.7% of enzyme activity after 0.1 mM NEM
treatment for 30 min). These results are consistent with our previous
work using HL-60 cell membranes as the enzyme source (6). This
differential effect of NEM and DTNB on enzyme activities suggests that
the sulfhydryl group of cysteine play a more important role in the
interaction of the enzyme with sphingosine, as compared with that of lysoplasmalogen.
In the present investigation, PAF-dependent transacetylase
was isolated from mitochondrial and microsomal membrane fractions. However, PAF-dependent transacetylase activity is also
present in cytosolic fraction (7). It is well known that some of the enzymes are distributed in both membrane and cytosols, and
translocation of the enzymes is regulated by modification of the
protein, such as phosphorylation (21) and myristoylation (22). For
example, phosphorylation of cytosolic phospholipase A2
causes translocation from the cytosol to the membrane (21). We have
demonstrated that PAF:acyllyso-GPC transacetylase is regulated through
phosphorylation/dephosphorylation (4). It is not known presently
whether phosphorylation/dephosphorylation is involved in determining
the cellular localization of PAF-dependent transacetylase
or PAF-AH II is regulated through phosphorylation/dephosphorylation. The availability of a homogeneous preparation of transacetylase protein
will facilitate the development of specific antibodies against this
enzyme. Specific antibodies against PAF-dependent transacetylase will be useful in clarifying the relationships of
the enzyme activities located in membrane and cytosol as well as
the mechanism of translocation of this enzyme between these two fractions.
Analysis of the partial amino acid sequences of five tryptic-digested
peptide fragments revealed that this enzyme shared sequence homology
with the previously reported bovine cytosolic PAF-AH II (20). Cytosolic
PAF-AH II was found to be N-myristoylated and could be
translocated from cytosol to membranes in oxidative stress-induced
cells (23). In addition, purified PAF-dependent transacetylase from rat kidney membranes contained PAF-AH activity in
the absence of a lipid acceptor. The activities of both
PAF-dependent transacetylase and PAF-AH II (24) were
inhibited by serine esterase inhibitors and sulfhydryl reagents.
Preliminary results (unpublished data in collaboration with Dr. K. Inoue's laboratory) also indicated that purified transacetylase and
transacetylases present in the mitochondria and microsomes cross reacts
with anti-human PAF-AH II monoclonal antibody. These results suggest
that transacetylase and PAF-AH II share similar structural requirement
for the active site and the same recognition site for the immunological
epitope. However, we have to wait for the result of cDNA cloning of
the transacetylase in order to compare the sequence of the
transacetylase with that of the PAF AH II. Information obtained from
the amino acid sequences of the analyzed peptides of the
PAF-dependent transacetylase should provide the necessary
means to identify the specific cDNA clones from the rat kidney
cDNA library. It is possible that transacetylase and PAF-AH II
share the same amino acid sequences, but are differentially posttranslationally modified.
Regardless of structural similarity or difference between
transacetylase and PAF-AH II, our finding that transacetylase has three
distinct catalytic activities raise important questions concerning the
regulation, biological implications, and consequences of this unique
enzyme. It is possible that PAF may exert some of its biological
effects through transacetylase without the need or presence of
participation of intracellular PAF receptor (s).
In addition to the PAF-dependent transacetylase, there are
other examples that an enzyme with transesterification activity also
possesses hydrolytic activity. Lecithin-cholesterol acyltransferase (LCAT), which normally transfers the acyl group of PC to cholesterol, also hydrolyzes PAF (25). PAF-AH activity of LCAT plays the role of
detoxification of oxidized PC, especially when the PAF-AH is absent or
inactivated (25). Thus, PAF-dependent transacetylase is
similar to LCAT and likely to be an enzyme that catalyzes diverse reactions and thus leads to different biological functions.
In conclusion, we have demonstrated that a single enzyme catalyzes
three kinds of reactions, namely PAF:lysoplasmalogen
(lysophospholipids) transacetylase, PAF:sphingosine transacetylase,
and PAF-acetylhydrolase. Depending on the differences of acceptor lipid
molecules, different lipid products such as PAF analogs of plasmalogens
(also acyl analogs of PAF and etc.), C2-ceramide, and
lyso-PAF will be generated by the transacetylase through these three
catalytic activities. These different lipid products possess different
cellular functions when compared with that of the original substrates
(i.e. PAF and sphingosine). For instance, sphingosine can
induce a potent and reversible inhibition of protein kinase C in
vitro and in cell cultures (26-29). Recently, Rodriguez-Lafrasse
(30) provided the first evidence to show that protein kinase C
inhibition is directly related to the sphingosine accumulation in
vivo. On the other hand, C2-ceramide has been
extensively used by many investigators as an unnatural, cell permeable
analog of long-chain acyl-ceramides to mimic the effects of various
inducers of sphingomyelin-ceramide signal pathways (8, 9). Multiple
experimental approaches suggest that long-chain acyl ceramides and
C2-ceramide play an important role in regulating cell cycle
arrest, apoptosis, differentiation, and cell senescence (8). However,
our previous data (6) demonstrated that PAF-dependent
transacetylase is the enzyme responsible for the biosynthesis of
C2-ceramide, and the cellular concentration of
C2-ceramide is in the range (micromolar) that could exert
significant biological effects. In addition, the signaling pathways
utilized by the sphingomyelinase differ from those of cell-permeable
ceramide anologs (31). Additionally, no detectable biological effects are observed in that neutral sphingomyelinase overexpressed cells (32).
These results suggest that C2-ceramide should be classified as a naturally occurring novel lipid mediator. Furthermore, acyl analogs of PAF have biological characteristics distinct from that of
PAF (4). Future research in this laboratory is directed toward the
elucidation of the mechanism(s) of how a single enzyme controls and
regulates three different kinds of enzymatic reactions. Additionally,
progress is being made to generate cDNA and polyclonal antibodies
against the transacetylase.