While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized.
Trang 15-lipoxygenase activation in polymorphonuclear
leukocytes and MonoMac6 cells
Dmitry A Aleksandrov1, Anna N Zagryagskaya1, Marina A Pushkareva1, Markus Bachschmid2, Marc Peters-Golden3, Oliver Werz4, Dieter Steinhilber4 and Galina F Sud’ina1
1 A.N Belozersky Institute of Physicochemical Biology, Moscow State University, Russia
2 Faculty of Biology, University of Konstanz, Germany
3 Pulmonary and Critical Care Medicine Division, University of Michigan, Ann Arbor, USA
4 Institute of Pharmaceutical Chemistry ⁄ ZAFES, University of Frankfurt, Germany
Mammalian 5-lipoxygenase (5-LO) converts
arachi-donic acid (AA) to 5-hydroperoxyeicosatetraenoic acid
(5-HPETE) and further to leukotriene A4 (LTA4) [1]
This substance is a key intermediate in the biosynthesis
of two leukotriene families that act as potent
media-tors of cell proliferation [2], apoptosis [3],
tumorigen-esis and inflammatory processes such as allergy,
atherosclerosis and asthma [4,5] 5-LO is activated by
intracellular Ca2+ influx that leads to translocation
and binding of 5-LO to the nuclear membrane [6–11]
It is commonly observed that the N-terminal domain
of the enzyme may function like the protein kinase C
C2 domain and facilitates Ca2+-mediated membrane binding [12–16]
There are two main physicochemical factors influen-cing translocation of 5-LO to the membrane First, the calcium-ion binding counteracts electrostatic repulsion
of anionic C2 domain of 5-LO at the surface of ani-onic membranes [15] Secondly, it may be regulated by membrane surface charge and membrane lipids It was shown that phosphatidyl choline (PC) head groups facilitate the 5-LO C2-like domain binding to mem-branes, and that Trp13, Trp75, Trp102 of 5-LO are involved in these protein–lipid interactions [15] The
Keywords
5-lipoxygenase; cholesterol sulfate;
leukotrienes; neutrophil
Correspondence
G F Sud’ina, A.N Belozersky Institute of
Physicochemical Biology, Moscow State
University, Russia
Fax: + 7 095 9393181
Tel: +7 095 9393184
E-mail: sudina@genebee.msu.ru
(Received 19 September 2005, revised
23 November 2005, accepted 5 December
2005)
doi:10.1111/j.1742-4658.2005.05087.x
5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrie-nes (LTs), biological mediators of host defense reactions and of inflamma-tory diseases While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized Here, we show such
a novel function of the most abundant sulfated sterol in human blood, cho-lesterol sulfate (CS), to suppress LT production in human polymorpho-nuclear leukocytes (PMNL) and Mono Mac6 cells We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity
in suppression of LT synthesis in PMNL Cholesteryl acetate was without effect Cholesterol increased the effect of CS on 5-LO product synthesis
CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL Addition of exogenous AA increased the threshold concentration
of CS required to inhibit LT synthesis The effect of cholesterol and its ani-onic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases⁄ sulfatases in 5-LO product synthesis
Abbreviations
AA, arachidonic acid; CA, cholesterol acetate; CP, cholesterol phosphate; CS, cholesterol sulfate; HBSS, Hank’s balanced salt solution; 5-HETE, 5-hydroxyeicosatetraenoic acid; LT, leukotrienes; LTB4, leukotriene B4; iso-LTB4, 5(S),12(S,R)-dihydroxy-all-trans-eicosatetraenoic acids; 5-LO, 5-lipoxygenase; MbCD, methyl-b-cyclodextrin; PMNL, polymorphonuclear leukocyte; ROS, reactive oxygen species.
