© 1995 American Heart Association, Inc.
Articles |
PAF-Degrading Acetylhydrolase Is Preferentially Associated With Dense LDL and VHDL-1 in Human Plasma
Catalytic Characteristics and Relation to the Monocyte-Derived Enzyme
From the Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, Greece (A.D.T., S.-A.P.K), and the Institut National de la Santé et de la Recherche, Unité de Recherches sur les Lipoprotéines et l'Athérogénèse, Hôpital de la Pitié, Paris, France (C.D., M.J.C., E.N.).
Correspondence to Ewa Ninio, INSERM, Unité de Recherches sur les Lipoprotéines et l'Athérogénèse, U-321, Pavillon Benjamin Delessert, Hôpital de la Pitié, 83, Bd de l'Hôpital, 75651 Paris Cedex 13, France.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract In human plasma, platelet activating factor (PAF)–degrading acetylhydrolase (acetylhydrolase) is principally transported in association with LDLs and HDLs; this enzyme hydrolyzes PAF and short-chain forms of oxidized phosphatidylcholine, transforming them into lyso-PAF and lysophosphatidylcholine, respectively. We have examined the distribution, catalytic characteristics, and transfer of acetylhydrolase activity among plasma lipoprotein subspecies separated by isopycnic density gradient ultracentrifugation; the possibility that the plasma enzyme may be partially derived from adherent monocytes has also been evaluated. In normolipidemic subjects with Lp(a) levels <0.1 mg/mL, acetylhydrolase was associated preferentially with small, dense LDL particles (LDL-5; d=1.050 to 1.063 g/mL) and with the very-high-density lipoprotein–1 subfraction (VHDL-1; d=1.156 to 1.179 g/mL), representing 23.9±1.7% and 20.6±3.2%, respectively, of total plasma activity. The apparent Km values for PAF of the enzyme associated with such lipoproteins were 89.7±23.4 and 34.8±4.5 µmol/L for LDL-5 and VHDL-1, respectively: indeed, the Km value for LDL-5 was some 10-fold higher than that of the light LDL-1, LDL-2, and LDL-3 subspecies, whereas the Km of VHDL-1 was some twofold greater than those of the HDL-2 and HDL-3 subspecies. Furthermore, when expressed on the basis of unit plasma volume, the Vmax of the acetylhydrolase associated with LDL-5 was some 150-fold greater than that in LDL-1 (d=1.019 to 1.023 g/mL). No significant differences in the pH dependence of enzyme activity or in sensitivity to protease inactivation, sulfydryl reagents, the serine protease inhibitor Pefabloc, or the PAF antagonist CV 3988 could be detected between apo B–containing and apo A-I–containing lipoprotein particle subspecies. Incubation of LDL-1 (Km=8.4±2.6 µmol/L) and LDL-2 (d=1.023 to 1.029 g/mL; Km=8.4±3.3 µmol/L) subspecies with LDL-5, in which acetylhydrolase had been inactivated by pretreatment with Pefabloc, demonstrated preferential transfer of acetylhydrolase to LDL-5. Acetylhydrolase transferred to LDL-5 from the light LDL subspecies exhibited a Km of 9.4±2.2 µmol/L, a value characteristic of the particle donors. Finally, acetylhydrolase (Km=23.4±7.6 µmol/L) released by adherent human monocytes in culture was found to bind preferentially to small, dense LDL subspecies upon incubation of Pefabloc-inactivated plasma with monocyte supernatant. We conclude that a form of acetylhydrolase with distinct catalytic properties is preferentially associated with small, dense LDL-5 and VHDL-1 particles in human plasma, suggesting that the surface properties of LDL-5 and VHDL-1 particles are distinct from those of other lipoproteins containing either apo B or apo A-I. This hypothesis is consistent with the transfer of active enzyme from the light LDL subspecies to LDL-5, as well as with the preferential binding of monocyte-derived acetylhydrolase to LDL-5. Finally, because the penetration of arterial intima by lipoproteins is inversely proportional to particle size, our data suggest that both small, dense apo B–containing lipoproteins (ie, LDL-5) and small apo A-I–rich lipoproteins (ie, VHDL-1) may play a key anti-inflammatory role in arterial tissue.
Key Words: inflammatory mediators • human blood monocytes • isopycnic density gradient ultracentrifugation • lipoprotein particle subspecies
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
PAF is a potent lipid mediator involved in inflammatory and allergic reactions1 that may play an important role in atherogenesis (reviewed in Reference 22 ). PAF is synthesized by various activated proinflammatory cells including endothelial cells, platelets, monocytes, and macrophages, all of which are known to play key roles in the development of the atheromatous plaque (reviewed in Reference 33 ). Indeed, PAF may be released from such cells and transported in plasma in association with albumin4 and in part with circulating lipoproteins.4 5
Acetylhydrolase (EC 3.11.48) was initially described as the Ca2+-independent enzyme that inactivates PAF by hydrolyzing its sn-2 acetate group and thus converting it into lyso-PAF.6 7 8 Several studies have suggested that acetylhydrolase plays a major role in the regulation of the pathophysiological effects of PAF (reviewed in Reference 99 ). Acetylhydrolase is present in plasma10 as well as in several cells and tissues, including monocytes and macrophages,11 platelets,12 erythrocytes,13 and spleen and liver cells.14 Recently, this enzyme has also been detected in bovine brain, from which it was purified15 and cloned.16 17 Differences in the biochemical and physicochemical properties of acetylhydrolase from various sources suggest that the enzyme is heterogeneous and can be classified into at least four different molecular species.14 Human plasma acetylhydrolase is associated mainly with LDL and HDL,10 and various studies suggest that its main cellular sources are monocyte-derived macrophages,18 platelets,12 and hepatocytes.19
The oxidative modification of LDL involves the hydrolysis of its content of oxidized phosphatidylcholine into lysophosphatidylcholine.20 Such hydrolysis has been reported to be mediated by a phospholipase A2–like activity intrinsic to LDL,21 but more recent studies suggest that it may instead be due to the LDL-associated acetylhydrolase.22 23 However, during oxidative modification of LDL, acetylhydrolase activity dramatically decreases, and thus oxidized LDL is devoid of its anti-inflammatory properties.23 24
Several investigators have shown that LDL and HDL are not structurally and metabolically homogeneous particles but instead consist of a spectrum of subspecies that differ in size, molecular weight, buoyant density, and composition.25 26 Furthermore, the structural and metabolic heterogeneity of LDL particles now appears related to their atherogenic potential.26 Indeed, a predominance of the small, dense LDL particles is known to predispose to coronary artery disease. Thus, dense LDL particles possess high susceptibility to oxidative modification27 28 and low binding affinity for the cellular LDL receptor.29 Similarly, plasma HDL are highly heterogeneous in their structure and metabolism, reflecting diverse pathways of formation and interconversion.30 31 Elevated levels of HDL are strongly correlated with low cardiovascular risk.3
Recently we have shown that the association of acetylhydrolase activity with LDL particles is heterogeneous over the density range of 1.030 to 1.048 g/mL.32 In the present study we investigated the distribution, catalytic characteristics, and transfer of acetylhydrolase activity among plasma apo B– and apo A-I–containing lipoprotein subspecies separated by a highly resolutive density gradient procedure.25 We demonstrate that acetylhydrolase is associated primarily with small, dense LDL of d=1.050 to 1.063 g/mL of elevated atherogenicity, but also with the apo A-I–containing VHDL-1 subclass of d=1.156 to 1.179 g/mL. Furthermore, acetylhydrolase released from human adherent monocytes in culture was found to bind preferentially to dense LDL and to VHDL-1. In addition, the catalytic properties of the enzyme in dense LDL and VHDL-1 are distinct from those of other lipoprotein subfractions, presenting a lower affinity (higher Km) for the substrate PAF but a higher maximal velocity (Vmax). The preferential association of acetylhydrolase with LDL-5 and VHDL-1 reflects an elevated binding affinity of the enzyme for the surface of both lipoprotein subspecies and confers on these particles a potential anti-inflammatory action.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
PAF (hexadecyl), obtained as a powder from Novabiochem, was dissolved at a final concentration of 5 mmol/L in ethanol (80% vol/vol). This solution was mixed with 1-O-hexadecyl-2-[3H-acetyl]-sn-glycero-3-phosphocholine (10 Ci/mmol; Du Pont–New England Nuclear) in various proportions, dried under a stream of nitrogen, and redissolved in a solution containing fatty acid–free BSA/saline (0.25%) to obtain [3H-acetyl]PAF solutions with concentrations ranging from 2 to 25 mmol/L. Polyunsaturated PC, fatty acid–free BSA, trypsin, iodoacetic acid, and dithiothreitol were from Sigma Chemical Co. Pefabloc SC (4-[2-aminoethyl benzenesulfonyl fluoride, Pefabloc) and BCA protein reagent were from Pierce, and CV 3988 was obtained from Takeda Chemical Industries Ltd. Liquid scintillation fluid (Optiphase Hi-Safe 3) was supplied by E.G.G. Berthold and RPMI 1640 was obtained from Biowhittaker.
