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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 64-74
Membrane Translocation of 15-Lipoxygenase in Hematopoietic
Cells Is Calcium-Dependent and Activates the Oxygenase Activity of
the Enzyme
By
Roland Brinckmann,
Kerstin Schnurr,
Dagmar Heydeck,
Thomas Rosenbach,
Gerhard Kolde, and
Hartmut Kühn
From the Institute of Biochemistry Pathology and Dermatological
Clinic, University Clinics Charité, Humboldt University, Berlin,
Germany.
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ABSTRACT |
Mammalian 15-lipoxygenases, which have been implicated in the
differentiation of hematopoietic cells are commonly regarded as
cytosolic enzymes. Studying the interaction of the purified rabbit
reticulocyte 15-lipoxygenase with various types of biomembranes, we
found that the enzyme binds to biomembranes when calcium is present in
the incubation mixture. Under these conditions, an oxidation of the
membrane lipids was observed. The membrane binding was reversible and
led to an increase in the fatty acid oxygenase activity of the enzyme.
To find out whether such a membrane binding also occurs in vivo, we
investigated the intracellular localization of the enzyme in stimulated
and resting hematopoietic cells by immunoelectron microscopy, cell
fractionation studies and activity assays. In rabbit reticulocytes, the
15-lipoxygenase was localized in the cytosol, but also bound to
intracellular membranes. This membrane binding was also reversible and
the detection of specific lipoxygenase products in the membrane lipids
indicated the in vivo activity of the enzyme on endogenous substrates.
Immunoelectron microscopy showed that in interleukin-4 -treated
monocytes, the 15-lipoxygenase was localized in the cytosol, but also
at the inner side of the plasma membrane and at the cytosolic side of
intracellular vesicles. Here again, cell fractionation studies
confirmed the in vivo membrane binding of the enzyme. In human
eosinophils, which constitutively express the 15-lipoxygenase, the
membrane bound share of the enzyme was augmented when the cells were
stimulated with calcium ionophore. Only under these conditions,
specific lipoxygenase products were detected in the membrane lipids.
These data suggest that in hematopoietic cells the cytosolic
15-lipoxygenase translocates reversibly to the cellular membranes. This
translocation, which increases the fatty acid oxygenase activity of the
enzyme, is calcium-dependent, but may not require a special docking
protein.
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INTRODUCTION |
LIPOXYGENASES (LOX) are lipid
peroxidizing enzymes that are categorized according to their positional
specificity of arachidonic acid oxygenation.1 5-LOXs are
involved in the biosynthesis of leukotrienes that constitute important
mediators of anaphylactic and inflammatory diseases.1 In
contrast to 5-LOXs, which strongly prefer free arachidonic acid as
substrate, mammalian 15-LOXs are capable of oxygenating not only free,
but also esterified polyenoic fatty acids,2-4 and even more
complex lipid protein assemblies such as biomembranes2,4,5
and lipoproteins.6 The enzyme is constitutively expressed
in reticulocytes of various species,7,8 in airway
epithelial cells, and in eosinophilic granulocytes.8 Human
peripheral monocytes do not express the 15-LOX, but when the cells are
cultured in the presence of interleukin-4 (IL-4) or IL-13, the
synthesis of the enzyme is induced.9,10 Although the
biological role of 15-LOXs is still under discussion, there is a
substantial body of experimental evidence for its implication in cell
development and differentiation.11,12 In rabbit
reticulocytes the expression of the enzyme parallels the maturational
decline of the cytochrome C oxidase, a marker enzyme of mitochondrial
respiration.12 At this stage of red blood cell
development, specific LOX products can be detected in the lipid
extracts of mitochondrial membranes and, to a lesser extent, in the
extracts of the plasma membranes indicating the in vivo activity of the
enzyme.13 Because the expression of the 15-LOX parallels
the inactivation of respiratory enzymes and the loss of cell membrane
receptors during the time course of reticulocyte maturation, an
involvement of the enzyme in the programmed breakdown of mitochondria
and in the remodeling process of the plasma membrane was
suggested.11,12 This hypothesis was later supported by
experiments on the in vitro maturation of rabbit reticulocytes in the
presence of 15-LOX inhibitors.12 A similar role of the
enzyme in remodeling of intracellular structures may be assumed for
monocyte/macrophage transition.9,11
For the 5-LOX of polymorphonuclear leukocytes and of alveolar
macrophages, which is involved in the synthesis of mediators of
inflammatory reactions,14,15 it has been shown that the
cytosolic enzyme binds to the cellular membranes when the cells were
stimulated with calcium ionophore16,17 suggesting a
calcium-dependent membrane association. Later on it was shown that the
enzyme translocates preferentially to the nuclear
envelope18,19 where it interacts with a special docking
protein, the five lipoxygenase activating protein (FLAP), which appears
to enhance the availability of the substrate fatty acid.20
In rat basophilic leukemia cells21,22 and alveolar
macrophages,23,24 the 5-LOX was localized inside the
nucleus and may also translocate to the nuclear envelope on cell
stimulation.24 In contrast to the well investigated
intracellular localization of the 5-LOX,16-24 little is
known about the subcellular distribution of the 15-LOX. The enzyme is
commonly referred to as cytosolic protein because it can be purified
from the cytosol of various mammalian cells7 and because
there is no obvious membrane binding sequence in its primary structure.
On the other hand, its capability of directly oxygenating
biomembranes2-5 and the recent finding that in more mature
reticulocytes the 15-LOX was predominantly detected in the stroma
fraction25 suggested a maturation-dependent membrane
association of the enzyme.
The lack of information on the subcellular localization of the 15-LOX
in mammalian cells prompted us to investigate the membrane association
behavior of this enzyme in reconstituted in vitro systems and in intact
cells. We found that in various hematopoietic cells the 15-LOX is
localized in the cytosol, but also bound to cellular membranes.
Membrane binding of the enzyme is calcium-dependent and may not require
a special docking protein.
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MATERIALS AND METHODS |
Chemicals.
The chemicals used were from the following sources: linoleic acid
(9Z,12Z-octadecadienoic acid) from Serva (Heidelberg,
Germany); human recombinant IL-4 from UBI (Lake Placid,
NY); phosphate-buffered saline (PBS) from Sigma (Deisenhofen,
Germany); immunochemicals from Dianova (Hamburg,
Germany); Percoll from Pharmacia (Freiburg,
Germany); Dextran 200 from Serva; authentic standards of
hydroxy fatty acids from Cayman Chem (Alexis Deutschland
GmbH, Grünberg, Germany); 3,3'-diaminobenzidine
tetrahydrochloride from Sigma (St Louis, MO); osmic acid
from Paesel (Frankfurt am, Germany); araldite from Serva;
uranyl acetate and lead citrate from Sigma. For the immunostainings, a
polyclonal antirabbit 15-LOX antibody (fast protein liquid
chromatography preparation of the IgG fraction), which was raised in
guinea pigs, was used. This antibody strongly cross-reacted with the
human 15-LOX and with the porcine leukocyte 12-LOX, but not with the
human platelet 12-LOX and with the human 5-LOX.