Trang 2anionic lipid
1-palmitoyl-2-oleoyl-glycero-3-phospho-glycerol decreased 5-LO activity [19] Just recently, it
was published by these authors that membrane fluidity
is a key modulator of membrane binding and activity
of 5-lipoxygenase [20] Earlier, it was demonstrated by
our group that cellular 5-LO activity is suppressed by
anionic lipid sulfatides [21] To further test the role of
anionic lipids as 5-LO inhibitors, we investigated
ani-onic derivatives of cholesterol Cholesterol and its
sul-fated product cholesterol sulfate (CS) are also known
as agents that increase membrane rigidity
Being an important constituent of cell membranes,
free cholesterol is converted to its ester in blood
plasma In view of the role of cholesterol lipids in
atherogenesis, we sought to determine the influence of
cholesterol and its derivatives on cellular 5-LO activity
We studied CS, cholesterol phosphate (CP) and
choles-terol acetate (CA), the latter being chosen as a
refer-ence ester compound The 5-lipoxygenase pathway has
recently been implicated as an important component in
the pathogenesis of atherosclerosis [22] It is likely that
neutrophils and monocytes largely dictate the levels of
LTs in the vasculature, and these mediators play an
important role in leukocyte adhesion to vessel walls,
ROS production and atherothrombosis The regulation
of LT synthesis by cholesterol and its derivatives in
polymorphonuclear leukocytes (PMNL) has not been
previously characterized
Among the two anionic cholesterol derivatives
inves-tigated here, CS is a natural compound CS is
synthes-ized by the sulfotransferase SULT2B1b from free
cholesterol It is quantitatively the most important and
abundant sterol sulfate in human plasma, where it is
present at concentrations of about 3 lgÆmL)1[23] The
concentration of CS may vary widely during disease
states [24,25] Infiltration of neutrophils and monocytes
into gastric mucosa is a hallmark of chronic gastritis
caused by Helicobacter pylori, with neutrophil
stimula-tion that results in the damage of the gastric
epithe-lium Human gastric fluid and gastric epithelia contain
up to 500 lg sulfatides and 700 lg CS per gram of dry
weight [26] CS exhibits a gastroprotective activity after
administration of sulfolipid-containing liposomes [26]
As a component of cell membranes, CS plays a
stabil-izing role [27], prevents osmotic lysis and supports cell
In this work, we report that CS suppresses LT syn-thesis in two cell types, PMNL and MM6
Results
Free cholesterol and its anionic derivatives inhibit cellular 5-LO product formation
We determined the effect of free cholesterol, CS, CP and CA on LT production in whole cells Isolated human PMNL were collected and suspended in PGC buffer with different cholesterol derivatives This mix-ture was preincubated for 30 min at 37C and leuko-triene production was stimulated for 10 min with 2 lm A23187 calcium ionophore The formation of LTB4, its trans- and epi-trans-isomers and 5-HETE was deter-mined The data are presented in Fig 1 The anionic derivative CS inhibited LT synthesis at concentrations
as low as 10 lgÆmL)1 (Fig 1A) Cholesterol had no significant effect at 10 lgÆmL)1, but inhibited at
25 lgÆmL)1 (Fig 1B) Both lipids were inactive in the presence of the cholesterol acceptor methyl-b-cyclodex-trin (MbCD) (Fig 1C) MbCD at 1 mm had no effect, but at the optimal concentration of 2 mm, it signifi-cantly increased AA release and LT synthesis
The synthetic anionic cholesterol derivative CP was
an even stronger inhibitor of LT synthesis than CS (Fig 1B) The naturally occurring anionic lipid CS was analyzed in more detail Combined exposure to cholesterol and CS at 25 lgÆmL)1 of lipids did not have a greater effect on 5-LO product synthesis than the exposure to 25 lgÆmL)1of CS alone (Fig 2) How-ever, when added with CS and AA, cholesterol signifi-cantly suppressed 5-LO product synthesis (lower panel), resulting in no difference between the sum of 5-LO products in the absence and presence of AA at