Fractionation of Plasma Lipoproteins
Subjects were healthy normolipidemic volunteers (all females)
who had fasted overnight and whose plasma Lp(a) levels were less than
0.1 mg/mL. Venous blood was collected into glass tubes containing EDTA
(3 mmol/L), from which plasma was rapidly separated by low-speed
centrifugation (1000g, 20 minutes) at 4°C.
Immediately after collection of plasma, gentamicin (50 µg/mL) and
EDTA (0.3 mmol/L) were added. Lipoproteins were fractionated by
isopycnic density gradient ultracentrifugation by
use of a Beckman SW41 Ti rotor at 40 000 rpm for 44 hours in a Beckman
XL 70 centrifuge at 15°C as described
previously.25 In brief, plasma density was increased to
1.21 g/mL by addition of dry solid KBr. Construction of a discontinuous
density gradient at ambient temperature was initiated by pumping 2 mL
of an NaCl-KBr solution of d=1.24 g/mL into the bottom of
the tube. The following solutions were then layered above: 3 mL of
plasma at 1.21 g/mL; 2 mL of an NaCl-KBr solution of d=1.063
g/mL; 2.5 mL of an NaCl-KBr solution of d=1.019 g/mL; and
2.5 mL of an NaCl solution of d=1.006 g/mL. All density
solutions contained 0.3 mmol/L EDTA and 50 µg/mL gentamicin at pH
7.4. After ultracentrifugation, 30 fractions (0.4
mL each) were collected by successive aspiration with a precision
pipette from the meniscus downwards. All fractions were dialyzed in
Spectrapor membrane tubing (exclusion limit, 12 000 to 14 000 D) at
4°C against 5 L of 10 mmol/L PBS containing 2 mmol/L EDTA at pH 7.4
for 6 hours and 5 L of HEPES buffer, pH 8, containing 4.2 mmol/L HEPES,
137 mmol/L NaCl, 2.6 mmol/L KCl, and 2 mmol/L EDTA for 12 hours. All
fractions were analyzed for their protein content by the BCA
method and their purity was evaluated by SDS-polyacrylamide gel
electrophoresis. Subsequently, equal volumes of certain gradient
fractions were pooled to constitute the lipoprotein subfractions, as
follows: fractions 1 and 2 (VLDL+IDL; d<1.019 g/mL); 3 and
4 (LDL-1; d=1.019 to 1.023 g/mL); 5 and 6 (LDL-2;
d=1.023 to 1.029 g/mL); 7 and 8 (LDL-3; d=1.029
to 1.039 g/mL); 9 and 10 (LDL-4; d=1.039 to 1.050 g/mL); 11
and 12 (LDL-5; d=1.050 to 1.063 g/mL); 14 and 15 (a subclass
of HDL2, denoted HDL-2; d=1.072 to
1.091 g/mL); 17 and 18 (a subclass of HDL3, denoted
HDL-3; d=1.100 to 1.120 g/mL), and 22 and 23 (VHDL-1;
d=1.156 to 1.179 g/mL). In some experiments, fractions 3
through 12 were pooled to reconstitute total LDL (d=1.019 to
1.063 g/mL) and 13 through 23 to reconstitute total HDL
(d=1.063 to 1.179 g/mL). Fractions 25 through 30 contained
neither lipoproteins nor acetylhydrolase activity and were therefore
discarded.
Electrophoretic Analysis
For evaluation of lipoprotein particle size and
heterogeneity, nondenaturing gradient gel
electrophoresis of native lipoprotein subfractions was performed in a
Pharmacia GE-2/4 LS electrophoresis apparatus loaded with
gels containing a 2% to 16% gradient (PAA 2/1, Pharmacia). An aliquot
of each subfraction (15 µg protein) was applied to the gel and
electrophoresis was carried out at 125 V for 24 hours at 4°C in a
Tris/borate buffer (0.09 mol/L Tris, 0.08 mol/L boric acid, and 0.003
mol/L EDTA at pH 8.35).33 A set of standard markers with
known hydrated diameters (latex beads, 380 Å; thyroglobulin, 170 Å;
ferritin, 122 Å; and catalase, 104 Å) was run on each slab as a
reference.
The apolipoprotein content of each gradient fraction was evaluated in SDS-polyacrylamide gels (5% to 19% gradient) as previously described.34 In brief, 10 µg of protein of each subfraction was dessicated and subsequently heated at 100°C for 5 minutes in 30 µL sample buffer containing SDS (1% SDS, 10 mmol/L Tris with 10 mmol/L dithiothreitol at pH 6.8). Twenty microliters of 50% sucrose/bromophenol blue was then added to each sample. Samples were loaded and gels electrophoresed at a current of 30 mA/slab at 15°C for 2 hours. A calibration curve was established by use of a series of protein standards, whose molecular weights ranged from 29 to 205 kD (Sigma), that were electrophoresed concomitantly.
Lipid and Lipoprotein Analysis
The lipid contents of lipoprotein subfractions were
analyzed by enzymatic methods with BioMerieux kits for total
cholesterol, free cholesterol, phospholipids,
and triglycerides.35 Cholesteryl ester mass
was calculated as 1.67 times the free cholesterol
mass.25 The protein content of lipoprotein subfractions
was determined by the BCA method. Lipoprotein mass was calculated for
each subfraction as the sum of the mass of the individual components
(free cholesterol, cholesteryl ester,
triglyceride, phospholipid, and protein). The lipoperoxide
and malondialdehyde contents of lipoprotein subfractions (9±2 and <1
nmol/mg of LDL protein, respectively; <1 and <1 nmol/mg of HDL
protein, respectively), determined as described earlier,36
showed the lipoproteins to be in their native state.37
Moreover, acetylhydrolase activity in lipoprotein subfractions was
stable at 4°C for at least 2 weeks; such activity corresponded
closely to that described earlier in native LDL before oxidation with
Cu2+.24
Effect of Ionic Strength on the Association of Acetylhydrolase
With Lipoproteins
Total LDL reconstituted from the respective gradient
subfractions or total HDL was treated separately for 2 hours at 37°C
by gentle mixing with three different concentrations of KBr
corresponding to densities of 1.063, 1.240, and 2.480 g/mL,
respectively. The densities of LDL and HDL were adjusted to 1.063 g/mL
and 1.210 g/mL, respectively, by addition of solid KBr, and
lipoproteins were then submitted to
ultracentrifugation for 10 hours in a Beckman NVT
65 rotor at 40 000 rpm at 14°C. Acetylhydrolase activity was
measured in each lipoprotein fraction both before and after treatment
at 37°C, as well as after the second ultracentrifugal isolation.
Isolation and Culture of Human Blood Monocytes
Monocytes were isolated from the blood of healthy,
normolipidemic volunteers (thrombopheresis residues) as previously
described.38 The cells were cultured and grown in
35x10-mm plastic tissue culture dishes (Primaria, Falcon) with RPMI
medium containing 40 µg/mL of gentamicin but devoid of serum. At
culture day 4, the supernatants were recovered, centrifuged
(500g for 10 minutes at 4°C), and stored under sterile
conditions at 4°C.
Acetylhydrolase Assay
Acetylhydrolase activity was measured in the gradient
subfractions and in supernatants of adherent monocytes in culture as
previously described,39 with some modifications. Protein
(4 µg) from the lipoprotein samples or 50 µL of the supernatant of
adherent monocytes in culture was mixed with buffer (pH 8.0) containing
4.2 mmol/L HEPES, 137 mmol/L NaCl, 2.6 mmol/L KCl, and 2 mmol/L EDTA in
a final volume of 90 µL. After preincubation at 37°C, the reaction
was initiated and performed for 10 minutes at 37°C by addition of 10
µL of [3H-acetyl]PAF (final concentration, 25 µmol/L;
specific activity, 16 890 dpm/nmol). In some experiments, the activity
of acetylhydrolase was measured in PBS at pH 5, pH 7, and pH 8.