Preparations and incubations.
The 15-LOX was purified to apparent electrophoretic homogeneity from a
reticulocyte rich blood cell suspension obtained from anemic rabbits by
a three-step purification procedure, which comprised fractionated
ammonium sulfate precipitation, hydrophobic interaction chromatography,
and anion exchange chromatography.6 Submitochondrial
particles (SMP), which constitute vesicles of mitochondrial membranes
were prepared from beef heart homogenates.26
For quantification of membrane binding, the pure 15-LOX was incubated
for 5 minutes at room temperature with EDTA washed SMP or with EDTA
washed erythrocyte ghosts in the presence or absence of 0.5 mmol/L
calcium. The membrane fraction was sedimented by ultracentrifugation
(100,000g), the supernatant was removed and the pellet was
resuspended in water (original volume). The resuspended membranes and
the supernatants were dialyzed overnight against water and the samples
were dried down in the Speedvac centrifuge. The protein residues were
redissolved in electrophoresis sample buffer (60 mmol/L Tris-HCl, pH
6.8, 2% sodium dodecyl sulfate [SDS], 10% glycerol, 5%
mercaptoethanol) and aliquots were applied to SDS electrophoresis.
After semidry blotting, the blots were probed with the anti-15-LOX
antibody.
Cell culture.
Reticulocytosis was induced in rabbits by daily removal of about 30 to
50 mL of blood for a period of 5 days. The hematocrit was checked every
day and if it dropped below 20%, bleeding was skipped that day. When
the reticulocyte content reached values between 20% to 25% (sixth or
seventh day of bleeding), blood was collected and the red blood cells
were prepared for immunoelectron microscopy and for the cell
fractionation studies. The cellular 15-LOX activity was checked by
incubating 0.1 mL of packed cells with 0.1 mmol/L arachidonic acid at
37°C for 15 minutes. After lipid extraction the 15-LOX products
(15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid [15-HETE]) were
quantified by reverse phase high performance liquid chromatography
(RP-HPLC) (see below).
Human peripheral monocytes were prepared from buffy coats obtained from
the local blood bank by density gradient centrifugation and adherence
to plastic dishes9 or by elutriation using a Beckman
elutriation system (Munich, Germany). The cells were
cultured in RPMI 1640 medium containing 10% fetal calf serum in the
absence or presence of 670 pmol/L IL-4. After 3 to 4 days in culture,
the cells were scraped off, washed twice with isotonic saline, and
prepared for immunoelectron microscopy.
Human eosinophils were purified from the blood of patients with a high
degree of eosinophilia (more than 10% eosinophilic granulocytes in the
differential blood counts). A total of 10 to 40 mL of heparinized blood
was withdrawn and the erythrocytes were sedimented through an isotonic
dextrane 200 solution. Afterwards the eosinophils were prepared by
centrifugation on a discontinuous Percoll density
gradient.27 The final cell preparations contained more than
90% eosinophils as indicated by Giemsa staining.
Immunoelectron microscopy.
The subcellular localization of the 15-lipoxygenase in vivo was
visualized by preembedding immunoperoxidase staining or by
postembedding immunogold labeling. For preembedding stainings, the
cells were fixed with Nakane's fixative for 10 minutes at room
temperature. After washing in phosphate-buffered saline (PBS), the
cells were incubated with the anti-15-LOX antibody for 2 hours at
4°C and subsequently with a peroxidase conjugated mouse antiguinea
pig IgG antibody (2 hours at 4°C). Positive reactions were
visualized with 3,3 -diaminobenzidine tetrahydrochloride
reaction. After immunostaining, the cells were postfixed in 1.3% osmic
acid (dissolved in PBS) for 2 hours at room temperature, dehydrated in
graded ethanol series, and embedded in Araldite. Ultrathin sections
were stained with uranyl acetate/lead citrate. Specificity of the
staining was checked using a nonimmune rabbit IgG preparation instead
of the anti-15-LOX antibody.
For immunogold stainings, the postembedding technique was used. For
this purpose, rabbit reticulocytes and human eosinophils were fixed in
Nakane's fixative for 10 minutes at room temperature. After washing in
PBS, the cells were dehydrated in graded ethanol series and afterwards
embedded in LR White. Ultrathin sections were mounted on
nickel grids and were incubated for 2 hours with the anti-15-LOX
antibody at 20°C. After washing, the sections were incubated with a
gold (particle diameter 20 µm) conjugated mouse antiguinea pig IgG
antibody. The sections were then dried and stained with uranyl
acetate/lead citrate. Here again, specificity of the staining was
checked using a nonimmune rabbit IgG preparation instead of the
anti-15-LOX antibody. Examination of the sections stained with both
the pre- and post-embedding method was performed with a Zeiss EM9S or a
EM412 electron microscope (Oberkochen, Germany).
Subcellular fractionation and immunoblot analysis.
Reticulocyte-rich blood was obtained from rabbits as described above.
The cells were spun down and the buffy coat was removed. After washing
twice in 5 vol of PBS, the cells were lysed by suspending them in 10
vol of ice-cold water containing dithioerythreitole (1 mmol/L),
phenylmethylsulfonyl fluoride (32 mg/L), and variable concentrations of
calcium chloride. To achieve complete cell disruption, the suspension
was sonicated with a microtip sonifier (Braun, Melsungen, Germany) for
15 seconds at 40 W. The homogenate was divided into two portions. Both
were centrifuged for 1 hour at 100,000g. The supernatant and
the pellet of the first portion were immediately prepared for
immunoblot analysis. The pellet of the second portion was resuspended
in the lysis buffer containing the appropriate calcium concentration
and the suspension was centrifuged again for 1 hour at
100,000g. Afterwards the pellet (washed membrane pellet) and
the supernatant (washing supernatant) of the second centrifugation were
prepared for immunoblot analysis. For SDS-electrophoresis, the membrane
pellets (unwashed and washed pellets) were dissolved in electrophoresis
sample buffer (60 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5%
mercaptoethanol) and boiled for 2 minutes. The supernatants were dried
down in a Speedvac centrifuge, the remaining proteins were dissolved in
the electrophoresis sample buffer, the samples were boiled for 2
minutes, and applied to SDS-polyacrylamide gel electrophoresis (PAGE).
