Báo cáo khoa học: Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT) activity in cortical and medullary human renal tissue docx

Acetyl-CoA:1-
O
-alkyl-
sn
-glycero-3-phosphocholine acetyltransferase
(lyso-PAF AT) activity in cortical and medullary human
renal tissue
Tzortzis N. Nomikos
1
, Christos Iatrou
2
and Constantine A. Demopoulos
1
1
National and Kapodistrian University of Athens, Faculty of Chemistry, Panepistimioupolis, and
2
Center for Nephrology
‘G. Papadakis’, General Hospital of Nikea-Pireaus, Athens, Greece
Platelet-activating factor (PAF) is one of the most potent
inflammatory mediators. It is biosynthesized by either the
de novo biosynthesis of glyceryl ether lipids or by remodeling
of membrane phospholipids. PAF is synthesized and catabo-
lized by various renal cells and tissues and exerts a wide range
of biological activities on renal tissue suggesting a potential
role during renal injury. The aim of this study was to identify
whether cortex and medulla of human kidney contain the
acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyl-
transferase (lyso-PAF AT) activity which catalyses the last
step of the remodeling biosynthetic route of PAF and is
activated in inflammatory conditions. Cortex and medulla
were obtained from nephrectomized patients with adeno-

carcinoma and the enzymatic activity was determined by a
trichloroacetic acid precipitation method. Lyso-PAF AT
activity was detected in both cortex and medulla and dis-
tributed among the membrane subcellular fractions. No
statistical differences between the specific activity of cortical
and medullary lyso-PAF AT was found. Both cortical and
medullary microsomal lyso-PAF ATs share similar bio-
chemical properties indicating common cellular sources.
Keywords: platelet-activating factor; biosynthesis; remode-
ling; acetyltransferase; human kidney.
Introduction
1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-
activating factor, PAF) [1] represents a class of highly active
lipid mediators. It is produced and released by various cells
such as leukocytes, lymphocytes, endothelial cells, neurons,
myocytes, hepatic cells and it is known to elicit a variety of
biological responses participating in the pathogenesis of
many inflammatory and noninflammatory diseases [2].
PAF is produced by two distinct biosynthetic pathways.
The first pathway, the de novo pathway, starts with the
acetylation of 1-O-alkyl-sn-glycero-3-phosphate (ALPA)
by the acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphate acetyl-
transferase (ALPA AT) (EC 2.3.1.105) followed by the
sequential action of a phosphohydrolase and a dithiothre-
itol-insensitive cholinephosphotransferase. The second
pathway, the remodeling pathway, involves the hydrolysis
of preexisting plasma membrane phospholipids to
1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) which
is then acetylated by the acetyl-CoA:1-O-alkyl-sn-glycero-
3-phosphocholine acetyltransferase (lyso-PAF AT; EC

2.3.1.67) leading to the formation of PAF. The de novo
pathway seems to be responsible for the constitutive
production of PAF at basal levels in cells, while the
remodeling one is thought to be responsible for the
increased production of PAF by inflammatory cells upon
stimulation. The latter pathway is regulated mainly by the
level and the degree of lyso-PAF AT and 85 kDa cPLA
2
activation. The latter enzyme hydrolyses plasma membrane
phospholipids serving the substrates for lyso-PAF AT [3].
Lyso-PAF AT is found in the microsomal fraction of
cells and tissues. It has a rather broad substrate specificity,
concerning the alkyl/acyl group at the sn-1 position and
the polar head group at the sn-3 position of the glycerol
backbone, it is Ca
2+
-dependent and is activated by
phosphorylation through the action of cAMP-dependent
protein kinases, calcium-calmodulin dependent protein
kinases, protein kinase C [4,5] and mitogen-activated
protein kinases [6,7]. According to our knowledge only
partial purification of lyso-PAF AT from rat spleens,
leading to a very low yield of pure enzyme, has been
reported [8,9].
The possibility that the kidney could produce PAF, even
under normal physiological conditions, has been proposed
in view of its activity in human urine [10,11]. PAF
production in the kidney is originated either by blood-born
cells (neutrophils, platelets and macrophages) or by resident
glomerular (messangial and endothelial) and medullary cells

[12,13]. Both biosynthetic pathways have been exhibited in
renal cells of different animal species and human and several
Correspondence to C. A. Demopoulos, 39 Anafis Str., Athens,
GR-113 64, Greece.
Fax: + 32 10 7274265, Tel.: + 32 10 7274265,
E-mail:
Abbreviations:ALPA,1-O-alkyl-sn-glycero-3-phosphate; ALPA AT,
acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphate acetyltransferase;
ESI, electron spray ionization; [H
3
]PAF, 1-O-hexadecyl-2-[H
3
]acetyl-
sn-glycero-3-phosphocholine; PAF, platelet-activating factor;
lyso-PAF AT, acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine
acetyltransferase; PAF-AH, platelet-activating factor acetylhydrolase.
Note: a web site is available at />Laboratories/FoodChem/CVS/demopoulos.htm
(Received 13 February 2003, revised 18 April 2003,
accepted 16 May 2003)
Eur. J. Biochem. 270, 2992–3000 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03676.x
of their enzymes have been characterized in both cultures
and tissues [14–18]. However, as in other cases, the
activation of lyso-PAF AT (to wit, of the remodeling
pathway) is mainly responsible for the rapid and increased
synthesis and release of PAF from the kidney, which
occurred after stimulation with inflammatory mediators
[19,20]. Increased levels of PAF have been found in blood,
urine and kidney tissue of animals and humans with renal
inflammatory diseases, such as glomerulonephritis and
could be or due to the enhanced biosynthesis or decreased