In selected experiments, lipoprotein subfractions or supernatant
of adherent monocytes in culture was preincubated for 30 minutes at
37°C in the presence of either 10 mmol/L CV 3988 or 0.1 mmol/L
Pefabloc before the addition of [3H-acetyl]PAF. The
reaction was stopped in an ice bath. Unreacted
[3H-acetyl]PAF was bound to an excess of BSA
(final concentration, 16.7 mg/mL) for 10 minutes and precipitated by
addition of trichloroacetic acid (final concentration, 8% vol/vol) as
described by Pinckard and Ludwig.40 The samples were then
centrifuged in an Eppendorf centrifuge for 5 minutes,
and the [3H]acetate released into the aqueous phase was
measured by liquid scintillation counting in Optiphase Hi-Safe 3.
Control assays, reflecting any nonenzymatic degradation of
[3H-acetyl]PAF, involved use of the
heat-denaturated enzyme from human serum (100°C for 10 minutes);
approximately 10% of the radiolabel was released compared with the
samples containing active enzyme. The results, after correction for
nonenzymatic degradation of PAF, are expressed as nanomoles of PAF
degraded each minute per milligram of lipoprotein protein, per
milliliter of plasma, or per milliliter of gradient fraction or as
nanomoles of PAF degraded per minute per milliliter of monocyte
supernatant.
Phosphatidylcholine Oxidation
Polyunsaturated PC (5 mg) was dispersed by sonication in 10 mL
of 10 mmol/L PBS at pH 7.4 and oxidized in the presence of 20 µmol/L
CuCl2 at 37°C for 24 hours. The oxidized aqueous
dispersion was extracted according to Bligh and Dyer41 and
subjected to thin-layer chromatography on
silica-gel 60 plates (Merck) with chloroform/methanol/water
(65:35:6, vol/vol/vol) as a solvent system. Lipids were identified
after brief exposure to iodine, and the band corresponding to the
retardation factor of PAF (located between the lysophosphatidylcholine
and sphingomyelin standards) was scraped off the plate and extracted
according to Bligh and Dyer, and the oxidized phospholipids were
collected in the chloroform phase. A portion of this extract was
submitted to phosphorus assay according to Bartlett42 as
modified by Marinetti.43 For the acetylhydrolase
inhibition studies, three different samples of the chloroform phase
were dried under a stream of nitrogen and the dried oxidized
phospholipids were redissolved in absolute ethanol; the concentrations
of the solutions of oxidized phospholipids used were 0.5, 1, and 2.5
mmol/L.
Kinetic Studies of Acetylhydrolase
The kinetic properties of acetylhydrolase associated with
lipoprotein subfractions or present in the supernatants of adherent
monocytes in culture were evaluated at 37°C for 10 minutes with
[3H-acetyl]PAF at final concentrations varying from 2.5
to 200 µmol/L. The inhibitory effect of the oxidized
polyunsaturated PC on [3H-acetyl]PAF degradation was also
studied with final concentrations of 12.5, 25, and 62.5 µmol/L of
oxidized polyunsaturated PC. The final [3H-acetyl]PAF
concentrations varied from 5 to 200 µmol/L, whereas the final ethanol
concentration in the reaction mixture was 2.5%. The
Km,
Vmax, and Ki
values were calculated with the Lineweaver-Burk representation
of the data.
Effects of Proteolysis and of Reducing Agents on
Acetylhydrolase Activity
Samples (lipoprotein subfractions or monocyte supernatants)
containing similar amounts of acetylhydrolase activity (0.2 nmol PAF
degraded/min) were preincubated at 37°C in a total volume of 200 µL
with HEPES buffer containing 5 mg/mL trypsin or 2 mmol/L iodoacetic
acid, or otherwise 1 mmol/L dithiothreitol for 60 minutes, 15 minutes,
or 15 minutes, respectively. The amount of acetylhydrolase activity
remaining in the sample was determined as described above by mixing of
90 µL of the sample with 10 µL of [3H-acetyl]PAF
(final concentration, 25 µmol/L) in a microcentrifuge
tube.
Association of Acetylhydrolase Released by Adherent Monocytes in
Culture With Lipoprotein Subfractions
Plasma or total LDL or total HDL particles were first incubated
for 30 minutes at 37°C in the presence of the serine esterase
inhibitor Pefabloc (1 mmol/L), and were extensively
dialyzed for 24 hours against PBS containing 2 mmol/L EDTA on
completion of incubation. The activity of acetylhydrolase was abolished
completely by this treatment (Reference 2424 , data not shown). Samples of
treated plasma or total LDL or total HDL were then mixed in a ratio of
1:1 (vol/vol) with aliquots of monocyte supernatant obtained from 4-day
cultured adherent monocytes containing acetylhydrolase activity of 2±1
nmol PAF degraded/min and were incubated for 60 minutes at 37°C. The
supernatant alone or a mixture of lipoproteins and cell supernatant was
fractionated by gradient ultracentrifugation as
described above. Thirty fractions were collected and acetylhydrolase
activity was determined in 50 µL of each fraction by use of
[3H-acetyl]PAF at a final concentration of 25 µmol/L;
the protein content of each fraction was determined as above.
Transfer of Acetylhydrolase Activity Between LDL
Subfractions
Initially, the acetylhydrolase activity of LDL-5 was inhibited
by treatment with Pefabloc, as described above, which was followed by
overnight dialysis in PBS containing 2 mmol/L EDTA. Afterwards, 1 mL of
such LDL-5 was mixed with 1 mL of native LDL-1 and 1 mL of native LDL-2
and incubated for 1 hour at 37°C. This mixture of LDL subfractions
was then fractionated by gradient
ultracentrifugation into 30 fractions of 0.4 mL;
the protein content and acetylhydrolase activity of each fraction was
then assayed as described above.
Statistical Analysis
Results are expressed as mean±SD. Mean values were compared by
Student's t test, with significance defined at a value of
P
.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Characterization of Lipoprotein Gradient Subfractions
The apo B and A-I contents of individual gradient fractions were evaluated by SDS-polyacrylamide gel electrophoresis. Fractions 1 through 12 (d<1.063 g/mL) contained almost exclusively apo B, whereas fraction 13 (d=1.063 to 1.072 g/mL) contained a mixture of apo B and apo A-I; in contrast, apo A-I was the major apoprotein in gradient fractions 14 through 23 (d=1.072 to 1.179 g/mL). Fraction 24 (d=1.179 to 1.190 g/mL) contained traces of albumin (results not shown). Gradient fractions were pooled to constitute the major lipoprotein classes as described in "Methods." As shown in Table 1
, the chemical
compositions as well as the profiles of lipoprotein mass corresponded
well with our previous findings in normolipidemic
subjects25 28 44 ; in addition, the particle size of each
subfraction as determined by nondenaturing gradient gel electrophoresis
(Table 1) confirmed our earlier data.28
|
Distribution of Acetylhydrolase Activity Among Lipoprotein
Subfractions
Initially, all 24 gradient fractions were analyzed
for acetylhydrolase activity. Subsequently, specific gradient fractions
were pooled to constitute the lipoprotein subclasses as described in
"Methods"; these subclasses were further analyzed for
acetylhydrolase activity. As shown in Fig 1A, fractions
11, 12, and 13 (d=1.050 to 1.072 g/mL) and fractions 22 and
23 (d=1.156 to 1.179 g/mL) displayed the highest
acetylhydrolase activity. The proportions of total plasma
acetylhydrolase activity expressed per milliliter of plasma (as shown
in Fig 1B) associated with these fractions were 3.3±1.6% for
fractions 1+2 (VLDL+IDL, d<1.019 g/mL), 23.9±1.7% for
fractions 11+12 (LDL-5, d=1.050 to 1.063 g/mL), 8.1±3.9%
for fraction 13 (a mixture of LDL-5 and HDL2,
d=1.063 to 1.072 g/mL), and 20.6±3.2% for fractions 22+23
(VHDL-1, d=1.156 to 1.179 g/mL). Among the LDL subfractions
(fractions 3 through 12, d=1.019 to 1.063 g/mL),
acetylhydrolase activity was primarily associated with fractions 11+12
(LDL-5), representing 60.4±16.6% of total LDL-associated
activity; among the HDL subspecies (fractions 13 to 23,
d=1.063 to 1.179 g/mL), acetylhydrolase was associated
predominantly with fractions 22+23 (VHDL-1) (35.5±10.9%).
|
indicates acetylhydrolase activity; 
, protein. B,
Bar graph shows mean±SD of acetylhydrolase activity in lipoprotein
subfractions from the plasma of three different subjects.