Human peripheral monocytes cultured for 3 to 4 days in the presence of
670 pmol/L IL-4 were scraped off the plastic dishes and washed twice
with PBS. Cell fractionation and preparation of the samples for
electrophoresis was performed as described for the reticulocytes.
After SDS-PAGE, the proteins were transferred to a Immobilon P membrane
by a semidry blotting procedure and the blot was incubated with the
anti-15-LOX antibody. After extensive washing, the membrane was
incubated with a peroxidase-labeled mouse antiguinea pig IgG antibody
and the blot was stained with 3,3-diaminobenzidine. As a reference
compound, the pure rabbit reticulocyte enzyme was used.
Lipoxygenase activity assays.
Linoleate oxygenase activity was measured recording the increase in
absorbance at 235 nm. The assay mixture consisted of a 0.1 mol/L sodium
phosphate buffer, pH 7.4 containing 0.2% sodium cholate and 0.250
mmol/L linoleic acid as substrate. Membrane oxygenase activity of the
15-LOX was assayed oxygraphically using a Gilson oxygraph
(Middelton, WI).
Quantification and structural elucidation of the oxygenated polyenoic
fatty acids formed during the in vitro and in vivo interaction of the
15-LOX with biomembranes was performed by three sequential steps of
HPLC on various types of columns. For the in vitro interaction of the
15-LOX with biomembranes, the following protocol was applied. After the
incubation period, the hydroperoxy lipids formed were reduced to the
corresponding hydroxy derivatives by the addition of sodium borohydride
(Serva). The lipids were extracted,28 hydrolyzed under
alkaline conditions, and the resulting free fatty acid derivatives were
analyzed by RP-HPLC on a Nucleosil C-18 column (Macherey/Nagel,
KS-system, 250 × 4 mm, 5 µm particle size) with a solvent
system of methanol/water/acetic acid (85/15/0.1; by vol) and a flow
rate of 1 mL/minute. The absorbances at 235 nm (detection of oxygenated
fatty acids) and at 210 nm (detection of polyenoic fatty acids) were
recorded simultaneously. For further structure elucidation of the
oxygenated fatty acids, the compounds comigrating with an authentic
standard of 13S-hydroxy-9Z,11E-octadecadienoic acid (13S-HODE) were
prepared and further analyzed by straight phase (SP)-HPLC and chiral
phase (CP)-HPLC. SP-HPLC, which separates the positional isomers of
hydroxy fatty acids, was performed on a Zorbax SIL column (DuPont,
Wilmington, DE; 250 × 4.6 mm, 5 µm particle size)
with a solvent system of n-hexane/2-propanol/acetic acid (100/2/0.1, by
vol) and a flow rate of 1 mL/minute. The enantiomer composition of the
hydroxy fatty acids was analyzed by CP-HPLC using a Chiralcel OD column
(Z.T. Baker, Deventer, The Netherlands; 250 × 4.6 mm, 5 µm
particle size) with a solvent system of hexane/2-propanol/acetic acid
(100/5/0.1, by vol) and a flow rate of 1 mL/minute. To quantify the in
vivo action of the 15-LOX in rabbit reticulocytes and human
eosinophils, the total lipids of the membrane preparations were
extracted under reducing conditions (presence of triphenylphosphine)
and the extracts were subjected to alkaline hydrolysis. After
acidification to pH 3, the hydrolysis mixture was
injected to RP-HPLC (see above) and the hydroxy fatty
acid/polyenoic fatty acid ratio was calculated as suitable measure for
the degree of oxidation of the membrane lipids. For more detailed
information on the structure of the oxidized lipids, SP-HPLC and
CP-HPLC was also performed.
Calcium concentrations in the incubation mixtures were determined by
atomic absorption spectroscopy.
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RESULTS |
Membrane association of the 15-LOX in vitro.
It has been reported2-5 that mammalian 15-LOXs are capable
of oxidizing biomembranes in vitro and in vivo suggesting that the
enzyme is capable of binding to the membranes. To find out whether
calcium is of importance for this membrane binding, we incubated the
purified rabbit 15-LOX with submitochondrial membranes (SMP) and
erythrocyte ghosts for 5 minutes in the presence and absence of
exogenously added calcium. After incubation, the membranes were spun
down and aliquots of the 100,000g pellet and of the supernatant
were analyzed by immunoblotting. From Fig
1, it can be seen that in the presence of
0.5 mmol/L calcium the enzyme was detected mainly in the membrane
fraction. Only small amounts of the enzyme (5% to 15%, three
different sets of experiments) were recovered in the supernatant. In
the absence of exogenously added calcium, the majority of the enzyme
was detected in the 100,000g supernatant. Nevertheless, even
under these conditions, a considerable share of the enzyme remained
membrane bound. This result may be due to the fact that the SMP did
contain significant amounts of calcium, which cannot be removed
completely, even by washing with EDTA. In fact, when the sample to
which no exogenous calcium was added was checked for endogenous
calcium, a concentration of 10.4 µmol/L was measured. When the
binding assays were performed in the presence of 1 mmol/L EDTA, which
complexes the calcium ions, more than 95% of the enzyme was found in
the supernatant (not shown). These data suggest that membrane binding
in the absence of calcium is minimal and that calcium-dependent
membrane association is a graded phenomenon. From Fig
2, it can be seen that a gradual increase
in the calcium concentration was accompanied by an increase in the
share of membrane bound enzyme pool. It appears to be of
particular importance that at calcium concentrations already in the
lower µmol/L range (10.4 µmol/L), more than 30% of the enzyme was
bound to the membranes. Such calcium concentrations may be reached in
the cytosol of activated cells and, thus, membrane association of the
15-LOX may be an integral part of cell activation.
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| Fig 1.
Calcium-dependent membrane binding of the rabbit
reticulocyte lipoxygenase in vitro. The purified rabbit 15-LOX (30
nanokatals/mL) was incubated for 5 minutes with erythrocyte ghosts (2
mg/mL) and SMP (1 mg/mL) in the presence (0.5 mmol/L) and absence of
calcium chloride. Sample work-up, electrophoresis, and immunoblotting
are described in Materials and Methods. (A) Supernatant in the presence
of calcium, (B) membrane pellet in the presence of calcium, (C)
supernatant in the absence of calcium, (D) membrane pellet in the
absence of calcium.
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| Fig 2.