degradation or to a combination of increased production
and decreased degradation of PAF in nephritic tissue
[21,22].
The latter proposal comes from our previous studies in
which we have demonstrated: (a) the existence of PAF
acetylhydrolase, PAF-AH, the degradative enzyme of PAF,
in human kidney tissue (cortex and medulla) [23]; (b) the
diminished activity of PAF-AH in renal tissue (mainly
cortex) received from patients with primary glomerulo-
nephritis compared to normal ones [24]; and (c) increased
levels of PAF in plasma and urine as well as increased PAF-
AH activity in serum in patients with primary glomerulo-
nephritis in comparison to normal volunteers [24].
As our previous works demonstrated the presence of the
degradative enzyme of PAF (PAF-AH) in human kidney
tissue, in this work an attempt is made to investigate the
presence of the biosynthetic enzyme of PAF, lyso-PAF AT,
in the same kind of tissue. Additionally, we characterized
and compared the main biochemical properties of the two
enzymatic activities (cortical and medullary lyso-PAF AT),
utilizing a modified trichloroacteic acid precipitation
method for the lyso-PAF AT assay. Our results demon-
strate the existence of lyso-PAF AT activity in both cortex
and medulla of human kidneys and show that cortical and
medullary lyso-PAF AT share similar biochemical proper-
ties indicating common cellular sources.
Materials and methods
Materials and instrumentation
All solvents were of analytical grade and supplied by Merck
(Darmstadt, Germany). HPLC solvents were from Rath-

burn (Walkerburn, Peebleshire, UK). The separation of the
lipid products of the assays was performed at room
temperature on a HP HPLC Series 1100 liquid chromato-
graphy model (Hewlett Packard, Waldbronn, German)
equipped with a 100-lL Rheodyne (7725 i.d.)
1
loop valve
injector, a degasser G1322A, a quat gradient pump G1311A
and a HP UV spectrophotometer G1314A as a detection
system. The spectrophotometer was connected to a Hewlett-
Packard (Hewlett-Packard, Waldbronn, German) model
HP-3395 integrator-plotter. The separation of lipids was
performed on a Partisil 10 lmC
18
column (250 · 4.6 mm
i.d.) from Analysentechnick (Wo
¨
ehlerstrasse, Mainz,
Germany) with an C
18
(20 · 4.0 mm i.d.) precolumn cart-
ridge. Chromatographic material used for TLC was silica
gel H-60 (Merck). The platelet aggregation was measured in
a Chrono-Log (Havertown, PA, USA) aggregometer cou-
pled to a Chrono-Log recorder (Chrono-Log). Electrospray
ionization (ESI) mass spectrometry experiments were per-
formed on a Q-Tof (Micromass UK Ltd, Manchester, UK)
orthogonal acceleration quadrupole time-of-flight mass
spectrometer equipped with nano-electrospray ionization.
Radioactivity was measured in a 1209 RackBeta-Flexivial

a-Counter (LKB-Pharmacia, Turku, Finland). A Virsonic
50 Ultrasonic Cell Disruptor was used for the homogeni-
zation of our samples (Virtis Co Gardiner, USA). All
centrifugations were performed in a Sorvall RC-5B refri-
gerated Superspeed centrifuge (Sigma) apart from the
centrifugation at 100 000 g, which was performed in a
Heraeus Christ, Omega 70 000 ultracentrifuge (Hanau,
Germany). For the precipitation of the BSA
2
pellet, a Sigma
201
M
microcentrifuge was used (Sigma, St. Louis, MO,
USA).
1-O-Hexadecyl-2-[H
3
]acetyl-sn-glycero-3-phosphocho-
line, [H
3
]PAF (specific activity 6 CiÆmmol
)1
) was purchased
from DuPont NEN (Boston, MA, USA). [H
3
]acetyl-CoA
(specific activity 200 mCiÆmmol
)1
) was obtained from ICN
(CostaMesa,CA,USA).UnlabeledPAF,lyso-PAF(1-O-
hexadecyl-2-lyso-sn-glycero-3-phosphocholine) and acetyl-

CoA were from Sigma Chemicals Co. 2,5-Diphenyloxazole
(PPO) and 1,4-bis(5-phenyl-2-oxazolyl)benzene (POPOP)
were purchased from BDH Chemicals (Dorset, UK).
4-[2-Aminoethyl]benzenesulfonyl fluoride (pefabloc) was
kindly offered by A. Tselepis, University of Ioannina,
Greece
3
. All other reagents were from Sigma Chemicals.
Human renal tissues
Human renal tissues were obtained from 20 nephrectomized
patients with adenocarcinoma. Immediately after the neph-
rectomy the kidneys were perfused with normal saline and
a speciment of the apparently normal parenchyma was
separated under stereoscopic microscopy into cortex and
medulla and placed immediately in cold saline. Subse-
quently, all homogenization and subcellular fractionation
procedures were completed in less than 3 h.
Homogenization of renal tissues and preparation
of subcellular fractions
Homogenization of cortical and medullary samples and
preparation of subcellular fraction is carried out by a
modification of the method described by Lenihan and Lee
[25]. Briefly, cortical and medullary tissues were rinsed with
ice-cold 0.25
M
sucrose, minced and homogenized with six
strokes of a motor-driven Potter-Elvehjem homogenizer
in 0.25
M
sucrose, 10 m

M
EDTA, 5 m
M
mercaptoethanol,
50 m
M
NaF, 50 m
M
Tris/HCl (pH 7.4) (homogenization
buffer). The final concentration of the tissue in the
homogenization buffer was 10% w/v. Further homogeni-
zation of the tissue by sonication (4 · 20 s with intervals of
1 min) was followed. The homogenates were centrifuged at
500 g for 10 min. The pellets were discarded, a small
portion of the supernatants was kept for protein and
lyso-PAF AT determination and the rest of them were
centrifuged at 20 000 g for 15 min. The resulting pellets
(mitochondria fraction) were suspended in 1 mL of 0.25
M
sucrose, 1 m
M
dithiothreitol, 50 m
M
Tris/HCl (pH 7.4)
(suspension buffer) (5–10 mg of proteinÆmL
)1
) while the
supernatants were centrifuged at 100 000 g for 1 h. The
100 000 g pellet (microsomal fraction) was suspended in
1 mL of suspension buffer (1–5 mg of proteinÆmL