*P<.01 compared with LDL-1 through LDL-4;
**P<.03 compared with HDL-2 and HDL-3.We next determined whether the specific distribution of acetylhydrolase among the LDL and HDL subfractions was influenced by exposure to high concentrations of KBr. Reconstituted total LDL and total HDL were separately treated with three different concentrations of KBr (corresponding to densities of 1.063, 1.240, and 2.480 g/mL) and again subjected to ultracentrifugation (see "Methods"). Measurement of acetylhydrolase activity before and after KBr treatment, as well as after ultracentrifugation, did not reveal any difference in either total enzyme activity or the density profile of enzyme activity. These findings indicated a lack of effect of high ionic strength on either the quantitative or qualitative features of lipoprotein-associated acetylhydrolase activity (data not shown).
Determination of the Kinetic Constants of Acetylhydrolase in
Lipoprotein Subfractions
We subsequently performed kinetic studies of acetylhydrolase
activity in the various lipoprotein subfractions. The Michaelis-Menten
kinetic curves were determined, and apparent
Km and Vmax values
were calculated with the Lineweaver-Burk representation of the
data. As shown in Table 2, the apparent
Km values of the first four LDL
subfractions (LDL-1 through LDL-4; d=1.019 to 1.050 g/mL)
were similar (Km 
9 µmol/L), whereas
LDL-5 (d=1.050 to 1.063 g/mL) exhibited a 10-fold higher
Km (89.7±23.4 µmol/L).
Vmax values in LDL subfractions 1 through 4 were
similar when expressed per milligram of protein (between 3 and 8 nmol
PAF degraded/mg protein), whereas LDL-5 exhibited an elevated
Vmax (188±40 nmol PAF degraded/mg protein).
When Vmax values were expressed per milliliter
of plasma, Vmax increased significantly in
parallel with the increase in the density of the LDL subspecies to
attain a peak in LDL-5 (18.45±3.96 nmol PAF degraded/mL plasma); such
an elevation in this kinetic parameter may reflect an
increase in the amount of enzyme protein associated with each LDL
particle. In total LDL (d=1.019 to 1.063 g/mL),
acetylhydrolase displayed a Km of
21.3±4.9 µmol/L, thereby reflecting an affinity for PAF that was
intermediate between that of the five LDL subspecies. Furthermore, when
expressed on the basis of unit plasma volume, the
Vmax of acetylhydrolase in total LDL (20.4±4.2
nmol PAF degraded/min) corresponded to the sum of the maximal
velocities of the five LDL subspecies, thereby providing an indication
of the amount of enzyme protein associated with the total spectrum of
LDL particles. VHDL-1 (d=1.156 to 1.179 g/mL) exhibited
higher Km and Vmax
values compared with those of the lighter HDL subfractions (HDL-2 and
HDL-3), the latter displaying similar Km
and Vmax values when expressed per milliliter of
plasma. These data may again suggest that elevated amounts of enzyme
protein are associated with VHDL-1 particles. Equally, the
Ki values for the inhibitory effect
of phospholipids derived from the oxidative fragmentation of
polyunsaturated PC on the acetylhydrolase-mediated hydrolysis of
[3H-acetyl]PAF were higher in the LDL-5 and VHDL-1
subfractions compared with those of LDL-1. These oxidized
phospholipids, which contain short chains at the sn-2
position of glycerol, are substrates for
acetylhydrolase,45 and as such they compete with PAF. Our
results (Table 2) revealed that these phospholipids competitively
inhibited [3H-acetyl]PAF hydrolysis by the subfractions
LDL-1, LDL-5, and VHDL-1, and that the apparent
Ki values of LDL-5 and VHDL-1 (35.5 and 19.1
µmol/L, respectively) were markedly higher compared with that of
LDL-1 (3.5 µmol/L).
|
Transfer of Acetylhydrolase Between LDL Subfractions
We next asked whether the differences observed in the catalytic
properties of acetylhydrolase among the lipoprotein subfractions might
arise from compositional and structural differences between the
particle subspecies or, alternatively, from differences in the form of
the enzyme.46 47 We first inactivated the
LDL-5–associated acetylhydrolase with Pefabloc, then incubated such
LDL-5 with a mixture of LDL-1 and LDL-2 containing the active enzyme.
Finally, the three LDL subfractions were separated by gradient
ultracentrifugation (see "Methods"). More
than 85±8% of the enzyme activity initially associated with the LDL-1
and LDL-2 subfractions was recovered associated with LDL-5 after
ultracentrifugation (Fig 2).
Determination of the kinetic constants of the enzyme transferred to
LDL-5 showed that the Km (9.4±2.1
µmol/L) was similar to that of the LDL particles from which it was
derived: ie, LDL-1 and LDL-2 (Km=8.4
µmol/L) (Tables 2
and 3).
|
|
Properties of Acetylhydrolase Associated With Lipoprotein
Subfractions
To evaluate whether the distinct catalytic properties of
acetylhydrolase among the respective subfractions arose from potential
differences in the physicochemical properties of the enzyme, we studied
the influence of pH on enzyme activity at pH 5, pH 7, and pH 8. We next
established the susceptibility of the enzyme to the proteolytic
activity of trypsin, as well as to treatment with the serine esterase
inhibitor Pefabloc,24 48 the specific PAF
antagonist CV 3988,49 and the sulfydryl
reagents iodoacetic acid and dithiothreitol. The activity of the enzyme
in each of the individual subfractions exhibited similar behavior in
the three pH regions, with maximal acetylhydrolase activity at pH 7
(100% to 115%) and minimal activity at pH 5 (75% to 80%)
compared with that determined at pH 8 (taken as 100%). In addition,
enzyme activity in all subfractions was almost completely inhibited by
0.1 mmol/L Pefabloc and by 10 mmol/L CV 3988 (less than 5% of
acetylhydrolase activity remained after each treatment). Furthermore,
acetylhydrolase activity in all subfractions was not markedly
influenced by treatment with trypsin or dithiothreitol (more than 75%
of acetylhydrolase activity remained after each treatment), and enzyme
activity was also totally recovered in all subfractions after
iodoacetic acid treatment.
Properties of the Acetylhydrolase Released by Adherent Monocytes
in Culture
Upon adherence culture of human peripheral blood
monocytes for 4 days in RPMI medium in the absence of serum, a
PAF-hydrolyzing activity was released into the supernatant; such
activity was characterized as that of acetylhydrolase because it did
not require Ca2+ (data not shown) and was
specifically inhibited by CV 3988 and Pefabloc (data not shown). The
physicochemical properties of the acetylhydrolase released by adherent
monocytes in culture resembled those of both the apo B– and the apo
A-I–containing lipoprotein subfractions because its activity was not
affected by treatment with iodoacetic acid and was only slightly
affected by treatment with trypsin or dithiothreitol (approximately
80% of acetylhydrolase activity remained after treatment). Kinetic
studies showed that the apparent Km of
this enzyme was 23.4±7.6 µmol/L.
Association and Distribution of the Acetylhydrolase Released by
Adherent Monocytes in Culture Among the Lipoprotein
Subfractions
The supernatant from adherent monocytes in culture was incubated
separately with plasma, reconstituted total LDL, or total HDL that had
been treated previously with Pefabloc to completely
inactivate acetylhydrolase; each incubation mixture was
then submitted to fractionation by gradient
ultracentrifugation and individual fractions were
assayed for acetylhydrolase activity. As shown in Fig 3A, the acetylhydrolase of monocyte supernatant was
distributed primarily in LDL gradient fractions 11 and 12 (ie, LDL-5)
upon incubation with whole plasma. However, a portion of
acetylhydrolase activity (>40% of total activity) did not associate
with lipoproteins in plasma and was isolated in the dense portion of
the gradient (fractions >24, d>1.179 g/mL, data not
shown). When the cell supernatant alone was submitted to
ultracentrifugal separation, the total acetylhydrolase activity was
almost exclusively isolated in the dense portion of the gradient
(>70% of total activity in fractions >24, d>1.179 g/mL)
(Fig 3D). In experiments in which the monocyte supernatant was
incubated with LDL alone, acetylhydrolase activity was distributed
primarily in LDL-5, whereas a portion (15% of total enzyme
activity) was recovered in the region of the gradient of
d>1.179 g/mL (Fig 3B). In the presence of HDL alone,
transfer of monocyte-derived acetylhydrolase was less efficient
(40% of total enzyme activity was recovered in the dense portion of
the gradient, d>1.179 g/mL), and a minor proportion of the
enzyme activity (26±8%) was recovered with VHDL-1 (Fig 3C).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the present study, we demonstrate for the first time that human plasma acetylhydrolase is preferentially associated with small, dense LDL-5 and VHDL-1 upon subfractionation of human plasma by gradient ultracentrifugation.25 The catalytic properties of the enzyme in LDL-5 and VHDL-1 were distinct from those in other apo B– and apo A-I–containing lipoprotein subfractions. The present results are consistent with our earlier study, which revealed heterogeneity in the distribution of acetylhydrolase among LDL subfractions with densities ranging from 1.030 to 1.048 g/mL32 ; this density interval excluded dense LDL of d=1.050 to 1.063 g/mL. Furthermore, we observed that approximately half of the total HDL-associated activity was localized to the dense VHDL-1 subfraction of d=1.156 to 1.179 g/mL containing almost exclusively apo A-I. This observation is in contrast with that previously reported by Ostermann et al,4 who proposed that only apo B–containing lipoproteins display acetylhydrolase activity. Acetylhydrolase activity is, however, associated with HDL in abetalipoproteinemia,50 indicating that the enzyme is not exclusively associated with lipoproteins in which apo B is the major protein component. Indeed, approximately 30% of plasma acetylhydrolase activity is associated with HDL in normolipidemic subjects.10 The distribution of plasma acetylhydrolase was not influenced by the ultracentrifugal fractionation procedure, because the enzyme did not dissociate from LDL and HDL particles during ultracentrifugation even when these lipoproteins had been previously incubated at high ionic strength, and indeed at ionic strengths significantly higher than those used in our gradient procedure. We postulate therefore that acetylhydrolase is associated with lipoproteins in a manner that involves interaction more efficient than mere electrostatic binding.