Membrane bound share of the 15-LOX depends on the calcium
concentration in the incubation mixture. The purified rabbit 15-LOX (30
nkat/mL) was incubated for 5 minutes with SMP (1 mg/mL) in the presence
of different calcium concentrations. The membranes were spun down and
the 15-LOX was determined in the supernatant by immunoblot analysis.
The blots were quantified with a Phoretix 1D Standard program. The LOX
content in the control incubation (no membranes) was set at 100%.
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Because membrane binding of the enzyme is a prerequisite for
oxygenation of the membrane lipids, the membrane oxygenase activity of
the 15-LOX in the presence and absence of calcium was assayed (Table
1). When submitochondrial particles were
incubated with the 15-LOX, an oxygen uptake of about 13 nmol/mg protein
was measured. When EDTA was present in the assay mixture, an almost
50% inhibition of the membrane oxygenase activity was observed. This
result may also be explained by the fact that small concentrations of
calcium were present in the assay buffer. Under the conditions of
maximal membrane binding (0.5 mmol/L calcium), the membrane oxygenase
activity was strongly stimulated. Similar results were obtained when
the formation of oxygenated fatty acids and the disappearance of
polyenoic fatty acids were assayed. Interestingly, when the lipids of
submitochondrial particles were extracted and used as 15-LOX substrate,
there was no stimulation of the oxygenase activity by calcium; even at
millimolar concentrations, we did not detect any increase in the oxygen
uptake and in the formation of specific LOX products (data not shown).
Summarizing these data one may conclude that membrane binding and
oxygenation of the membrane lipids by the purified 15-LOX requires
calcium and that membrane proteins and/or an intact membrane
structure are necessary for the stimulatory effects of calcium.
Besides activation of the membrane oxygenase activity, calcium-mediated
membrane binding of the 15-LOX leads to an activation of the free fatty
acid oxygenase activity of the enzyme. When the oxygenation of linoleic
acid was assayed, we observed a strong increase in the formation of
conjugated dienes (13-HPODE) when biomembranes and calcium were present
(Table 2). Under the conditions of maximal
membrane binding of the enzyme (0.5 mmol/L calcium in the incubation
mixture), its linoleic acid oxygenase activity was eightfold increased.
Because calcium alone (absence of biomembranes) only slightly augmented
the rate of linoleic acid oxygenation, the increase in the linoleic
acid oxygenase activity may be due to a calcium-mediated
membrane/enzyme interaction. Although the mechanism of this phenomenon
has not been investigated, it might be possible that membrane
association of the enzyme leads to a better availability of the free
fatty acid substrate.
Intracellular localization of 15-LOX in hematopoietic cells.
In rabbit reticulocytes the 15-LOX is expressed at a very high level.
It has been estimated that 1 mL of packed cells contain about 4 mg of
the enzyme.7 Recent cell fractionation studies suggested a
membrane association of the enzyme in mature
reticulocytes.25 We performed immunogold electron
microscopy studies of rabbit reticulocytes (Fig
3) and found that the enzyme is localized
in the cytosol and at the cytoplasmic side of intracellular membranes.
A strong 15-LOX labeling was consistently observed at the mitochondria
(arrow). Because mammalian reticulocytes do not contain a nucleous, we
could not check a hypothetical localization of the enzyme at the
chromatin or at the nuclear envelope.
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| Fig 3.
Immunogold labeling of the 15-lipoxygenase in rabbit
reticulocytes. Cells were prepared and stained as described in the
Materials and Methods section, 24,300-fold magnification. Inset,
staining with a nonimmuno-IgG preparation; 27,100-fold magnification.
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For independent evidence of the intracellular localization of the
15-LOX, cell fractionation studies and immunoblot analyses were
performed. When the cells were lysed at different calcium
concentrations and the membrane and cytosol fractions were separated by
centrifugation, we consistently found the enzyme in both cellular
compartments (Fig 4). If the cells were
lysed at a calcium concentration of 10 3 mol/L, an
almost equal distribution of the enzyme between the membranes and the
cytosol were observed (Fig 4, lanes A and B). At lower
calcium concentrations, the cytosolic shares were more pronounced (Fig
4, lanes E and F, I and K). To check the reversibility of
the membrane binding, the membrane pellet was washed at various calcium
concentrations. When washing was performed at 103
mol/L calcium, no enzyme could be removed from the membranes (Fig
4, lanes C and D). However, at lower calcium
concentrations (106 mol/L), small but detectable
amounts of the enzyme were washed away (Fig 4, lanes G
and H). Decreasing the calcium concentrations to a level which
represents the cytosolic calcium concentrations in most resting
mammalian cells (Fig 4, lanes L and M), a significant
share of the enzyme was removed from the membrane pellet.
It must be stressed that the buffer used for the fractionation studies
was not completely free of calcium; the endogenous calcium
concentration varied between 0.1 and 0.5 µmol/L in the different sets
of experiments. Thus, the real calcium concentration for this
experiment was in this range. These data suggest that membrane binding
of the 15-LOX is at least in part reversible and that the shares of
membrane bound and cytosolic 15-LOX may be variable depending on the
intracellular calcium concentration. Considering the fact that
calcium-mediated membrane binding of the 15-LOX increases both the
membrane oxygenase activity and the free fatty acid oxygenase activity
of the enzyme, the cytosolic calcium concentration may be regarded as
endogenous regulator of the intracellular 15-LOX activity.
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| Fig 4.
Cell fractionation studies of rabbit reticulocytes at
various calcium concentrations. Cell preparation, hemolysis, sample
work-up, and immunoblotting are described in the Materials and Methods
section. (A) Cytosol fraction; (B) membrane pellet; (C) supernatant of
washing (washed at 103 mol/L calcium); (D) washed
membrane pellet (washed at 103 mol/L calcium); (E)
cytosol fraction; (F) membrane pellet; (G) supernatant of washing
(washed at 106 mol/L calcium); (H) washed membrane
pellet (washed at 106 mol/L calcium); (I) cytosol; (K)
membrane pellet; (L) supernatant of washing (washed at 5 ×
108 mol/L calcium); (M) washed membrane pellet (washed
at 5 × 108 mol/L calcium).