25 lgÆmL-1CS + 25 lgÆmL)1 cholesterol At fixed concentrations of CS, MbCD significantly increased 5-LO product synthesis in the absence of added AA (Fig 2, upper panel), thus increasing the threshold concentration of CS to inhibit LT synthesis Similarly, the sensitivity to CS decreased in the presence of AA (Fig 2, lower panel) From Fig 2 it can be seen that agents like cholesterol, which increase membrane rigid-ity, enhanced the inhibitory effects of CS on 5-LO
Trang 3product formation, and that agents that decrease
mem-brane rigidity (MbCD, AA) attenuated the effect of
CS on 5-LO product synthesis In a wide concentration
range, CS significantly inhibited 5-LO product
synthe-sis in PMNL and MM6 cells also in the presence of
AA in the medium (Fig 3)
CS down-regulates AA-release in PMNL
Experiments performed with PMNL labeled with
radioactive 14C-AA revealed the mode of inhibition
of LT production by CS Stimulation of PMNL with
ionophore induced the release of 14C-activity into the
supernatant and the formation of radioactively
labe-led 5-LO products After a 30-min incubation of
PMNL with CS, the release of 14C-AA and 14
C-labelled 5-LO products was suppressed in a
concen-tration-dependent manner (Fig 4A) Interestingly, the
release of endogenous 14C-AA was inhibited in a similar way in the presence or absence of 20 lm AA
in the medium Figure 4B presents the total product synthesis under the same assay conditions using labe-led and exogenously added nonlabelabe-led AA, and demonstrates the crucial alteration of the dose– response curve in the presence of exogenously added
AA This fact demonstrates that CS is likely to inhi-bit LT production at the level of substrate availabil-ity The data demonstrate that AA added together with CS masks the effect of CS up to 50 lgÆmL)1
CS When no AA is added in preincubations, CS effectively suppressed (50% inhibition) AA release and 5-LO product formation at concentrations of
50 lgÆmL)1 CS Experiments performed in the pres-ence of exogenous AA showed that 5-LO was inhib-ited by 50% at about 100 lgÆmL)1 CS in Mono Mac6 cells (Fig 3) and in PMNL (Fig 4B)
A
B
C
Fig 1 Comparative effects of cholesterol (chol) and its esters on 5-LO product synthe-sis in PMNL PMNLs were preincubated for
30 min at 37 C with indicated lipids, and then stimulated for 10 min with 2 l M
A23187 calcium ionophore (A) and (B) Lipid concentration is 10 lgÆmL)1(A) and
25 lgÆmL)1(B) (C) MbCD was added together with indicated lipids taken at
25 lgÆmL)1 The LT synthesis and the total 5-LO product release (as measured by radio-activity) are presented as percentage of the control *P < 0.05; **P < 0.01, when corres-ponding data compared to control.
Trang 4Cytochalasin D and chlorpromazine inhibit the effect of CS on LT synthesis in PMNL
We hypothesized that the effect of CS depended on its internalization and trafficking to ER This vesicular transport is sensitive to a class of amphipathic amines such as U18666A [35] and chlorpromazine [36,37] Chlorpromazine (CPZ), as well as an inhibitor of endocytosis, cytochalasin D (Cyto D), completely pre-vented CS-induced inhibition of LT synthesis in PMNL (Fig 5)
Inhibitory effect of CS in a 5-LO cell-free assay
We investigated the influence of CS on 5-LO activity under cell-free conditions 5-LO isolated from PMNL was dissolved in PGC buffer containing AA and differ-ent concdiffer-entrations of CS CS in a concdiffer-entration range
of 10–100 lgÆmL)1 inhibited the production of LTs and 5-HETE (Fig 6)
Fig 2 Cumulative effect of CS and
choles-terol (chol) in joined incubations, without
(above) or with 20 l M AA (below) PMNLs
were preincubated for 30 min at 37 C with
the indicated lipids, with or without AA, and
then stimulated for 10 min with 2 l M
cal-cium ionophore A23187 MbCD was added
at 0.5 m M *P < 0.05; **P < 0.01, when
data are compared with the control when
no lipid added (*)P < 0.05, when data are
compared with corresponding control at the
fixed concentration of CS.