In an effort to account for the preferential distribution of plasma
acetylhydrolase in dense LDL and VHDL-1, the apparent
Km and Vmax values
of the enzyme were determined. We observed that acetylhydrolase
associated with dense LDL and with dense VHDL-1 exhibited markedly
higher Km and Vmax
values compared with those in other apo B– and apo A-I–containing
subfractions, respectively. Because the acetylhydrolase was not
purified from lipoprotein subfractions, Vmax may
reflect the amount of enzyme associated with each particle subspecies.
Furthermore, acetylhydrolase associated with LDL-5 and VHDL-1 displayed
a markedly lower affinity for the substrate (higher
Km) compared with other lipoprotein
subspecies. In this context, it is relevant that the catalytic behavior
of acetylhydrolase is highly influenced by lipoprotein composition and
structure.4 50 Among the LDL subspecies, dense LDL are
distinguished by a low binding affinity for the cellular LDL
receptor,29 their diminished resistance to oxidative
stress,28 and the cleavage pattern seen in apo B 100 upon
limited proteolysis.51 These findings are
consistent with the hypothesis that the surface organization of
dense LDL particles is distinct. To address the possibility that
differences in the apparent Km values of
acetylhydrolase among lipoprotein subspecies might arise from
dissimilarities in their composition and structure, we measured the
kinetic constants of acetylhydrolase after transfer from LDL-1 and
LDL-2 subfractions to LDL-5, in which the enzyme had been previously
inactivated. We found that the
Km of the transferred enzyme did not
differ from that exhibited by LDL-1 and LDL-2 alone, thereby providing
evidence that the specific compositional and structural features of
LDL-5 did not influence enzyme activity. This observation is further
supported by the fact that the Km values
of acetylhydrolase associated with VLDL+IDL and LDL-1 through LDL-4
resembled each other, although such particles are distinct in both
composition and structure (Table 1).
It has been previously established that acetylhydrolase derived from different cellular sources is not homogeneous but consists of distinct forms that differ in their physicochemical behavior.14 Furthermore, recent data have revealed the existence of three isoforms of the bovine brain acetylhydrolase, one of which (isoform II) exhibits different behavior at pH 5 compared with isoforms Ia and Ib.15 To identify possible differences in the biochemical properties of acetylhydrolase distributed among the various lipoprotein subfractions, we studied the susceptibility of the enzyme to digestion with trypsin and to treatment with iodoacetic acid or dithiothreitol, as well as its behavior as a function of pH. No significant differences between the effects of such treatments on enzyme activity in the various subfractions were detected. These findings are in agreement with those of previous reports showing that the human plasma acetylhydrolase is not sensitive to trypsin, is not affected by sulfydryl reagents, and exhibits optimal activity at neutral pH.8 14 Furthermore, the enzyme present in small, dense subfractions of LDL and VHDL-1, as well as in other subfractions, was completely inhibited by the specific PAF antagonist CV 3988 and the serine esterase inhibitor Pefabloc. Thus, on the basis of these criteria, acetylhydrolase activity associated with LDL-5 and VHDL-1 showed similar behavior in response to classic biochemical treatments compared with that in the other subfractions.
Because adhering monocytes and macrophages are potential sources of acetylhydrolase in plasma,* we next determined whether the enzyme released from these cells would preferentially associate with dense lipoprotein subspecies containing apo B or apo A-I. We used cultured human peripheral blood monocytes, which are known to release acetylhydrolase during differentiation into macrophages.18 We observed that this enzyme is resistant to treatment with trypsin and was not affected by treatment with iodoacetic acid and dithiothreitol, as previously reported.14 18 The Km of the monocyte-released acetylhydrolase was similar to that in total LDL of d=1.019 to 1.063 g/mL. After incubation of monocyte supernatant with plasma or total LDL, or total HDL including VHDL-1, each of which had been previously treated with Pefabloc to inactivate acetylhydrolase and subsequently reisolated by gradient ultracentrifugation, the bulk of lipoprotein-associated acetylhydrolase activity was localized to the small, dense LDL-5 subfractions. This result is consistent with the suggestion that monocytes act primarily as donors of LDL-associated acetylhydrolase. The marked differences that we observed in the catalytic properties of acetylhydrolase among apo B– and apo A-I–containing lipoprotein subfractions lead us to postulate that human lipoproteins transport a family of closely related polymorphic proteins that are endowed with acetylhydrolase activity but are distinct in catalytic properties. We are presently purifying acetylhydrolase from distinct lipoprotein subfractions and monocyte supernatant to shed light on this key question. Further experiments are now required to evaluate the potential contribution of other cell types to the plasma pool of lipoprotein-associated acetylhydrolase.
In conclusion, our results show that the bulk of human plasma acetylhydrolase activity is associated with dense lipoprotein subspecies containing either apo B100 (ie, LDL-5) or apo A-I (ie, VHDL-1) as their major apolipoprotein. In addition, the lipoprotein-associated enzyme in these subspecies exhibits distinct catalytic features. Moreover, the transfer of acetylhydrolase from light LDL (LDL-1 and LDL-2) indicates that the enzyme possesses a higher affinity for the surface of LDL-5 particles, although its Km for PAF remains unaltered. Furthermore, the acetylhydrolase released from adherent monocytes in culture also associated preferentially with LDL-5 particles. It is of special interest that the LDL-5 and VHDL-1 subspecies also carry the majority of TFPI in plasma.44 Indeed, the comparable distribution among lipoprotein subspecies of acetylhydrolase and of TFPI, which both possess antithrombotic properties, is of key relevance to the pathophysiology of atherosclerosis. In this context, it is noteworthy that the penetration of the arterial wall by lipoprotein particles is inversely proportional to their size52 ; because LDL-5 and VHDL-1 are the smallest particles among the apo B– and apo A-I–containing families respectively, they may facilitate the efficient delivery of acetylhydrolase and TFPI to arterial tissue. Clearly, then, both LDL-5 and VHDL-1 in their native states may exert both anti-inflammatory and anticoagulant effects in the arterial intima. In the case of LDL-5, however, such activities are progressively inactivated by oxidation,23 24 36 a process to which dense LDL-5 particles are highly susceptible.27 28
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
These studies were supported by INSERM, by the Franco-Hellenic program PLATON, and by the research grant of the European Community (PL 931790). C. Dentan was the recipient of a research fellowship from the French Ministry of Research and Technology. We thank Dr M. Guérin for help in gradient preparation, and we are indebted to C. Debets-Albertini (Centre Départemental de Transfusion Sanguine, Créteil, France) for the generous gift of thrombopheresis residues.
|
Footnotes |
|---|
*Note added in proof. Tjoelker LW, Wilder C, Eberhardt C, Stafforini DM, Dietsch G, Schimpf B, Hooper S, Trong HL, Cousens LS, Zimmerman GA, Yamada Y, McIntyre TM, Prescott SM, Gray PW. Anti-inflammatory properties of a platelet-activating factor acetylhydrolase. Nature. 1995;374;549-552.
Received April 5, 1995; accepted May 9, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Benveniste J. Paf-acether: an ether phospholipid with biological activity. In: Karnovsky ML, Leaf A, Bolis LC, eds. Progress in Clinical and Biological Research: Biological Membranes—Aberrations in Membrane Structure and Function. New York, NY: AR Liss; 1988:73-85.