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To find out whether the in vivo membrane binding of the 15-LOX is
associated with an intracellular action of the enzyme, in vivo activity
assays were performed. It has been reported13 that the
membranes of rabbit reticulocytes contain specific lipoxygenase
products indicating the in vivo activity of the enzyme. We confirmed
these data in the present study (Fig 5) and
calculated a hydroxy fatty acid/polyenoic fatty acid ratio of the
membrane lipids of 0.8% suggesting that in the membranes one of 100
polyenoic fatty acid residues was present as oxygenated derivative. In
rabbit erythrocytes, which lack a 15-LOX, this ratio was about 100-fold
lower (0.01%). Because an increase in the intracellular calcium
concentration was supposed to increase the share of membrane bound
enzyme, an augmentation of the oxygenated polyenoic fatty acids in the
membrane lipids was expected. To test this hypothesis, the following
experiment was performed. Young rabbit reticulocytes (sixth and seventh
day of a bleeding anemia) were prepared and divided into three
portions. One sample was hemolyzed with 2 vol of water and the
membranes were recovered by centrifugation. The other two samples were
incubated in PBS (containing calcium and magnesium) in the presence or
in the absence of 4 µmol/L calcium ionophore A23187. After 4 hours,
the cells were lysed and the membranes were prepared. After extraction
of the membrane lipids, transmethylation was performed and the
resulting fatty acid methyl esters were analyzed by RP-HPLC. From Table
3, it can be seen that incubation of the
cells in the absence of A23187 did not lead to an increase in the
hydroxy fatty acid content of the membrane. However, if ionophore was
present, a 100% increase in the content of lipoxygenase products was
observed. These data suggest that an increase in the cytosolic calcium
level stimulates the 15-LOX activity on the membrane ester lipids.
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| Fig 5.
15-LOX products occur in the membranes of rabbit
reticulocytes. Reticulocyte-rich blood cell suspensions were prepared
from a rabbit on the seventh day of the bleeding period. The cells were
lysed with two vol of distilled water, the membranes were spun down,
and the membrane lipids were extracted. After transmethylation with
sodium methoxide, the resulting fatty acid methylesters were analyzed
by RP-HPLC with a solvent system methanol/water/acetic acid (85/15/0.1,
by vol) and a flow rate of 1 mL/min. Upper trace: recording at 210 nm
(detection of polyenoic fatty acids), lower trace: recording at 235 nm
(detection of oxygenated polyenoic fatty acids. Each trace was
normalized with respect to the highest peak of the partial
chromatogram. Inset: ultraviolet (UV)-spectrum of the
products comigrating with an authentic standard of 13-HODE. In the
membranes of rabbit erythrocytes, which were analyzed as controls,
these products were not detected (data not shown). 13-HODE,
13-hydroxy-9Z,11E-octadecadienoic acid; LA, linoleic acid; AA,
arachidonic acid.
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Table 3.
Increased Intracellular Calcium Concentrations
Facilitate the Intracellular 15-LOX Activity With Endogenous
Substrates
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Human peripheral monocytes do not express the 15-LOX.9,10
However, if the cells are cultured in the presence of IL-4 or IL-13,
the enzyme is induced as indicated by immunohistochemical staining,
activity assays, and Western blot analysis.8,9 In our
hands, human monocytes cultured for 4 days in the presence of IL-4,
exhibited a linoleic acid oxygenase activity of 2.2 µg 13S-HODE
formation/106 cells. Immunohistochemical staining with an
anti-15-LOX antibody indicated the expression of the enzyme in
IL-4-treated cells and its absence in untreated monocytes (data not
shown). Interestingly, we consistently observed that only a
subpopulation (10% to 40%) of the monocytes was stained lipoxygenase
positive. The reasons for this inhomogeneity are unclear, but a
maturation dependence of the lipoxygenase expression may be assumed.
Ultrastructural examinations of peripheral monocytes kept in culture
for 4 days in the absence of IL-4 showed typical monocyte
characteristics including a round-shaped nucleus and an organized
cytoplasm containing small lysosomes (Fig
6A). As expected from earlier
studies,9 no specific staining with an anti-15-LOX
antibody was observed (not shown). In contrast, staining of
IL-4-treated monocytes with this antibody indicated a strong positive
reaction at the cytosolic side of the cell membrane and at the
membranes of intracellular vacuoles (Fig 6B). We did not detect major
amounts of the 15-LOX at the nuclear envelope or inside the nucleus.
Microscopic inspections of several monocyte preparations led to the
impression that there is a decreasing outside-inside gradient of 15-LOX
distribution in the cytoplasm of IL-4-treated monocytes. The highest
concentration of the enzyme was consistently observed just underneath
the plasma membrane, whereas the enzyme was virtually indetectable in
the surrounding nucleus.
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| Fig 6.
Intracellular distribution of the 15-lipoxygenase in
IL-4-treated monocytes. Cell preparation, immunoperoxidase staining,
and electron microscopy are described in Materials and Methods. (A)
Control monocytes (no IL-4 treatment) incubated with a nonimmune rabbit
IgG preparation; 10,500-fold magnification; (B) IL-4-treated monocyte,
9,200-fold magnification. Here the cells were stained with a polyclonal
anti-15-LOX antibody (IgG fraction). Arrows indicate the localization
of the enzyme at the membranes of subcellular vesicles. Arrowheads
indicate the localization at the cytosolic side of the plasma
membrane.
|
|
Cell fractionation studies at high calcium concentrations
(106 mol/L) indicated an approximate 1:1
distribution of the 15-LOX between the 100,000g supernatant and
the pellet (Fig 7, lanes A and B).
Quantification of the immunoblots showed that 42% of the enzyme was
localized in the cytosol, whereas 58% was found at the membranes.
Lysing the cells at lower calcium concentrations (5 ×
108 mol/L) led to an increase in the cytosolic share
of the 15-LOX (42% at 106 mol/L v 63% at 5
× 10-8 mol/L) and to a concomitant drop of the
membrane bound share (Fig 7, lanes E and F). Washing the
membrane pellets with the lysis buffer at both calcium concentrations
(Fig 7, lanes C and D, as well as G and H) removed the
enzyme partly from the membranes indicating the reversibility of the
membrane binding. These data suggest that modification of the cytosolic
calcium concentrations in the range between 106
mol/L and 5 × 108 mol/L may regulate the
relative shares of membrane bound and cytosolic 15-LOX. To test this
hypothesis in a whole cell system, we attempted to estimate the
membrane bound share of the 15-LOX in IL-4-treated monocytes that were
incubated for 5 minutes in the presence of the calcium ionophore A23187
by immunoelectron microscopy. Unfortunately, the cells became fragile
and did not survive the work-up procedure.
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| Fig 7.