)1
). Total
Ó FEBS 2003 Lyso-PAF acetyltransferase activity in human kidney (Eur. J. Biochem. 270) 2993
membranes were obtained by centrifugation of the 500 g
supernatant at 100 000 g for 1 h. All fractions were
aliquoted and stored at – 30 °C. All homogenization and
fractionation procedures were carried out at 0 °C.
Lyso-PAF AT activity assay
Unless stated otherwise, subcellular fractions of cortex and
medulla, containing 10–40 lg of total protein, were incu-
bated with 4 nmol of lyso-PAF and 40 nmol of [H
3
]acetyl-
CoA (100 BqÆnmol
)1
)for30minat37°C in a final volume
of 200 lLof50m
M
Tris/HCl buffer (pH 7.4) containing
0.25 mg mL
)1
BSA. The final volume of the suspension
buffer in all incubation mixtures was 50 lL. By the end of
the incubation time, 2 lLofBSA100 mgÆmL
)1
were added
and the reaction was stopped by addition of 64 lLofa40%
cold trichloroacetic acid solution. The reaction mixture was
kept in ice for 30 min and centrifuged at 10 000 g for 2 min.
The supernatant was discarded and the pellet containing the

[H
3
]PAF bound to the denaturated BSA is dissolved in
the scintillation cocktail (dioxane-base) and the radioactivity
was determined by liquid scintillation counting. Matching
controls were run in the absence of lyso-PAF in order to
subtract the radioactivity of the endogenously produced
[H
3
]PAF.
Characterization of the lipid products of the lyso-PAF
AT assay
In order to identify the lipid products of the lyso-PAF AT
assay, the enzymatic reactions were terminated by lipid
extraction using the method of Bligh and Dyer [26],
modified by the addition of 1
M
HCl to the methanol.
The lipid products were separated on TLC plates precoated
with Silica Gel H using a solvent system of chloroform–
methanol–ethanoic acid–water (100 : 57 : 16 : 8, by vol-
ume). The distribution of the label was determined by zonal
scraping and measuring the radioactivity by liquid scintil-
lation spectrometry. Standards of [H
3
]PAF and [H
3
]acetyl-
CoAwererunwiththesamples.
Lipid products were further analyzed by HPLC on a

reversed-phase C
18
using an isocratic elution system consis-
ted of methanol 90% and water 10% (v/v) and a flow rate of
1mLÆmin
)1
. Fractions of 0.5 mL were collected and their
radioactivity was measured by liquid scintillation counting.
The retention time of authentic [H
3
]PAF and [H
3
]acetyl-CoA
was compared with the retention time of the radioactive
peaks obtained from the separation of assay products.
The above procedures were also applied for the extraction
and chromatographic analysis of the radioactive products
bound to the BSA precipitates after the addition of
trichloroacetic acid to the reaction mixture.
The radioactive product, co eluted with authentic PAF at
either TLC or HPLC separation, was further characterized
by bioassay using the washed rabbit platelet aggregation
method as described previously [1]. We estimated the
biological activity of the product by measuring its aggre-
gatory activity towards washed rabbit platelets and com-
paring it with the aggregatory activity of known
concentrations of synthetic PAF. The biological activity of
the hydrolyzed product, obtained by mild alkaline hydro-
lysis as well as the biological activity of the reacetylated
product obtained by acetylation of the hydrolyzed product

was also estimated as described previously [27]. Finally, the
percentage inhibitory activity of 0.7 m
M
creatine phosphate/
creatine phosphate kinase, 10 l
M
4,5
indomethacin and 0.1 l
M
4,5
BN 52021 towards the biologically active product was
compared with their respective activity towards standard
PAF of the same aggregatory activity with the product.
The biologically active lipid was also analyzed by ESI
mass spectrometry. Samples were dissolved in a small
volume of HPLC grade methanol–water (70 : 30, v/v)
0.01
M
in ammonium acetate. Electrospray samples are
typically introduced into the mass analyzer at a rate of
4.0 lLÆmin
)1
. The positive and negative ions, generated by
charged droplet evaporation, entered the analyzer through
an interface plate and a 100-mm orifice, while the declus-
tering potential was maintained between 60 and 100 V to
control the collisional energy of the ions entering the mass
analyzer. The emitter voltage was typically maintained at
4000 V.
Analytical methods

Protein was determined by the method of Lowry et al.[28].
Statistical analysis
Unless otherwise stated, data are expressed as mean
values ± SD. Differences between groups were assessed
by Mann–Whitney U-test. A P-value <0.05 was considered
as significant.
Results
Characterization of the trichloroacetic acid method
for the determination of lyso-PAF AT activity
Preliminary experiments were carried out in order to
determine the best experimental conditions for the quanti-
tative recovery of [
3
H]-PAF, the product of the lyso-PAF
AT assay, to the BSA precipitate. [
3
H]-PAF of known
specific activity (20 000 c.p.m., 20 l
M
)
6
was incubated with
various concentrations of BSA, ranging from 0 to
8mgmL
)1
,in50m
M
Tris/HCl buffer (pH 7.4) and the
radioactivity of the pellet obtained by the addition of
trichloroacetic acid was measured. Practically, all [

3
H]-PAF
is obtained in the protein pellet at BSA concentrations of
0.5 mgÆmL
)1
and higher while only 0.6% of [
3
H]-acetyl-
CoA was coprecipitated with [
3
H]-PAF the rest of which
remained in the supernatant. This indicates an efficient
separation of the substrate, [
3
H]-acetyl-CoA and the
reaction product, [
3
H]-PAF. Incubation of [
3
H]-PAF with
different concentrations (0–0.5 mgÆmL
)1
) of various subcel-
lular fractions of human kidney, such as the 100 000 g
pellet, the 100 000 g supernatant, the 20 000 g pellet and the
homogenates could not alter the recovery of [
3
H]-PAF in
the BSA pellet indicating that apart from BSA no other
endogenous protein could affect the precipitation of PAF

after the addition of trichloroacetic acid. None of the other
constituents of the lyso-PAF AT assay, such as the type of
buffer solution, the type and concentration of the enzymatic
preparation, the temperature and the pH had any major
effect on the recovery of the [
3
H]-PAF at the BSA pellet.
2994 T. N. Nomikos et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Characterization of the assay products
The products of the lyso-PAF AT assay, with either cortex
or medulla microsomes, were extracted by the Bligh-Dyer
method and separated by TLC. The main radioactive
product comigrated with authentic PAF at R
F
0.2. When
the lyso-PAF AT assay was carried out in the absence of
lyso-PAF, a minimal formation of [
3
H]-PAF was observed
suggesting that either endogenous lyso-PAF or lyso-PC may
be used as acetyl acceptors. A small radioactive peak at R
F
0.8 was also observed in the presence or absence of lyso-
PAF, which is probably due to by products of the reaction
between [
3
H]-acetyl-CoA and microsomal constituents. The
same profile was obtained when the products of the assay
were analyzed by HPLC (Fig. 1).
The distribution of the radioactivity between the BSA