2. Koltai M, Hosford D, Bourgain RH, Braquet P. The role of PAF in blood cell-vessel wall interactions and atherosclerosis. Thromb Haemorrh Disorders. 1990;2:47-60.
3. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
4. Ostermann G, Kostner GM, Gries A, Malle E, Till U. The contribution of individual lipoproteins to the degradation of platelet-activating factor in human serum. Haemostasis. 1989;19:160-168. [Medline] [Order article via Infotrieve]
5. Benveniste J, Nunez D, Duriez P, Korth R, Bidault J, Fruchart JC. Preformed paf-acether and lyso paf-acether are bound to blood lipoproteins. FEBS Lett. 1988;226:371-376. [Medline] [Order article via Infotrieve]
6. Farr RS, Cox CP, Wardlow ML, Jorgensen R. Preliminary studies of an acid-labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF). Clin Immunol Immunopathol. 1980;15:318-330. [Medline] [Order article via Infotrieve]
7.
Blank ML, Lee T, Fitzgerald V, Snyder F. A
specific acetylhydrolase for
1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and
platelet-activating lipid). J Biol
Chem. 1981;256:175-178.
8.
Stafforini DM, Prescott SM, McIntyre TM. Human
plasma platelet-activating factor acetylhydrolase: purification
and properties. J Biol Chem. 1987;262:4223-4230.
9. Evangelou AM. Platelet-activating factor (PAF): implications for coronary heart and vascular diseases. Prostaglandins Leukot Essent Fatty Acids. 1994;50:1-28. [Medline] [Order article via Infotrieve]
10.
Stafforini DM, McIntyre TM, Carter ME, Prescott SM.
Human plasma platelet-activating factor acetylhydrolase:
association with lipoprotein particles and role in the degradation of
platelet-activating factor. J Biol
Chem. 1987;262:4215-4222.
11.
Elstad MR, Stafforini DM, McIntyre TM, Prescott SM,
Zimmerman GA. Platelet-activating factor acetylhydrolase
increase during macrophage differentiation: a novel mechanism
that regulates accumulation of platelet-activating
factor. J Biol Chem. 1989;264:8467-8470.
12. Korth R, Bidault J, Palmantier R, Benveniste J, Ninio E. Human platelets release a paf-acether: acetylhydrolase similar to that in plasma. Lipids. 1993;28:193-199. [Medline] [Order article via Infotrieve]
13.
Stafforini DM, Rollins EN, Prescott SM, McIntyre TM.
The platelet-activating factor acetylhydrolase from
human erythrocytes: purification and properties. J
Biol Chem. 1993;268:3857-3865.
14. Stafforini DM, Prescott SM, Zimmermann GA, McIntyre TM. Platelet-activating factor acetylhydrolase in human tissues and blood cells. Lipids. 1991;26:979-985. [Medline] [Order article via Infotrieve]
15.
Hattori M, Arai H, Inoue K. Purification and
characterization of bovine brain platelet-activating factor
acetylhydrolase. J Biol Chem. 1993;268:18748-18753.
16. Hattori M, Adachi H, Tsujimoto M, Arai H, Inoue K. Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor acetylhydrolase. Nature. 1994;370:216-218. [Medline] [Order article via Infotrieve]
17.
Hattori M, Adachi H, Tsujimoto M, Arai H, Inoue K.
The catalytic subunit of bovine brain
platelet-activating factor acetylhydrolase is a novel type of
serine esterase. J Biol Chem. 1994;269:23150-23155.
18.
Stafforini DM, Elstad MR, McIntyre TM, Zimmerman GA,
Prescott SM. Human macrophages secrete
platelet-activating factor acetylhydrolase.
J Biol Chem. 1990;265:9682-9687.
19.
Tarbet EB, Stafforini DM, Elstad MR, Zimmerman GA,
McIntyre TM, Prescott SM. Liver cells secrete the plasma form of
platelet-activating factor acetylhydrolase.
J Biol Chem. 1991;266:16667-16673.
20. Steinbrecher UP, Parthasarthy S, Leake DS, Witztum JL, Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Natl Acad Sci U S A. 1984;81:3383-3387.
21.
Parthasarathy S, Steinbrecher UP, Barnett J, Witztum
JL, Steinberg D. The essential role of phospholipase A2 activity
in endothelial cell-induced modification of low
density lipoprotein. Proc Natl Acad Sci U S A. 1985;82:3000-3004.
22. Steinbrecher UP, Pritchard PH. Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase. J Lipid Res. 1989;30:305-315. [Abstract]
23. Liapikos TA, Antonopoulou S, Karabina SA, Tsoukatos DC, Demopoulos CA, Tselepis AD. Platelet-activating factor formation during oxidative modification of low-density lipoprotein when PAF-acetylhydrolase has been inactivated. Biochim Biophys Acta. 1994;1212:353-360. [Medline] [Order article via Infotrieve]
24.
Dentan C, Lesnik P, Chapman MJ, Ninio E.
PAF-acether–degrading acetylhydrolase in plasma LDL is
inactivated by copper- and cell-mediated
oxidation. Arterioscler Thromb. 1994;14:353-360.
25. Chapman MJ, Goldstein S, Lagrange D, Laplaud PM. A density gradient ultracentrifugal procedure for isolation of the major lipoprotein classes from human serum. J Lipid Res. 1981;22:339-358. [Abstract]
26. Krauss RM. Heterogeneity of plasma low-density lipoproteins and atherosclerosis risk. Curr Opin Lipidol. 1994;5:339-349.[Medline] [Order article via Infotrieve]
27.
De Graaf J, Hak-Lemmers HLM, Hectors MPC, Demacker PNM,
Hendricks JCM, Stalenhoef AFH. Enhanced susceptibility to in
vitro oxidation of the dense low-density lipoprotein subfraction in
healthy subjects. Arterioscler Thromb. 1991;11:298-306.
28. Dejager S, Bruckert E, Chapman MJ. Dense low density lipoprotein subspecies with diminished oxidative resistance predominate in combined hyperlipidemia. J Lipid Res. 1993;34:295-308. [Abstract]
29. Nigon F, Lesnik P, Rouis M, Chapman J. Discrete subspecies of human low-density lipoproteins are heterogeneous in their interaction with the LDL receptor. J Lipid Res. 1991;32:1741-1753. [Abstract]
30. Anderson DW, Nichols AV, Forte TM, Lindgren FT. Particle distribution of serum high density lipoproteins. Biochim Biophys Acta. 1977;493:55-68. [Medline] [Order article via Infotrieve]
31.
Patsch WP, Schonfeld G, Gotto AM, Patsch J, Patsch JR.
Characterization of human high density lipoproteins by zonal
ultracentrifugation. J Biol
Chem. 1980;255:3178-3185.
32. Karabina SA, Liapikos TA, Grekas G, Goudevenos J, Tselepis AD. Distribution of PAF-acetylhydrolase activity in human plasma low-density lipoprotein subfractions. Biochim Biophys Acta. 1994;1213:34-38. [Medline] [Order article via Infotrieve]
33. Nichols AV, Krauss RM, Musliner TA. Non-denaturing polyacrylamide gradient gel electrophoresis. Methods Enzymol. 1986;128:417-431. [Medline] [Order article via Infotrieve]
34. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophaga T4. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]
35. Chapman MJ, Laplaud PM, Luc G, Forgez P, Bruckert E, Goulinet S, Lagrange D. Further resolution of the low-density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation. J Lipid Res. 1988;29:442-458. [Abstract]
36.
Lesnik P, Dentan C, Vonika A, Moreau M, Chapman MJ.
Tissue factor pathway inhibitor activity with LDL is
inactivated by cell- and copper-mediated
oxidation. Arterioscler Thromb Vasc Biol. 1995;15:1121-1130.
37. El-Saadani M, Esterbauer H, El-Sayed M, Goher M, Nassar AY, Jürgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res. 1989;30:627-630. [Abstract]
38.
Rouis M, Nigon F, Lafuma C, Hornebeck W, Chapman MJ.
Expression of elastase activity by human
monocyte-macrophages is modulated by cellular
cholesterol content, inflammatory mediators, and phorbol
myristate acetate.
Arteriosclerosis. 1990;10:246-255.
39. Palmantier R, Dulioust A, Maiza H, Benveniste J, Ninio E. Biosynthesis of paf-acether: paf-acether output in murine peritoneal macrophages is regulated by the level of acetylhydrolase. Biochem Biophys Res Commun. 1989;162:475-482. [Medline] [Order article via Infotrieve]
40. Pinckard RN, Ludwig JC. Determination of PAF-2 acylhydrolase activity. Fed Proc. 1986;45:856. Abstract.
41. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911-917.