Cell fractionation studies of IL-4-treated monocytes at
various calcium concentrations. Cell preparation, sample work-up, and
immunoblotting are described in Materials and Methods. Cell lysis was
performed at two calcium concentrations (106 mol/L for A
through D and 5 × 108 mol/L for E through H). After
lysis, the membranes were spun down at 100,000g. The membrane
pellet and the supernatant of the 100,000g centrifugation,
which was considered the cytosol, were prepared for electrophoresis as
described in the Materials and Methods section. (A) Membrane pellet;
(B) cytosol; (C) washing supernatant (washed at 106
mol/L calcium); (D) washed membrane pellet (washed at
106 mol/L calcium); (E) membrane pellet; (F) cytosol;
(G) washing supernatant (washed at 5 × 108 mol/L
calcium); (H) washed membrane pellet (washed at 5 ×
108 mol/L calcium).
|
|
As third cellular system, human eosinophils were used to study the
intracellular localization of the 15-LOX. From Fig
8A, it can be seen that the enzyme is
localized at the cytosolic site of the plasma membrane and in a rather
diffuse pattern in the cytoplasm. After stimulation of the cells with
calcium ionophore, an increased 15-LOX labeling was found (Fig 8B and
C). This increase, which was consistently observed in the eosinophils
of different preparations, may be explained by the fact that ionophore
leads to an enrichment of the enzyme at the intracellular membranes and
thus the antigen is picked up more efficiently by the antibody.
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| Fig 8.
Intracellular localization of the 15-LOX in human
eosinophilic granulocytes. Cell preparation and immunogold labeling was
performed as described in the Materials and Methods section. (A)
Unstimulated eosinophilic granulocyte (12,700-fold magnification); (B)
unstimulated eosinophilic granulocyte (30,400-fold magnification); (C)
eosinophilic granulocyte stimulated with 4 µmol/L calcium ionophore A
23187 (29,600-fold magnification).
|
|
If ionophore treatment increases the membrane bound share of the 15-LOX
in eosinophils, it should be possible to detect higher amounts of
specific LOX products in the membrane lipids. To check this assumption,
we extracted the membrane lipids of human eosinophils treated with and
without calcium ionophore, hydrolyzed the lipid extracts and analyzed
the resulting free fatty acid derivatives by HPLC. As shown in Fig
9, products which comigrate with authentic
standards of 15-HETE were detected in RP-HPLC in the membranes of
ionophore-treated cells. These products were characterized by a
conjugated diene chromophore with an absorbance maximum at 235 nm (Fig
9). In contrast, the hydrolyzed lipid extracts obtained from cells that
were not treated with ionophore did not contain oxygenated fatty acids.
Analysis of the enantiomer composition (Fig 9, inset)
showed as strong preponderance of 15S-HETE over its 15R-enantiomer
indicating that the products are formed via 15-LOX reaction and not by
nonenzymatic lipid peroxidation. These data are consistent with the
finding that calcium ionophore may increase the membrane bound share of
15-LOX in human eosinophils.
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| Fig 9.
HPLC analysis of oxygenated lipids present in the
cellular membranes of human eosinophilic granulocytes. After
preparation (see Materials and Methods), human eosinophils were
incubated for 10 minutes in the absence and presence of 4 µmol/L
calcium ionophore (extracellular calcium concentration of 2 mmol/L).
The cells were spun down, the membrane lipids were extracted, and the
extracts were hydrolyzed under alkaline conditions. HPLC analysis of
the resulting free fatty acid derivatives was performed as described in
the Materials and Methods section. Inset: enantiomer composition of
15-HETE isolated from the membrane lipids determined by CP-HPLC.
|
|
| |
DISCUSSION |
Mammalian 15-LOX, which can be prepared from natural and recombinant
sources, have been well characterized with respect to their protein
chemical, enzymatic,7,29-33 and molecular
biologic34,35 properties. In contrast, little is known
about the subcellular localization of the enzyme. 15-LOXs have been
regarded as cytosolic enzymes because they do not contain any obvious
membrane binding structural element and because they can be prepared
from the cytosol of rabbit reticulocytes7 and human
eosinophils.36 However, recent cell fractionation studies
and concomitant activity assays suggested that in mature rabbit
reticulocytes, the 15-LOX can also be detected in the membrane
fraction.25 The immunoelectron microscopical data presented
here indicate that in intact cells, too, a cytosolic and a membrane
bound pool of the 15-LOX can be differentiated. Moreover, we found a
similar distribution of the enzyme between the cytosol and the
membranes for other 15-LOX-expressing cells such as IL-4-treated
monocytes and human eosinophils.
Our data on the calcium-dependent membrane association of the 15-LOX in
the reconstituted in vitro system suggested that in intact cells the
enzyme may behave similarly. It is, however, very difficult to show
directly that the 15-LOX is redistributed from the cytosol to the
membranes in intact cells when the cytosolic calcium concentration is
increased. Neither the immunogold technique nor the immunoperoxidase
staining can be quantified exactly. However, there is indirect evidence
suggesting a membrane translocation of the enzyme. In freshly prepared
eosinophils, we did not detect any 15-LOX products in the membrane
lipids. However, when the cells were incubated with ionophore, such
products were found. Considering the fact that the enzyme itself is not
activated by calcium, one may conclude that the formation of these
products is due to an enhanced intracellular binding of the enzyme to
the membranes. A similar conclusion may be drawn from the data reported
in Table 3 for rabbit reticulocytes.
It is of particular importance for the regulation of the intracellular
15-LOX activity that membrane translocation of the enzyme activates its
fatty acid oxygenase activity. Thus, calcium-mediated membrane binding
may be regarded as an additional regulatory element of the
intracellular LOX activity. Other elements of regulation of cellular
15-LOX activity have also been reported. For instance, human peripheral
monocytes do not express the enzyme, but when cultured in the presence
of IL-4, large amounts of 15-LOX can be detected.9 However,
in these cells, the intracellular activity of the enzyme on endogenous
substrates, such as the membrane phospholipids, is rather
limited.9 Moreover, we found that in 15-LOX-transfected
human promonocytic cells (U937) and in a 15-LOX-transfected rabbit
smooth muscle cell line, no 15-LOX products can be detected in the
cellular membranes, suggesting that the enzyme, which is expressed in
large quantities, may not be active on endogenous ester lipid
substrates (unpublished data). Thus, one may conclude that the
intracellular activity of 15-LOXs does not necessarily parallel the
expression of the enzyme. Most probably, the expressed enzyme requires
additional intracellular activation. Although the in vivo activation
mechanisms are not clear and may differ from cell to cell,
calcium-dependent membrane association may be involved. As indicated in
our in vitro studies, membrane binding of the 15-LOX is of dual
consequences: (1) it enhances the specific activity with free fatty
acids as substrate and (2) it is a necessary precondition for the
membrane oxygenase activity. In mature reticulocytes in which the
intracellular calcium concentration is in the micromolar
range,37,38 a large share of the enzyme is membrane bound
and specific lipoxygenase products were detected in the membrane
lipids.