pellet and the supernatant, after the addition of trichloro-
acetic acid to the incubation mixture, was also determined.
Incubation of cortical or medullary microsomes with
[
3
H]-acetyl-CoA in the absence of lyso-PAF or incubation
of [
3
H]-acetyl-CoA with lyso-PAF in the absence of micro-
somes resulted in the precipitation of 0.8% of the radio-
activity to the BSA pellet. All the precipitated radioactivity
was found in the water phase of the Bligh–Dyer extraction of
the pellet indicating that it was due to the small fraction of
[
3
H]-acetyl-CoA that was precipitated to the BSA pellet
under the assay conditions. Incubation of microsomes with
both lyso-PAF and [
3
H]-acetyl-CoA resulted in a six- to
sevenfold increase of radioactivity in the pellet, most of which
was distributed to the organic phase of the Bligh–Dyer
extraction of the pellet and it comigrated with authentic PAF
after TLC analysis. No [
3
H]-PAF was found in the super-
natant of the incubation mixture after the addition of
trichloroacetic acid indicating that all [
3
H]-PAF produced in

the lyso-PAF AT assay is precipitated in the BSA pellet.
The lipid product, comigrated with PAF, and could
activate the aggregation of washed rabbit platelets. The
biological activity of the product was proportional to its
radioactivity indicating that the radioactive product and the
biologically active compound are the same molecule.
Moreover, inhibitors of platelet aggregation such as
BN-52021, indomethacin and the enzymatic system of
creatinine phosphate and creatinine phosphate kinase
exerted the same inhibitory effect on both standard PAF
and the lipid product of similar aggregatory activity. Mild
alkaline hydrolysis of the product resulted to a complete loss
of its biological activity, which is re-obtained by reacetyla-
tion of the hydrolyzed product.
The positive ESI spectra of the assay product showed
[M + Na]
+
at m/z 546. The ions at m/z 184 and 147 cor-
responding to fragments of the phosphocholine moiety were
also present (Fig. 2). All the above findings show that the
only product of the lyso-PAF AT assay is indeed C
16
-PAF.
Effect of BSA on the microsomal lyso-PAF AT activity
of human cortex and medulla
As shown in Fig. 3, a plateau of maximum activity, for both
cortical and medullary lyso-PAF ATs, was observed for
BSA concentrations ranging from 0 mgÆmL
)1
to

0.5 mgÆmL
)1
. BSA concentrations above 0.5 mgÆmL
)1
have
a slight inhibitory effect on both acetyltransferases activities.
We routinely used 0.25 mgÆmL
)1
BSA for the determina-
tions of lyso-PAF AT activity. However, this concentration
is insufficient for the quantitative precipitation of [
3
H]-PAF
so an excess of BSA (final concentration 2 mg mL
)1
)was
added at the end of the incubation time in order to achieve
the maximum recovery of PAF at the BSA precipitate.
Effect of Tris concentration and pH on the microsomal
lyso-PAF AT activity of human cortex and medulla
We studied the dependence of the enzyme activity with pH
at 20, 50, 100 m
M
7
of Tris-buffered solution. The pH–activity
profile was bell-shaped for both enzymes and the optimum
pH was in the range of 7.4–7.7. Lyso-PAF AT activity was
also dependent on Tris concentration showing maximum
activity at 50 m
M

8
of Tris. Subsequent experiments were
carried out with 50 m
M
9
Tris/HCl (pH 7.4).
Kinetics of PAF formation
The kinetics of PAF synthesis in relation to time and
microsomal protein are shown in Fig. 4. Linearity for up to
20 min of incubation and 0.2 mgÆmL
)1
of cortex micro-
somalproteinandforupto20minand0.12mgÆmL
)1
of
medulla microsomal protein was observed. Apart from the
experiments concerning the influence of substrate concen-
trations on lyso-PAF AT activity, we routinely used 0.1–
0.2 mgÆmL
)1
of microsomal protein for the lyso-PAF AT
assays and incubated the reaction mixture for 30 min in
order to achieve the maximum yield of the reaction product.
Dependence of lyso-PAF AT activity on divalent cations
Cortical and medullary microsomes were incubated with
various concentrations of exogenous added CaCl
2
and
MgCl
2

in the presence or absence of EDTA. Results are
shown in Table 1. Low CaCl
2
and MgCl
2
concentrations,
upto10
)5
M
did not have any significant effect on lyso-PAF
AT activity while higher concentration inhibited lyso-PAF
Fig. 1. Separation of the lipid products produced by the incubation of
cortical microsomes (0.192 mg proteinÆmL
)1
)with[H
3
]acetyl-CoA in the
absence of lyso-PAF (m) or in the presence of lyso-PAF (e). The
retention time of authentic [H
3
]PAF (d)and[H
3
]lyso-PAF (s)was
compared with the retention time of the radioactive peaks obtained
from the separation of the assay products. The extraction and separ-
ation of the products was carried out as described in materials and
methods.
Ó FEBS 2003 Lyso-PAF acetyltransferase activity in human kidney (Eur. J. Biochem. 270) 2995
AT activity in a dose-dependent manner. Mg
2+

was a more
potent inhibitor of both cortical and medullary lyso-PAF
AT than Ca
2+
. Both divalent cations showed an increased
inhibitory activity against medulla lyso-PAF AT. The
chelating agent, EDTA (1 m
M
) inhibited the cortex and
medulla acetyltransferase activity by 80 and 90%, respect-
ively, in the absence of divalent cations. Addition of 10
)3
M
CaCl
2
totally reversed the inhibition caused by EDTA. This
reversion was more significant for the cortical lyso-PAF AT
activity than the medullary one.
Influence of various compounds on lyso-PAF AT activity
The influence of various compounds on the activity of both
cortical and medullary lyso-PAF AT activity was tested
Fig. 2. Positive ion electrospray mass spectrum
of the lyso-PAF AT assay product. The isola-
tion of the assay product and the electrospray
analysis was performed as described in Mate-
rials and methods.
Fig. 3. Effect of BSA concentration on microsomal lyso-PAF AT.
Microsomal fractions of human cortex (0.14 mgÆmL
)1
)(s)orhuman