Fig 3 CS inhibits 5-LO product synthesis in MM6 and PMNL in a
concentration-dependent manner MM6 and PMNL were
preincu-bated for 30 min at 37 C with CS at the indicated concentrations
with 20 l M AA, and then stimulated for 10 min with 1 l M A23187
calcium ionophore The data presented as the sum of LTs and
5-HETE.
Trang 5CS suppresses 5-LO binding to the nuclear
membrane in PMNL
The influence of CS on intracellular 5-LO localization
in human PMNL was investigated after cell
stimula-tion with calcium ionophore A23187 Cells were
preincubated in PGC buffer with 100 lgÆmL)1 of CS for 15 min at 37C Ionophore was then added and the cells were incubated for an additional 10 min The cells were then centrifuged and lyzed Membrane and cytosolic fractions were separated and analyzed by SDS⁄ PAGE and western blotting technique The results are presented in Fig 7 In intact cells, 5-LO was mostly located in the cytosolic fraction Calcium ionophore induced membrane association of 5-LO CS prevented the translocation and binding of 5-LO to the membranes to a significant extent (Fig 7)
Discussion
We reveal a novel role of CS as a regulatory molecule
in the production of LTs We propose that CS affects
LT production by several mechanisms The liberation
of free arachidonic acid is often the initial, rate-limit-ing step in the biosynthesis of eicosanoids Cytosolic PLA2 (cPLA2), like 5-LO, is a C2 domain-containing
A
B
Fig 4 CS suppresses AA release (A) and the 5-LO product
forma-tion (B) in PMNL in a concentraforma-tion dependent manner (A)
Thirty-minute incubation of PMNL with CS decreases A23187-induced
release of 14 C-labelled 5-LO products and free AA This effect
occurs without (empty columns) and with exogenously added
20 l M AA (hatched columns) (B) The dependence of 5-LO product
synthesis on the concentration of CS Data are given in the
pres-ence (hatched columns) and in the abspres-ence (empty columns) of
20 l M AA in the medium.
Fig 5 The CS effect on LT synthesis is inhibited by cytochalasin D
and chlorpromazine The inhibitory influence of 25 lgÆmL)1CS on
LT formation in PMNL was abolished in the presence of 10 l M
cytochalasin D (Cyto D) and 30 l M chlorpromazine (CPZ).
Fig 6 CS suppresses 5-LO product synthesis in a cell-free assay 5-LO was isolated as described in the Experimental procedures and diluted into NaCl ⁄ P i ⁄ EDTA buffer CS was added for 15 min, and then LT synthesis was induced by addition of Ca2+at a final con-centration 2 m M
Fig 7 CS inhibits 5-LO translocation to membranes in human PMNL A, Ionophore A23187; CS, cholesterol sulfate, 100 lgÆmL)1;
M, membrane (nuclear) fraction; C, cytosolic fraction PMN were incubated and stimulated as described in Experimental procedures The result is representative for three experiments.