42.
Bartlett GR. Phosphorus assay in column
chromatography. J Biol
Chem. 1959;234:466-468.
43. Marinetti GV. Chromatographic separation, identification and analysis of phosphatides. J Lipid Res. 1962;3:1-17.
44.
Lesnik P, Vonika A, Guérin M, Moreau M, Chapman
MJ. Anticoagulant activity of tissue factor pathway
inhibitor in human plasma is preferentially associated with
dense subspecies of LDL and HDL and with Lp(a).
Arterioscler Thromb. 1993;13:1066-1075.
45.
Stremler KE, Stafforini DM, Prescott SM, McIntyre TM.
Human plasma platelet-activating factor acetylhydrolase:
oxidatively fragmented phospholipids as substrates.
J Biol Chem. 1991;266:11095-11103.
46. Kinoshita M, Krul ES, Schonfeld G. Modification of the core lipids of low-density lipoproteins produces selective alterations in the expression of apo B-100 epitopes. J Lipid Res. 1990;31:701-708. [Abstract]
47. Baumstark MW, Kreutz W, Berg A, Frey I, Keul J. Structure of human low-density lipoprotein subfractions, determined by X-ray small angle scattering. Biochim Biophys Acta. 1990;1037:48-57. [Medline] [Order article via Infotrieve]
48. Mintz GR. An irreversible serine protease inhibitor. Biopharm. 1993;6:34-38.
49. Terashita ZI, Tsushima S, Yoshioka Y, Nomura H, Inada Y, Nishikawa K. CV 3988: a specific antagonist of platelet-activating factor (PAF). Life Sci. 1983;32:1975-1982. [Medline] [Order article via Infotrieve]
50.
Stafforini DM, Carter ME, Zimmerman GA, McIntyre TM,
Prescott SM. Lipoproteins alter the catalytic behavior of the
platelet-activating factor acetylhydrolase in human
plasma. Proc Natl Acad Sci U S A. 1989;86:2393-2397.
51.
Chen GC, Liu W, Duchateau P, Allaart J, Hamilton RL,
Mendel CM, Lau K, Hardman DA, Frost PH, Malloy MJ, Kane JP.
Conformational differences in human apolipoprotein B-100 among
subspecies of low-density lipoproteins (LDL): association of
altered proteolytic accessibility with decreased receptor binding of
LDL subspecies from hypertriglyceridemic
subjects. J Biol Chem. 1994;269:29121-29128.
52.
Stender S, Zilversmit DB. Transfer of plasma
lipoprotein components and of plasma proteins into aortas of
cholesterol-fed rabbits: molecular size as determinant
of plasma lipoprotein influx.
Arteriosclerosis. 1981;1:38-49.
This article has been cited by other articles:
 |
|
 |
|
  M. S. Kostapanos, H. J. Milionis, T. D. Filippatos, L. G. Christogiannis, E. T. Bairaktari, A. D. Tselepis, and M. S. Elisaf Dose-dependent Effect of Rosuvastatin Treatment on HDL-subfraction Phenotype in Patients With Primary Hyperlipidemia Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2009; 14(1): 5 - 13. [Abstract] [PDF]
|
|
|
|
 |
|
 D. C. Tsoukatos, I. Brocheriou, V. Moussis, C. P. Panopoulou, E. D. Christofidou, S. Koussissis, S. Sismanidis, E. Ninio, and S. Siminelakis Platelet-activating factor acetylhydrolase and transacetylase activities in human aorta and mammary artery J. Lipid Res., October 1, 2008; 49(10): 2240 - 2249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Gardner, E. C. Reichert, M. K. Topham, and D. M. Stafforini Identification of a Domain That Mediates Association of Platelet-activating Factor Acetylhydrolase with High Density Lipoprotein J. Biol. Chem., June 20, 2008; 283(25): 17099 - 17106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Mohler III, C. M. Ballantyne, M. H. Davidson, M. Hanefeld, L. M. Ruilope, J. L. Johnson, A. Zalewski, and for the Darapladib Investigators The Effect of Darapladib on Plasma Lipoprotein-Associated Phospholipase A2 Activity and Cardiovascular Biomarkers in Patients With Stable Coronary Heart Disease or Coronary Heart Disease Risk Equivalent: The Results of a Multicenter, Randomized, Double-Blind, Placebo-Controlled Study J. Am. Coll. Cardiol., April 29, 2008; 51(17): 1632 - 1641. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Kono, T. Inoue, Y. Yoshida, H. Sato, T. Matsusue, H. Itabe, E. Niki, J. Aoki, and H. Arai Protection against Oxidative Stress-induced Hepatic Injury by Intracellular Type II Platelet-activating Factor Acetylhydrolase by Metabolism of Oxidized Phospholipids in Vivo J. Biol. Chem., January 18, 2008; 283(3): 1628 - 1636. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. G. Saougos, A. P. Tambaki, M. Kalogirou, M. Kostapanos, I. F. Gazi, R. L. Wolfert, M. Elisaf, and A. D. Tselepis Differential Effect of Hypolipidemic Drugs on Lipoprotein-Associated Phospholipase A2 Arterioscler Thromb Vasc Biol, October 1, 2007; 27(10): 2236 - 2243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. D. Kolodgie, A. P. Burke, K. S. Skorija, E. Ladich, R. Kutys, A. T. Makuria, and R. Virmani Lipoprotein-Associated Phospholipase A2 Protein Expression in the Natural Progression of Human Coronary Atherosclerosis Arterioscler Thromb Vasc Biol, November 1, 2006; 26(11): 2523 - 2529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. V. Mitsios, M. P. Vini, D. Stengel, E. Ninio, and A. D. Tselepis Human Platelets Secrete the Plasma Type of Platelet-Activating Factor Acetylhydrolase Primarily Associated With Microparticles Arterioscler Thromb Vasc Biol, August 1, 2006; 26(8): 1907 - 1913. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Gazi, E. S. Lourida, T. Filippatos, V. Tsimihodimos, M. Elisaf, and A. D. Tselepis Lipoprotein-Associated Phospholipase A2 Activity Is a Marker of Small, Dense LDL Particles in Human Plasma Clin. Chem., December 1, 2005; 51(12): 2264 - 2273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Androulakis, H. Durand, E. Ninio, and D. C. Tsoukatos Molecular and mechanistic characterization of platelet-activating factor-like bioactivity produced upon LDL oxidation J. Lipid Res., September 1, 2005; 46(9): 1923 - 1932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Ninio, K. Winkler, M. M. Hoffmann, A. B. Grawitz, M. Nauck, B. R. Winkelmann, H. Scharnagl, W. Marz, and B. O. Bohm Letter Regarding Article by Winkler et al, "Platelet-Activating Factor Acetylhydrolase Activity Indicates Angiographic Coronary Artery Disease Independently of Systemic Inflammation and Other Risk Factors: The Ludwigshafen Risk and Cardiovascular Health Study" * Response Circulation, August 23, 2005; 112(8): e108 - e109. [Full Text] [PDF]
|
|
|
|
|
|
A. Zalewski and C. Macphee Role of Lipoprotein-Associated Phospholipase A2 in Atherosclerosis: Biology, Epidemiology, and Possible Therapeutic Target Arterioscler Thromb Vasc Biol, May 1, 2005; 25(5): 923 - 931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Winkler, B. R. Winkelmann, H. Scharnagl, M. M. Hoffmann, A. B. Grawitz, M. Nauck, B. O. Bohm, and W. Marz Platelet-Activating Factor Acetylhydrolase Activity Indicates Angiographic Coronary Artery Disease Independently of Systemic Inflammation and Other Risk Factors: The Ludwigshafen Risk and Cardiovascular Health Study Circulation, March 1, 2005; 111(8): 980 - 987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Iribarren, M. D. Gross, J. A. Darbinian, D. R. Jacobs Jr, S. Sidney, and C. M. Loria Association of Lipoprotein-Associated Phospholipase A2 Mass and Activity With Calcified Coronary Plaque in Young Adults: The CARDIA Study Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 216 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hockerstedt, M. Jauhiainen, and M. J. Tikkanen Lecithin/Cholesterol Acyltransferase Induces Estradiol Esterification in High-Density Lipoprotein, Increasing Its Antioxidant Potential J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5088 - 5093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Ninio, D. Tregouet, J.-L. Carrier, D. Stengel, C. Bickel, C. Perret, H. J. Rupprecht, F. Cambien, S. Blankenberg, and L. Tiret Platelet-activating factor-acetylhydrolase and PAF-receptor gene haplotypes in relation to future cardiovascular event in patients with coronary artery disease Hum. Mol. Genet., July 1, 2004; 13(13): 1341 - 1351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. D Tsironis, J. V Mitsios, H. J Milionis, M. Elisaf, and A. D Tselepis Effect of lipoprotein (a) on platelet activation induced by platelet-activating factor: role of apolipoprotein (a) and endogenous PAF-acetylhydrolase Cardiovasc Res, July 1, 2004; 63(1): 130 - 138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Lie, R. de Crom, T. van Gent, R. van Haperen, L. Scheek, F. Sadeghi-Niaraki, and A. van Tol Elevation of plasma phospholipid transfer protein increases the risk of atherosclerosis despite lower apolipoprotein B-containing lipoproteins J. Lipid Res., May 1, 2004; 45(5): 805 - 811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Tsimihodimos, A. Kostoula, A. Kakafika, E. Bairaktari, A. D. Tselepis, D. P. Mikhailidis, and M. Elisaf Effect of Fenofibrate on Serum Inflammatory Markers in Patients With High Triglyceride Values Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2004; 9(1): 27 - 33. [Abstract] [PDF]