medulla (0.12 mgÆmL
)1
)(d) were incubated with various concentra-
tions of BSA and the lyso-PAF AT activity was determined as described
in the materials and methods section. Results are expressed in per-
centage related to control (incubation inthe absence of BSA) and are the
averages of two experiments with different enzyme preparations.
Fig. 4. Time course of [H
3
]PAF formation as a function of cortical and
medullary microsomal protein concentration. (A)Kineticsof[H
3
]PAF
formation using 0.04 (d), 0.1 (s)and0.2(h)mgproteinÆmL
)1
of
human cortex microsomes. (B) Kinetics of [H
3
]PAF formation using
0.02 (d), 0.06 (s) and 0.12 (h)mgproteinÆmL
)1
of human medulla
microsomes. Lyso-PAF AT activity was determined as described
in Materials and methods. Results are representative of three
experiments.
2996 T. N. Nomikos et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(Table 2). Neither NaF, a phosphatase inhibitor, nor
phenylmethylsulfonyl fluoride, a serine esterase inhibitor,
had any significant effect on both enzymatic activities. The
same results were obtained after the incubation of cortical

and medullary microsomes with the reducing agents
dithiothreitol and mercaptoethanol. On the other hand,
lyso-PAF AT activity was completely abolished by the
incubation of microsomes with 5,5¢-dithio-bis(2-nitroben-
zoic acid) (DTNB), a potent inhibitor of -SH enzymes.
Pefabloc, a very potent, irreversible inhibitor of the PAF
degradative enzyme, PAF-AH, had no significant effect on
lyso-PAF AT activities, indicating that the possible presence
of PAF-AH in the lyso-PAF AT assay does not influence
the product formation.
Substrates of lyso-PAF AT
When the activity of lyso-PAF ATs was determined at lyso-
PAF concentrations ranging from 2 to 100 l
M
at a fixed
concentration of acetyl-CoA (200 l
M
) both cortical and
medullary lyso-PAF AT exhibited classical Michaelis–
Menten kinetics up to 60 l
M
. Higher concentrations of
lyso-PAF resulted in a drop of the activity possibly due to the
detergent effects of the substrate against the enzyme. Simple
saturation kinetics were also observed up to 400 l
M
of acetyl-
CoA when the activity of the lyso-PAF ATs was determined
at acetyl-CoA concentrations ranging from 25 to 800 l
M

at a
fixed concentration of lyso-PAF (20 l
M
). When the concen-
tration of acetyl-CoA exceeded 400 l
M
a reduction of the
lyso-PAF AT activity was also observed (Fig. 5). The kinetic
parameters derived from these experiments are summarized
in Table 3. In all experiments, the K
M,app
and V
max,app
values
for medullary lyso-PAF AT were higher than the respective
values of cortical lyso-PAF AT. However, a statistical
analysis for the comparison of the values could not be
conducted because of the small number of samples.
Subsequently, the specificity of the microsomal enzymes
for ester/ether substrates was investigated. Microsomes of
Table 1. Effect of divalent cations and EDTA on cortical and medullary lyso-PAF AT activity. Lyso-PAF AT was assayed in the presence of varying
concentration of CaCl
2
,MgCl
2
and EDTA as described in Materials and methods. Results are expressed in percent related to non added control
(100%) are the averages of two determinations in different enzyme preparations.
Addition Cortex microsomes Medulla microsomes
CaCl
2

10
)6
M
100.0 96.9
CaCl
2
10
)5
M
109.2 88.6
CaCl
2
10
)4
M
89.9 96.4
CaCl
2
10
)3
M
94.5 61.5
CaCl
2
10
)2
M
80.3 59.8
EDTA 1 m
M

24.8 10.8
EDTA 10 m
M
nd
a
nd
EDTA 1 m
M
+ CaCl
2
10
)3
M
138.4 93.3
EDTA 1 m
M
+ CaCl
2
10
)2
M
101.5 67.2
MgCl
2
10
)6
M
83.9 77.3
MgCl
2

10
)5
M
110.9 96.8
MgCl
2
10
)4
M
107.8 88.2
MgCl
2
10
)3
M
82.2 80.5
MgCl
2
10
)2
M
79.2 39.2
a
nd, no lyso-PAF AT activity was detected.
Table 2. Effect of various chemicals on cortical and medullary lyso-PAF AT activity. Lyso-PAF AT was assayed in the presence of varying
concentration of chemicals as described in Materials and methods. Results expressed in percent related to non-added control (100%) are the average
of two determinations in different enzyme preparations.
Addition Cortex microsomes Medulla microsomes
NaF (5 m
M

) 95.9 104.2
NaF (50 m
M
) 95.5 122.0
Phenylmethylsulfonyl fluoride (1 m
M
) 100.2 ND
b
Phenylmethylsulfonyl fluoride (5 m
M
) 113.5 65.6
Dithiothreitol (1 m
M
) 120.3 128.3
Dithiothreitol (5 m
M
) 97.2 104.5
Mercaptoethanol (1 m
M
) 90.6 117
Mercaptoethanol (5 m
M
) 76.8 82.7
Pefabloc (0.1 m
M
) 90.1 134.2
DTNB (0.1 m
M
) 16.4 2.3
DTNB (0.5 m

M
) 5.4 nd
DTNB (1 m
M
)nd
a
nd
a
nd, not detected.
b
ND, not determined.
Ó FEBS 2003 Lyso-PAF acetyltransferase activity in human kidney (Eur. J. Biochem. 270) 2997
either cortex or medulla were incubated with [H
3
]-acetyl-
CoA and lyso-PAF or the ester analog lyso-phosphatidyl-
choline (lyso-PC) and the kinetics of the product formation
was determined. The velocities of both acetyltransferases
were twice as high in the presence of lyso-PAF (10–20 l
M
)
as in that of their ester analogs. In fact, no radioactive
product was recovered in the BSA precipitate when
microsomes were incubated with lyso-phosphatidylcholine
concentrations exceeding 20 l
M
. In order to rule out the
possibility of the degradation of lyso-phosphatidylcholine
by non specific PLA
1