Trang 6CS affects LT production at the level of substrate
availability (Fig 4) We observed inhibition of the
A23187-induced 5-LO binding to the membrane
frac-tion in CS-treated cells (Fig 7), which can reduce
5-LO function due to lack of colocalization with the
substrate AA that is released from the membrane
These two effects of CS, on AA release and 5-LO
binding to the membrane, may be linked in light of
new published data [41], suggesting that AA can
pro-mote membrane association CS also inhibited LT
syn-thesis in a cell-free assay (Fig 6) This can arise from
a direct effect of CS on 5-LO The CS structure is
sim-ilar to that of tirucallic acid, for which direct binding
to the 5-LO protein was shown [42]
Cholesterol is especially abundant in the plasma
mem-brane of mammalian cells The importance of
maintain-ing the cholesterol–phospholipid (C⁄ PL) ratio is
illustrated by studies which show a marked alteration in
the activity of transmembrane proteins and cell function
following alterations in the C⁄ PL ratio [43] The effects
of cholesterol on membrane and cell function are
pre-sumably related to its ability to modulate the
biophysi-cal properties of membranes Insertion of cholesterol
derivatives would therefore strikingly influence the
membrane properties and exert complex effects on
several parameters that influence LT biosynthesis: AA
release, 5-LO translocation and 5-LO enzyme activity
Recent publications investigated the regulation of
5-LO activity by lipids and it was found that
structur-ally diverse lipids showed a similar behavior: both the
acidic lipid cardiolipin and the zwitterionic
1-palmi-toyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
faci-litated 5-LO activation in contrast to PIP and
ceramide [19] We tested the effect of modification of
the polar hydroxy group of cholesterol on cellular
5-LO activity Cholesterol acetate was the most
favora-ble substitute Increasing or decreasing free cholesterol
was found to affect 5-LO activity, with clear inhibition
at high cholesterol concentrations The anionic
deriva-tives CS and CP inhibited LT synthesis
ER membranes have a low C⁄ PL ratio in
compar-ison with plasma membranes and are consequently the
most ‘fluid’ membranes in the cell Cholesterol
addi-tion is known to decrease membrane fluidity Inclusion
of cholesterol reduces the permeability of membranes,
attraction [45] Incorporation of CS stabilized mem-brane vesicles and decreased the elasticity of the lipid bilayer [46] Our results on the inhibition of cellular 5-LO by cholesterol and its derivatives fit well with the recently published concept that membrane fluidity is a key modulator of the activity of 5-lipoxygenase [20] Chlorpromazine known to decrease ER cholesterol content abolished the effect of CS in our assay Cholesterol sulfate has been known to be a normal constituent of blood platelets and to modulate platelet function [47] The sulfotransferase SULT2B1b is selec-tively expressed in human platelets and 566 pmol of
CS can be found per 109untreated platelets [48] Addi-tion of the sulfate donor 3¢-phosphoadenosine-5-phos-phosulfate (PAPS) induced a 300-fold increase in platelet CS content HDL and specifically apoA-I sta-bilized and maintained the level of platelet SULT2B1b mRNA [48] We speculate that platelets might present their CS to PMNL, and inhibition of LT synthesis may be a consequence of such a lipid exchange Physiological concentrations of platelets inhibited LT biosynthesis in human neutrophils in an adhesion-dependent manner [49,50] After degranulation by thrombin under nonaggregating conditions, platelets lost their inhibitory activity [50] CS might be a medi-ator of PMNL–platelet interaction and platelet-medi-ated inhibition of leukotriene synthesis in PMNL
It is a published report that a cytoprotective effect
of CS exists in gastric ulcer models [26] Considering the role of PMNL in gastric inflammation and gastric epithelial damage, one might propose that inhibition
of LT synthesis by CS contributes to the suppressive effect of CS in gastric inflammation