It should be stressed that membrane binding alone may not be sufficient
for the activation of the intracellular 15-LOX. It has been reported
before that the native ferrous 15-LOX is enzymatically silent and
requires oxidation to a ferric enzyme to become catalytically active.
In vitro studies on various 15-LOX indicated that trace amounts of
hydroperoxy lipids are sufficient to initiate the oxidation of the
enzyme to the activated ferric form.39,40 Thus, in addition
to the intracellular calcium concentrations, the cellular peroxide tone
may be regarded as modulator of the cellular 15-LOX
activity.11
For the human 5-LOX, it has been reported that the enzyme translocates
to the nuclear envelope18,19 when polymorphonuclear
leukocytes were stimulated with calcium ionophore A23187. There the
enzyme interacts with the five lipoxygenase activating protein and thus
becomes activated.20,41,42 In resting rat basophilic
leukemia cells and alveolar macrophages, the 5-LOX was detected in the
euchromatin region of the nucleus and on ionophore stimulation, it also
translocates to the nuclear envelope.21,22 Because of these
findings, we specifically examined the nucleus, and in particular, the
nuclear envelope of IL-4-treated monocytes for the occurrence of the
15-LOX, but were not able to detect the enzyme there. The same
conclusion was drawn when the electron micrographs of eosinophilic
granulocytes were evaluated. Even after ionophore stimulation, we did
not detect the enzyme at the nuclear envelope. In contrast, our
findings suggest a calcium-dependent translocation of the 15-LOX from
the cytosol to the cellular and to subcellular membranes. According to
our knowledge, the nuclear envelope constitutes a major side of the
synthesis of eicosanoids involved in allergy and inflammatory
reactions. Because the 15-LOX is not present at the nuclear envelope,
it may not be involved in the formation of inflammatory mediators.
As mentioned above, translocation of the 5-LOX to the nuclear envelope
requires the special anchoring protein, five lipoxygenase activating
protein (FLAP). In contrast, we suppose that such a docking protein may
not be implicated in the membrane binding of the 15-LOX because of two
experimental findings: (1) In vitro, the 15-LOX binds to various kinds
of biomembranes (rat liver and beef heart mitochondrial membranes, rat
liver endoplasmic membranes, human inside-out and right side-out
erythrocyte ghosts). If a special docking protein were involved in
membrane binding of the enzyme, it would have to be present in all
types of biomembranes. This, however, appears to be unlikely. (2) To
test a specific 15-LOX/docking protein interaction, we performed
cross-linking studies. After loading SMPs with 15-LOX in the presence
of 1 mmol/L calcium, we attempted to cross-link the 15-LOX with a
hypothetical docking protein. However, in immunoblots, we were unable
to detect a distinct high molecular weight band that cross-reacted with
the anti-15-LOX antibody.
The molecular mechanism of the translocation process of the 15-LOX to
biomembranes remains to be investigated. This study suggests that
membrane binding is reversible and requires calcium. Preliminary
calcium binding studies on the purified 15-LOX indicated that the
enzyme is capable of binding calcium (unpublished data). However,
databank searches in which the amino acid sequence of the 15-LOX was
compared with the sequences of calcium binding proteins did not show
typical calcium binding motifs in the primary structure of the 15-LOX.
In particular, we found that 15-LOXs lack any obvious calcium-binding
domain.43,44 On the other hand, computer-assisted modeling
of the three-dimensional structure of the rabbit 15-LOX, which was
based on the x-ray coordinates of the soybean enzyme,45,46
showed several putative calcium binding structures at the surface of
the enzyme. Whether these structures are required for
calcium-associated membrane binding is currently under investigation.
Because a specific docking protein appears not to be involved in
membrane binding of the 15-LOX, calcium ions may bridge unspecifically
between negatively charged residues (lipids or proteins) of the
membrane and acidic amino acid residues at the surface of the LOX
protein.
| |
FOOTNOTES |
Submitted April 9, 1997;
accepted August 26, 1997.
Supported in part by research grants from Deutsche
Forschungsgemeinschaft Ku 961/1-2 (Bonn, Germany).
Address reprint requests to Hartmut Kühn, MD, OSci,
Institute for Biochemistry, University Clinics (Charité),
Humboldt University, Hessische Str. 3-4, 10115 Berlin,
Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
REFERENCES |
1.
Yamamoto S,
Smith WL:
Molecular biology of the arachidonic acid cascade.
J Lipid Med Cell Signal
12:195,
1995[Medline]
[Order article via Infotrieve]
2.
Schewe T,
Halangk W,
Hiebsch C,
Rapoport SM:
A lipoxygenase in rabbit reticulocytes which attacks mitochondrial membranes.
FEBS Lett
60:149,
1975[Medline]
[Order article via Infotrieve]
3.
Jung G,
Yang DC,
Nakao A:
Oxygenation of phosphatidylcholine by human polymorphonuclear leukocyte.
Biochem Biophys Res Commun
130:559,
1985[Medline]
[Order article via Infotrieve]
4.
Murray JJ,
Brash AR:
Rabbit reticulocyte lipoxygenase catalyzes specific 12(S) and 15(S) oxygenation of arachidonyl-phosphatidylcholine.
Arch Biochem Biophys
265:514,
1988[Medline]
[Order article via Infotrieve]
5.
Kühn H,
Belkner J,
Wiesner R,
Brash AR:
Oxygenation of biological membranes by the pure reticulocyte lipoxygenase.
J Biol Chem
265:18351,
1990[Abstract/Free Full Text]
6.
Belkner J,
Wiesner R,
Rathmann J,
Barnett J,
Sigal E,
Kühn H:
Oxygenation of lipoproteins by mammalian lipoxygenases.
Eur J Biochem
213:251,
1993[Medline]
[Order article via Infotrieve]
7.
Rapoport SM,
Schewe T,
Wiesner R,
Halangk W,
Ludwig P,
Janicke-Höhne M,
Tannert C,
Hiebsch C,
Klatt D:
The lipoxygenase of reticulocytes. Purification, characterization and biologial dynamics of the lipoxygenase, its identity with the respiratory inhibitors of reticulocytes.
Eur J Biochem
96:545,
1979[Medline]
[Order article via Infotrieve]
8.
Nadel J,
Conrad DJ,
Veki IF,
Schuster A,
Sigal E:
Immunohistochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes and airway cells.