, the experiment was repeated in the
presence of phenylmethylsulfonyl fluoride (1 m
M
and
5m
M
), a well-known PLA
1
inhibitor. No increase of the
radioactivity recovered in the BSA pellet was observed
suggesting a low specificity of acetyltransferases for ester
analogs.
The ability of lyso-PAF ATs to acetylate ALPA, which is
substratefortheacetyltransferaseofthede novo biosyn-
thetic route, was also tested. Cortex or medulla microsomes
were incubated with either lyso-PAF (20 l
M
), ALPA
(20 l
M
) or with a mixture of lyso-PAF (20 l
M
)andALPA
(20 l
M
) and the products of the reactions were extracted by
a modified Bligh–Dyer extraction and separated by TLC.
The radioactivity of the areas with the same R
f
as PAF,

AAPA and alkylacetylglycerol, was determined. The results
showed that PAF was the only product of the incubation of
the microsomes with lyso-PAF. No lipidic products were
found when ALPA served as the substrate in the lyso-PAF
ATs assay. Moreover, addition of ALPA to the lyso-PAF
ATs assay had no significant effect on the production of
PAF indicating that ALPA cannot serve as substrate for the
lyso-PAF AT of cortex or medulla.
Subcellular localization of lyso-PAF ATs activity
The specific activity of lyso-PAF AT was determined in all
subcellular fraction obtained by the subcellular fraction-
ation of the tissues. Microsomes exhibited the higher lyso-
PAF AT activity. The total activity of the enzyme in all
subcellular fractions was also determined and the subcellu-
lar distribution of the activity was calculated considering the
total activity of the 500 g supernatant as 100% (Table 4).
The acetyltransferase activity was distributed between the
membrane fractions of the 20 000 g and the 100 000 g
pellets (microsomes). The recoveries of lyso-PAF ATs
activity for cortex and medulla were 27.8 ± 8.5% (n ¼ 5)
and 28.4 ± 8.1% (n ¼ 5), respectively. No statistical
difference between the cortical and medullary lyso-PAF
AT specific activities of the same fractions was observed.
Discussion
This study demonstrates the existence of an acetyltrans-
ferase activity, capable of transferring the acetyl moiety of
acetyl-CoA to lyso-PAF, in both cortical and medullary
human renal tissue. As far as we know this is the first study
concerning with the biochemical characterization of lyso-
PAF AT in human renal tissues. The determination of the

acetyltransferase activity was carried out by a modified
trichloroacetic acid precipitation method described previ-
ously [29]. Assessment of the method indicates that it can be
used for the routine determination of lyso-PAF AT activity
Fig. 5. Influence of substrate concentration on microsomal lyso-PAF
AT activity of human cortex and medulla. (A)Activityofcortical(s)
and medullary (d) lyso-PAF AT as a function of lyso-PAF at fixed
concentrations (200 l
M
)ofacetyl-CoA.(B)Activityofcortical(s)and
medullary (d) lyso-PAF AT as a function of acetyl-CoA at fixed
concentrations (20 l
M
) of lyso-PAF. Lyso-PAF AT activity was
determined as described in Materials and methods. Results are rep-
resentative of four experiments.
Table 3. Kinetic parameters of cortical and medullary microsomal lyso-PAF AT. Kinetic data were obtained from experiments shown in Fig. 3.
Results are the averages of two determinations or the mean ± SD of four determinations in different enzyme preparations.
Enzyme preparation
Substrates
Lyso-PAF Acetyl-CoA
K
M,app
(l
M
) V
max
(nmolÆmin
)1
Æmg

)1
) K
M,app
(l
M
) V
max
(nmolÆmin
)1
Æmg
)1
)
Cortex, microsomes 15.6 ± 2.6 (n ¼ 4) 2.1 ± 0.5 (n ¼ 4) 90.4 ± 14.7 (n ¼ 4) 1.49 ± 0.25 (n ¼ 4)
Medulla, microsomes 24.9 (n ¼ 2) 3.72 (n ¼ 2) 100.6 (n ¼ 2) 2.92 (n ¼ 2)
2998 T. N. Nomikos et al. (Eur. J. Biochem. 270) Ó FEBS 2003
as [H
3
]PAF, the only radioactive product of the acetyl-
transferase reaction, is quantitatively precipitated to the
BSA pellet, readily separating from [H
3
]acetyl-CoA, which
remains in the supernatant. This method is faster and more
convenient than the TLC methods routinely used for the
determination of lyso-PAF AT activity [30].
Both cortical and medullary lyso-PAF AT activities share
similar biochemical characteristics indicating that they are
originated from common cellular sources. As previous
studies have shown that mesangial and endothelial cells
possess the higher acetyltransferase activities in rat and

human renal tissue [13,15], we can hypothesize that the
lyso-PAF AT activity found, in this study, in homogenates
of renal cortex and medulla is mainly derived by this kind
of cells.
The lyso-PAF AT activity is associated with the mem-
branous fractions of the renal cells. No lyso-PAF AT
activity could be detected in the cytoplasmic fraction. The
higher specific activity of lyso-PAF AT is found in the
microsomes. Microsomal lyso-PAF AT showed an opti-
mum pH in the range of 7.4–7.7, similar to that found for
the acetyltransferases of other tissues [5]. Lyso-PAF AT is
also sensitive to the concentration of Tris in the buffer
solution showing a maximum activity at 50 m
M
.This
indicates that it is important to determine the concentration
of Tris solution in order to achieve the best experimental
conditions for the determination of acetyltransferase.
BSA concentrations up to 0.5 mgÆmL
)1
had no signifi-
cant effect on lyso-PAF AT activity while higher concen-
trations inhibited lyso-PAF activity dose-dependently. The
above results are in conflict with previous studies in human
polymorphonuclear neutrophils showing an activation of
microsomal lyso-PAF AT at BSA concentrations of
0.5 mgÆmL
)1
and higher. The activating effect of BSA was
attributed to its ability to bind PAF and free the enzyme