Regarding the role of CS in inflammation and athero-genesis, other activities of CS should also be considered Key events in atherogenesis are increased ROS genera-tion and lipid peroxidagenera-tion Thus, the role of CS in ROS production should be elucidated
Altogether, our findings show that CS is a potent inhibitor of LT production and that different mecha-nisms are involved in the inhibitory process These observations provide us with new insights and approa-ches to modify inflammatory processes by means of cholesterol amount and its conversion to anionic deriv-atives
Trang 7Experimental procedures
Materials
RPMI-1640 medium was from Gibco (Grand Island, NY,
USA), and fetal bovine serum (FBS) was obtained from
Boehringer Mannheim (Mannheim, Germany) CP was
kindly provided by E Volkov (Moscow State University,
Belozersky Institute of Physicochemical Biology) Insulin
was a gift from Aventis (Frankfurt, Germany) Human
transforming growth factor-b1 (TGF-b1) was purified from
outdated platelets as described [51] Calcitriol was kindly
provided by H Wiesinger (Schering AG, Berlin, Germany)
5-lipoxygenase polyclonal antiserum was kindly provided
by O Radmark (Karolinska Institute, Stockholm, Sweden)
CS, CA, calcium ionophore A23187, sucrose, and AA were
from Sigma Chemical Co (Deisenhofen, Germany)
High-pressure liquid chromatography (HPLC) solvents were from
Merck (Darmstadt, Germany)
Cells
MM6 cells were maintained in RPMI 1640 medium with
glutamine supplemented with 10% fetal calf serum,
100 lgÆmL)1 streptomycin, 100 UÆmL)1 penicillin, 1 mm
sodium pyruvate, nonessential amino acids, 1 mm
oxalace-tic acid, and 10 lgÆmL)1 bovine insulin All cultures were
seeded at a density of 2· 105
cellsÆmL)1 MM6 cells were treated with 2 ngÆmL)1 TGF-b1 and 50 nm vitamin D3
(VD3) for 4 days Cells were harvested by centrifugation
(200 g for 10 min at room temperature) and washed once
in NaCl⁄ Pi, pH 7.4
Human PMNL were isolated from freshly drawn
citrate-anticoagulated donor blood Leukocyte-rich plasma was
prepared by Dextran sedimentation Granulocytes were
obtained from leukocyte-rich plasma by centrifugation on
Ficoll–Paque and hypotonic lysis of erythrocytes PMNL
were resuspended (5· 106
cellsÆmL)1; purity > 96–97%) in phosphate-buffered saline (NaCl⁄ Pi) plus 1 mgÆmL)1
glu-cose (PG buffer) or alternatively, in PG and 1 mm CaCl2
(PGC buffer) as indicated For incubations, cells were
finally resuspended in PGC buffer
The viability of cells was tested by trypan blue exclusion,
and more than 95% of cells were viable up to a
concentra-tion of 100 lgÆmL)1 CS and 40 lgÆmL)1 CP; more than
90% were viable at a concentration of 200 lgÆmL)1CS
5-LO isolation and cell-free assay
PMNL (20–80· 106) were suspended in 1 mL of buffer 1
[50 mm KH2PO4, 0.1 m NaCl, 2 mm EDTA, 1 mm
dithio-threitol, 0.5 mm phenylmethylsulfonyl fluoride, and
60 lgÆmL)1 soybean trypsin inhibitor (STI), pH 7.1] and
sonicated on ice for three times 10 s Cell sonicates were
centrifuged at 10 000 g for 10 min at 4C to remove nuclei
and unbroken cells The supernatant was removed and cen-trifuged again at 100 000 g for 60 min at 4C The pellet was rinsed with 1 mL of buffer 2 (20 mm KH2PO4, 2 mm EDTA, 1 mm dithiothreitol, pH 7.1) to remove residual cytosol The washed pellet (membrane fraction) was then resuspended in 1 mL of buffer 1 by sonication on ice twice for 10 s containing 0.2 m Tris⁄ HCl (pH 7.5), 1.6 mm EDTA, 1.8 mm ATP, aliquot of cytosol and respective con-centrations of CS These mixtures were preincubated at
37C for 15 min, and the reaction was initiated by the addition of CaCl2(3 mm in the media) and AA (20 lm in the media) After 15 min, the reaction was stopped by addi-tion of 1 mL methanol 30 lL 1m HCl and 200 ng of pros-taglandin B1 (PGB1) were added This mixture was prepared and analyzed by HPLC as further described
Determination of 5-LO product formation in cells PMNL (10· 106
) and MM6 cells (3· 106
) were finally resuspended in 1 mL (MM6) or 6 mL (PMNL) of PGC buffer and preincubated at 37C with indicated additives