|
|
|
|
|
|
K. Winkler, C. Abletshauser, I. Friedrich, M. M. Hoffmann, H. Wieland, and W. Marz Fluvastatin Slow-Release Lowers Platelet-Activating Factor Acetyl Hydrolase Activity: A Placebo-Controlled Trial in Patients with Type 2 Diabetes J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1153 - 1159. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Blankenberg, D. Stengel, H. J. Rupprecht, C. Bickel, J. Meyer, F. Cambien, L. Tiret, and E. Ninio Plasma PAF-acetylhydrolase in patients with coronary artery disease: results of a cross-sectional analysis J. Lipid Res., July 1, 2003; 44(7): 1381 - 1386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Tsimihodimos, A. Kakafika, A. P. Tambaki, E. Bairaktari, M. J. Chapman, M. Elisaf, and A. D. Tselepis Fenofibrate induces HDL-associated PAF-AH but attenuates enzyme activity associated with apoB-containing lipoproteins J. Lipid Res., May 1, 2003; 44(5): 927 - 934. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mertens, P. Verhamme, J. K. Bielicki, M. C. Phillips, R. Quarck, W. Verreth, D. Stengel, E. Ninio, M. Navab, B. Mackness, et al. Increased Low-Density Lipoprotein Oxidation and Impaired High-Density Lipoprotein Antioxidant Defense Are Associated With Increased Macrophage Homing and Atherosclerosis in Dyslipidemic Obese Mice: LCAT Gene Transfer Decreases Atherosclerosis Circulation, April 1, 2003; 107(12): 1640 - 1646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Boisfer, D. Stengel, D. Pastier, P. M. Laplaud, N. Dousset, E. Ninio, and A.-D. Kalopissis Antioxidant properties of HDL in transgenic mice overexpressing human apolipoprotein A-II J. Lipid Res., May 1, 2002; 43(5): 732 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Tsimihodimos, S.-A. P. Karabina, A. P. Tambaki, E. Bairaktari, G. Miltiadous, J. A. Goudevenos, M. A. Cariolou, M. J. Chapman, A. D. Tselepis, and M. Elisaf Altered distribution of platelet-activating factor- acetylhydrolase activity between LDL and HDL as a function of the severity of hypercholesterolemia J. Lipid Res., February 1, 2002; 43(2): 256 - 263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Tsimihodimos, S.-A. P. Karabina, A. P. Tambaki, E. Bairaktari, J. A. Goudevenos, M. J. Chapman, M. Elisaf, and A. D. Tselepis Atorvastatin Preferentially Reduces LDL-Associated Platelet-Activating Factor Acetylhydrolase Activity in Dyslipidemias of Type IIA and Type IIB Arterioscler Thromb Vasc Biol, February 1, 2002; 22(2): 306 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Holvoet, K. Peeters, S. Lund-Katz, A. Mertens, P. Verhamme, R. Quarck, D. Stengel, M. Lox, E. Deridder, H. Bernar, et al. Arg123-Tyr166 Domain of Human ApoA-I Is Critical for HDL-Mediated Inhibition of Macrophage Homing and Early Atherosclerosis in Mice Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1977 - 1983. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Tselepis, S.-A. P. Karabina, D. Stengel, R. Piedagnel, M. J. Chapman, and E. Ninio N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL J. Lipid Res., October 1, 2001; 42(10): 1645 - 1654. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. MERTENS and P. HOLVOET Oxidized LDL and HDL: antagonists in atherothrombosis FASEB J, October 1, 2001; 15(12): 2073 - 2084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Quarck, B. De Geest, D. Stengel, A. Mertens, M. Lox, G. Theilmeier, C. Michiels, M. Raes, H. Bult, D. Collen, et al. Adenovirus-Mediated Gene Transfer of Human Platelet-Activating Factor-Acetylhydrolase Prevents Injury-Induced Neointima Formation and Reduces Spontaneous Atherosclerosis in Apolipoprotein E-Deficient Mice Circulation, May 22, 2001; 103(20): 2495 - 2500. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Barlage, D. Fröhlich, A. Böttcher, M. Jauhiainen, H. P. Müller, F. Noetzel, G. Rothe, C. Schütt, R. P. Linke, K. J. Lackner, et al. ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response J. Lipid Res., February 1, 2001; 42(2): 281 - 290. [Abstract] [Full Text]
|
|
|
|
|
|
Bart De Geest, D. Stengel, M. Landeloos, M. Lox, L. Le Gat, D. Collen, P. Holvoet, and E. Ninio Effect of Overexpression of Human Apo A-I in C57BL/6 and C57BL/6 Apo E-Deficient Mice on 2 Lipoprotein-Associated Enzymes, Platelet-Activating Factor Acetylhydrolase and Paraoxonase : Comparison of Adenovirus-Mediated Human Apo A-I Gene Transfer and Human Apo A-I Transgenesis Arterioscler Thromb Vasc Biol, October 1, 2000; 20 (10): e68 - e75. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. THEILMEIER, B. DE GEEST, P. P. VAN VELDHOVEN, D. STENGEL, C. MICHIELS, M. LOX, M. LANDELOOS, M. J. CHAPMAN, E. NINIO, D. COLLEN, et al. HDL-associated PAF-AH reduces endothelial adhesiveness in apoE-/- mice FASEB J, October 1, 2000; 14(13): 2032 - 2039. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. Montrucchio, G. Alloatti, and G. Camussi Role of Platelet-Activating Factor in Cardiovascular Pathophysiology Physiol Rev, October 1, 2000; 80(4): 1669 - 1699. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Kudchodkar, A. G. Lacko, L. Dory, and T. V. Fungwe Dietary Fat Modulates Serum Paraoxonase 1 Activity in Rats J. Nutr., October 1, 2000; 130(10): 2427 - 2433. [Abstract] [Full Text]
|
|
|
|
|
|
T. M. Forte, M. N. Oda, L. Knoff, B. Frei, J. Suh, J. A. K. Harmony, W. D. Stuart, E. M. Rubin, and D. S. Ng Targeted disruption of the murine lecithin:cholesterol acyltransferase gene is associated with reductions in plasma paraoxonase and platelet-activating factor acetylhydrolase activities but not in apolipoprotein J concentration J. Lipid Res., July 1, 1999; 40(7): 1276 - 1283. [Abstract] [Full Text]
|
|
|
|
|
|
A. D. Tselepis, J. A. Goudevenos, A. P. Tambaki, L. Michalis, C. S. Stroumbis, D. C. Tsoukatos, M. Elisaf, and D. A. Sideris Platelet aggregatory response to platelet activating factor (PAF), ex vivo, and PAF-acetylhydrolase activity in patients with unstable angina: effect of c7E3 Fab (abciximab) therapy Cardiovasc Res, July 1, 1999; 43(1): 183 - 191. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Chancharme, P. Therond, F. Nigon, S. Lepage, M. Couturier, and M. J. Chapman Cholesteryl Ester Hydroperoxide Lability Is a Key Feature of the Oxidative Susceptibility of Small, Dense LDL Arterioscler Thromb Vasc Biol, March 1, 1999; 19(3): 810 - 820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. Tsoukatos, M. Arborati, T. Liapikos, K. L. Clay, R. C. Murphy, M. J. Chapman, and E. Ninio Copper-Catalyzed Oxidation Mediates PAF Formation in Human LDL Subspecies : Protective Role of PAF:Acetylhydrolase in Dense LDL Arterioscler Thromb Vasc Biol, December 1, 1997; 17(12): 3505 - 3512. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||