from the product of the reaction, which has inhibitory
effects on lyso-PAF AT. The same researchers had also
shown that microsomal fraction is much more effective than
BSA in binding PAF or lyso-PAF [31]. In our assays, it
seems that the microsomal proteins can bind PAF effect-
ively preventing it from inhibiting lyso-PAF AT activity and
the addition of BSA up to 0.5 mgÆmL
)1
hasnoeffectsonthe
lyso-PAF AT activity. However, higher BSA concentrations
could prevent the enzyme to act on the substrate and an
inhibitory action is observed.
Addition of divalent cations in the incubation mixture
resulted in a dose-dependent inhibition of lyso-PAF AT. A
complete loss of lyso-PAF AT activity was also observed in
the presence of chelating agents which was totally reversed
by the addition of CaCl
2
, thus a direct inhibitory effect of
EDTA on the enzyme should be ruled out. It seems that the
endogenous microsomal stores of Ca
2+
are adequate for
the proper function of the enzyme, which is inhibited by the
chelation of the endogenous Ca
2+
by EDTA. The same
mode of action has been observed for the lyso-PAF AT
from rat spleen [9] and mouse macrophages [32].
The substrate specificity of lyso-PAF determines the

composition and the biological activity of the molecular
mixture of PAF analogs that are produced under inflam-
matory conditions. Lyso-PAF AT specifically acts on ether
analogs of PAF while its activity on ester analogs (acyl-
PAF) is diminished by almost 70%. These results are in
agreement with previous studies showing that the major
PAF species synthesized by rat glomerular mesangial cells is
the ether analog of PAF [33]. The inability of lyso-PAF AT
to act on ALPA indicates that the lyso-PAF AT activity is
distinct from the acetylating activity of the de novo
biosynthetic route.
In conclusion, an acetylating activity, capable of trans-
ferring an acetyl group from acetyl-CoA to lyso-PAF, was
demonstrated in cortical and medullary human renal tissues
for the first time. The biochemical properties of both cortical
and medullary acetylating activities are similar with the
biochemical properties of lyso-PAF ATs characterized in
other tissues or cells. The existence of a lyso-PAF AT
activity in human renal tissues indicates that human kidneys
are able to produce PAF through the remodeling pathway.
However, the relative contribution of this pathway to the
increased synthesis of PAF under pathological conditions
needs further elucidation.
Acknowledgements
This work was supported by grants from the General Secreteriat for
Research and Technology, Ministry of Development.
References
1. Demopoulos, C.A. & Pinckard, R.N. & Hanahan, D.J. (1979)
Platelet-activating factor: evidence for 1-O-alkyl-2-acetyl-sn-
Table 4. Subcellular distribution and specific activities of lyso-PAF AT in human cortex and medulla. Lyso-PAF AT activity was determined as

described in materials and methods. Results of subcellular distribution are expressed as percent related to the total activity of lyso-PAF AT in the
500 g supernatant (100%). Results are the mean ± SD of 4–14 determinations in different enzyme preparations.
Fraction
Cortex Medulla
Specific activity
(nmolÆmin
)1
Æmg
)1
)
Distribution of
total activity (%)
Specific activity
(nmolÆmin
)1
Æmg
)1
)
Distribution of
total activity (%)
500 g supernatant 0.37 ± 0.20 (n ¼ 6) 100 0.43 ± 0.31 (n ¼ 7) 100
20 000 g pellet 0.54 ± 0.33 (n ¼ 5) 15.6 ± 4.5 (n ¼ 5) 0.77 ± 0.42 (n ¼ 9) 18.3 ± 5.1 (n ¼ 5)
100 000 g supernatant nd
a
nd nd ND
b
100 000 g pellet 1.14 ± 0.53 (n ¼ 14) 12.1 ± 3.5 (n ¼ 5) 1.45 ± 0.67 (n ¼ 13) 10.6 ± 3.8 (n ¼ 5)
Total membrane fraction 0.80 ± 0.39 (n ¼ 4) ND 1.24 ± 0.28 (n ¼ 4) ND
a
nd, not detected.

b
ND, not determined.
Ó FEBS 2003 Lyso-PAF acetyltransferase activity in human kidney (Eur. J. Biochem. 270) 2999
glyceryl-3-phosphorylcholine as the active component (a new class
of lipid chemical mediators). J. Biol. Chem. 254, 9355–9358.
2. Prescott, S.M., Zimmerman, G.A., Stafforini, D.M. & McIntyre,
T.M. (2000) Platelet-activating factor and related lipid mediators.
Annu. Rev. Biochem. 69, 419–445.
3. Snyder, F. (1995) Platelet-activating factor and its analogs,
metabolic pathways related intracellular processes. Biochim. Bio-
phys. Acta 1254, 231–249.
4. Lee, T.C., Vallari, D.S. & Snyder, F. (1992) 1-Alkyl-2-lyso-sn-
glycero-3-phosphocholine acetyltransferase. Methods Enzymol.
209, 396–401.
5. Snyder, F. (1995) Platelet-activating factor, the biosynthetic and
catabolic enzymes. Biochem. J. 305, 689–705.
6. Nixon, A.B., O’Flaherty, J.T., Salyer, J.K. & Wykle, R.L. (1999)
Acetyl-CoA: 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acet-
yltransferase is directly activated by p38 kinase. J. Biol. Chem. 274,
5469–5473.
7. Baker, P.R., Owen, J.S., Nixon, A.B., Thomas, L.N., Wooten, R.,
Daniel, L.W., O’Flaherty, J.T. & Wykle, R.L. (2002) Regulation
of platelet-activating factor synthesis in human neutrophils by
MAP kinases. Biochim. Biophys. Acta 1592, 175–184.
8. Gomez-Cambronero, J., Velasco, S., Sanchez-Crespo, M.,
Vivanco, F. & Mato, J.M. (1986) Partial purification and char-
acterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:
acetyl-CoA acetyltransferase from rat spleen. Biochem. J. 237,
439–445.
9. Seyama, K. & Ishibashi, T. (1987) Biochemical characterization