as described The lipids were added as ethanol solutions (cholesterol, CA and CP) or ethanol⁄ dimethyl sulfoxide (1 : 3) for CS After 30 min at 37C the reaction was star-ted by the addition of 2 lm ionophore A23187 After
10 min, the reaction was stopped with an equal volume of methanol with 200 ng PGB2 as internal standard for PMNL or 200 ng PGB1as internal standard for MM6 To water⁄ methanol extracts were added 30 lL 1 m HCl and
500 lL NaCl⁄ Piper 1 mL incubation After centrifugation (10 min, 800 g), the samples were applied to C-18 solid-phase extraction columns, which were conditioned first with methanol, then with water The columns were washed with water, 25% methanol; 5-LO metabolites were extracted with methanol and were analyzed by HPLC as described [52] using UV light detection at 235 and 280 nm The respective molar absorptions were used for calculation Cysteinyl LTs (LTC4, D4, and E4) were not determined
AA-release assay Labeled PMNL were prepared by incubating cells for 1.5 h
at 1· 107⁄ mL in 45 mL PGC buffer with 5.0 lCi of 14
C-AA At the end of the incubation, 5 mL of 1% bovine serum albumin in Dulbecco’s NaCl⁄ Pi was added for next 0.5 h, then the cells were centrifuged and resuspended at
107⁄ mL in Hank’s balanced salt solution (HBSS) ⁄ 10 mm Hepes Medium with or without cholesterol sulfate was equilibrated at 37C, the cells were added at 2 · 106⁄ mL and incubated for 30 min at 37C Then cells were stimula-ted with 2 lm A23187 for 10 min The incubations were stopped by addition of an equal volume of methanol at )20 C, with prostaglandin B2 as an internal standard The denatured cell suspensions were centrifuged and the water⁄ methanol extracts were removed in the supernatants
Trang 8Subcellular fractionation by detergent lysis
Isolated human PMNL (3· 107) were resuspended in
1 mL PGC buffer The cells were preincubated in the
presence or absence of additives at 37C before the
addi-tion of ionophore A23187 at the indicated concentraaddi-tions
After another 5-min incubation period at 37C, the
samples were dulled on ice for 3–5 min and centrifuged
(200 g, 5 min, 4C) Pellets were then suspended in
500 lL, ice-cold NP-40 lysis buffer (10 mm Tris⁄ HCl,
pH 7.4; 10 mm NaCl; 3 mm MgCl2; 1 mm EDTA; 0.1%
NP-40; 1 mm phenylmethylsulfonyl fluoride; 60 lgÆmL)1
soybean trypsin inhibitor (STI), and 10 lgÆmL)1
leupep-tin), vortexed (3· 6 s), kept on ice for 10 min, and
centrifuged (800 g, 10 min, 4C) Resultant supernatants
(non-nuclear fractions) were transferred to a new tube,
and the pellets (nuclear fractions) were resuspended in
500 lL ice-cold NP-40 lysis buffer Nuclei were disrupted
by sonication (3· 6 s) Aliquots of nuclear and
non-nuclear fractions were immediately mixed with the
load-ing buffer, heated for 6 min at 95C, and analyzed for
5-LO protein by SDS⁄ PAGE and western blotting
Immunoblot analysis of subcellular fractions
Twenty-five microliters of each fraction were mixed with
4 lL glycerol⁄ 0.1% bromophenol blue (1 : 1, v ⁄ v) and
ana-lyzed by SDS⁄ PAGE on 10% gel After electroblotting,
nitrocellulose membranes were blocked with 5% nonfat dry
milk in 50 mm Tris-HCI, pH 7.4 and 100 mm NaCl (TBS)
for 1 h at room temperature Membranes were washed and
incubated with anti-5-LO antiserum overnight at 4C
Then, membranes were washed with TBS and incubated
with a 1 : 1000 dilution of alkaline phosphatase-conjugated
anti-rabbit IgG for 2 h at room temperature After washing
with TBS, and TBS with 0.1% NP40, 5-LO protein was
visualized with the alkaline phosphatase substrates
nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate
in detection buffer (100 mm Tris-HCI, pH 9.5, 100 mm
NaCl, 5 mm MgCl2)
Statistics
Results are given as mean ± sd from at least three
inde-pendent experiments Statistical evaluation of the data was
performed by one-way anova followed by Dunnett’s
study was supported by the grant from FEBS Fellow-ship Program given to D Aleksandrov for his work in the Institute of Pharmaceutical Chemistry, Frankfurt-am-Main, Germany
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