of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyl-
transferase in rat spleen microsomes. Lipids 22, 185–189.
10. Billah, M.M. & Johnston, J.M. (1983) Identification of phos-
pholipid platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-
3-phosphocholine) in human amniotic fluid and urine. Biochem.
Biophys. Res. Commun. 113, 51–58.
11. Sanchez-Crespo, M., Inarrea, P., Alvarez, V., Alonso, F., Egido, J.
& Hernando, L. (1983) Presence in normal human urine of a
hypotensive and platelet-activating phospholipid. Am.J.Physiol.
244, F706–F711.
12. Schlondorff, D., Goldwasser, P., Neuwirth, R., Satriano, J.A. &
Clay, K.L. (1986) Production of platelet-activating factor in glo-
meruli and cultured glomerular mesangial cells. Am.J.Physiol.
250, F1123–F1127.
13. Kester, M., Nowinski, R.J., Holthofer, H., Marsden, P.A. &
Dunn M.J. (1994) Characterization of platelet-activating factor
synthesis in glomerular endothelial cell lines. Kidney Int. 46,
1404–1412.
14. Neuwirth, R., Ardaillou, N. & Schlondorff, D. (1989) Extra and
intracellular metabolism of platelet-activating factor by cultured
mesangial cells. Am. J. Physiol. 256, F735–F741.
15. Pirotzky,E.,Ninio,E.,Bidault,J.,Pfister,A.&Benveniste,J.,
1984) Biosynthesis of platelet-activating factor. VI. Precursor of
platelet-activating factor and acetyltransferase activity in isolated
rat kidney cells. Lab. Invest. 51, 567–572.
16. Zanglis, A. & Lianos, E.A. (1987) Synthesis of alkyl-ether gly-
cerophospholipids in rat glomerular mesangial cells, evidence for
alkyldihydroxyacetone phosphate synthase activity. Biochem.
Biophys. Res. Commun. 144, 666–673.
17. Woodard, D.S., Lee, T.C. & Snyder, F. (1987) The final step

in the de novo biosynthesis of platelet-activating factor.
Properties of a unique CDP-choline, 1-alkyl-2-acetyl-sn-glycerol
choline-phosphotransferase in microsomes from the renal
inner medulla of rats. J. Biol. Chem. 262, 2520–2527.
18. Karasawa, K., Qiu, X.Y. & Lee, T.C. (1999) Purification and
characterization from rat kidney membranes of a novel platelet-
activating factor (PAF)-dependent transacetylase that catalyzes
the hydrolysis of PAF, formation of PAF analogs, and C-2-cer-
amide. J. Biol. Chem. 274, 8655–8661.
19. Lianos, E.A. & Zanglis, A. (1992) Effects of complement activa-
tion on platelet-activating factor and eicosanoid synthesis in rat
mesangial cells. J. Lab. Clin. Med. 120, 459–464.
20. Biancone, L., Tetta, C., Turello, E., Montrucchio, G., Iorio, E.L.,
Servillo, L., Balestrieri, C. & Camussi, G. (1992) Platelet-activat-
ing factor biosynthesis by cultured mesangial cells is modulated by
proteinase inhibitors. J. Am. Soc. Nephrol. 2, 1251–1261.
21. Camussi, G., Salvidio, G. & Tetta, C. (1989) Platelet-activating
factor in renal diseases. Am. J. Nephrol. 9 (Suppl. 1), 23–26.
22. Lopez-Novoa, J.M. (1999) Potential role of platelet activating
factor in acute renal failure. Kidney Int. 55, 1672–1682.
23. Antonopoulou, S., Demopoulos, C.A., Iatrou, C., Moustakas, G.
& Zirogiannis, P. (1994) Platelet-activating factor acetylhydrolase
(PAF-AH) in human kidney. Int. J. Biochem. 26, 1157–1162.
24. Iatrou, C., Moustakas, G., Antonopoulou, S., Demopoulos, C.A.
& Ziroyiannis, P. (1996) PAF levels and PAF-acetylhydrolase
activities in patients with primary glomerulonephritis. Nephron 72,
611–616.
25. Lenihan, D.G. & Lee, T.C. (1984) Regulation of platelet activating
factor synthesis, modulation of 1-alkyl-2-lyso-sn-glycero-3-phos-
phocholine, acetyl-CoA acetyltransferase by phosphorylation and

dephosphorylation in rat spleen microsomes. Biochem. Biophys.
Res. Commun. 120, 834–839.
26. Bligh, E.G. & Dyer, W.J. (1959) A rapid method of total
lipid extraction and purification. Can. J. Biochem. Physiol. 37,
911–917.
27. Karantonis, H.C., Antonopoulou, S. & Demopoulos, C.A. (2002)
Antithrombotic lipid minor constituents from vegetable oils:
comparison between olive oils and others. J. Agric. Food Chem.
50, 1150–1160.
28. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J.
(1951) Protein measurement with the folin phenol reagent. J. Biol.
Chem. 193, 265–275.
29. Yamazaki, R., Sugatani, J., Fujii, I., Kuroyanagi, M., Umehara,
K., Ueno, A., Suzuki, Y. & Miwa, M. (1994) Development of a
novel method for determination of acetyl-CoA: 1-alkyl-2n-gly-
cero-3-phosphocholine acetyltransferase activity and its applica-
tion to screening for acetyltransferase inhibitors. Biochem.
Pharmacol. 47, 995–1006.
30. Lee, T.C., Vallari, D.S. & Snyder, F. (1992) 1-Alkyl-2-lyso-
sn-glycero-3-phosphocholine acetyltransferase. Methods Enzymol.
209, 396–401.
31. Ninio, E., Joly, F. & Bessou, G. (1988) Biosynthesis of PAF-
acether. XI. Regulation of acetyltransferase by enzyme-substrate
imbalance. Biochim. Biophys. Acta 963, 288–294.
32. Ninio, E., Mencia-Huerta, J.M., Heymans, F. & Benveniste, J.
(1982) Biosynthesis of platelet-activating factor. I. Evidence for
an acetyltransferase activity in murine macrophages. Biochim.
Biophys. Acta 710, 23–31.
33. Lianos, E.A. & Zanglis, A. (1987) Biosynthesis and metabolism of
1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular

mesangial cells. J. Biol. Chem. 262, 8990–8993.
3000 T. N. Nomikos et al. (Eur. J. Biochem. 270) Ó FEBS 2003