J Clin Invest
87:1139,
1991
9.
Conrad DJ,
Kühn H,
Mulkins M,
Highland E,
Sigal E:
Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase.
Proc Natl Acad Sci USA
89:217,
1992[Abstract/Free Full Text]
10. Nassar GM, Morrow JD, Roberts II LJ, Lakkis FG, Badr KF:
Induction of 15-lipoxygenase by interleukin-13 in human blood
monocytes. J Biol Chem 269:27631, 1994
11.
Kühn H:
Biosynthesis, metabolization and biological importance of the primary 15-lipoxygenase metabolites 15-hydro(pero)xy-5Z,8Z,11Z,13E-eicosatetraenoic acid and 13-hydro(pero)xy-9Z,11E-octadecadienoic acid.
Prog Lipid Res
35:203,
1996[Medline]
[Order article via Infotrieve]
12. Rapoport SM, Schewe T, Thiele BJ: Maturational breakdown of
mitochondria and other organelles in reticulocytes, in Harris JR (ed):
Blood Cell Biochemistry (vol 1). New York, NY, Plenum, 1990, p 151
13.
Kühn H,
Belkner J,
Wiesner R:
Subcellular distribution of lipoxygenase products in rabbit reticulocyte membranes.
Eur J Biochem
191:221,
1990[Medline]
[Order article via Infotrieve]
14.
Samuelsson B,
Dahlen SE,
Lindgren JA,
Rouzer CA,
Serhan CN:
Leukotrienes and lipoxins; structures and biological effects.
Science
237:1171,
1987[Abstract/Free Full Text]
15.
Lewis RA,
Austen KF,
Soberman RJ:
Leukotrienes and other products of the 5-lipoxygenase pathway. Biochemistry and relation to pathobiology in human diseases.
N Engl J Med
323:645,
1990[Medline]
[Order article via Infotrieve]
16.
Rouzer CA,
Kargman S:
Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A 23187.
J Biol Chem
263:10980,
1988[Abstract/Free Full Text]
17.
Pueringer RJ,
Bahns CC,
Monick MM,
Hunninghake GW:
A23187 stimulates translocation of the 5-lipoxygenase from the cytosol to membrane in human alveolar macrophages.
Am J Physiol
262:L454,
1992[Abstract/Free Full Text]
18. Woods JW, Evans JF, Ethier D, Scott S, Vickers PJ, Hearn L,
Heibein J A, Charleson S, Singer II: 5-lipoxygenase and 5-lipoxygenase
activating protein are localized in the nuclear envelope of activated
human leukocytes. J Exp Med 178:1935, 1993
19.
Peters-Golden M,
McNish RW:
Redistribution of 5-lipoxygenase and cytosolic phospholipase A2 to the nuclear fraction upon macrophage activation.
Biochem Biophys Res Commun
196:147,
1993[Medline]
[Order article via Infotrieve]
20.
Vickers PJ:
5-lipoxygenase activating protein (FLAP).
J Lipid Med Cell Signal
12:185,
1995[Medline]
[Order article via Infotrieve]
21.
Brock TG,
Paine R,
Peters-Golden M:
Localization of 5-lipoxygenase to the nucleus of unstimulated rat basophilic leukemia cells.
J Biol Chem
269:22059,
1994[Abstract/Free Full Text]
22.
Brock TG,
McNish RW,
Peters-Golden M:
Translocation of leukotriene synthetic capacity of nuclear 5-lipoxygenase in rat basophilic leukemia cells and alveolar macrophages.
J Biol Chem
270:21652,
1995[Abstract/Free Full Text]
23. Coffey M, Peters-Golden M, Fantone III GC, Sporn PHS: Membrane
association of active 5-lipoxygenase in resting cells: Evidence for
novel regulation of the enzyme in rat alveolar macrophages. J Biol Chem
267:570, 1992
24.
Woods JW,
Coffey MJ,
Brock TG,
Singer II,
Peters-Golden M:
5-lipoxygenase is located in the euchromatin of the nucleus in resting alveolar macrophages and translocates to the nuclear envelope upon cell activation.
J Clin Invest
95:2035,
1995
25.
Watson A,
Doherty FJ:
Calcium promotes membrane association of reticulocyte lipoxygenase.
Biochem J
298:377,
1994
26.
Crane FL,
Glenn JL,
Green DE:
Studies on the electron transfer system. The electron transfer particle.
Biochim Biophys Acta
22:475,
1956
27.
Gärtner I:
Separation of human eosinophils in density gradients of polyvinylpyrrolidone-coated silica gel (Percoll).
Immunology
40:133,
1980[Medline]
[Order article via Infotrieve]
28.
Bligh EG,
Dyer WJ:
A rapid method of total lipid extraction and purification.
Can J Biochem Biophysiol
37:911,
1959
29.
Kühn H,
Barnett J,
Grunberger D,
Baecker P,
Chow J,
Nguyen B,
Bursztyn-Pettegrew H,
Chan H,
Sigal E:
Overexpression, purification and characterization of human recombinant 15-lipoxygenase.
Biochim Biophys Acta
1169:80,
1993[Medline]
[Order article via Infotrieve]
30.
Gan QF,
Witkop GL,
Sloane DL,
Straub KM,
Sigal E:
Identification of a specific methionine in mammalian 15-lipoxygenases which is oxygenated by the enzyme product 13-HPODE: Dissociation of sulfoxide formation from self-inactivation.
Biochemistry
34:7069,
1995[Medline]
[Order article via Infotrieve]
31.
Gan QF,
Browner MF,
Sloane DL,
Sigal E:
Defining the arachidonic acid binding site of human 15-lipoxygenase.
J Biol Chem
271:25412,
1996[Abstract/Free Full Text]
32.
Sloane DL,
Leung R,
Barnett J,
Craik CS,
Sigal E:
Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: The side-chain geometry of amino acids 417 and 418 determines positional specificity.
Protein Eng
8:275,
1996[Abstract/Free Full Text]
33.
Borngräber S,
Kuban RJ,
Anton M,
Kühn H:
Phenylalanine 353 is a primary determinant for the positional specificity of mammalian 15-lipoxygenases.
J Mol Biol
264:1145,
1996[Medline]
[Order article via Infotrieve]
34.
Kühn H,
Thiele BJ:
Molecular biology of 15-lipoxygenase.
J Lipid Med Cell Signal
12:157,
1995[Medline]
[Order article via Infotrieve]
35.
Sigal E:
The molecular biology of mammalian arachidonic acid metabolism.
Am J Physiol
260:L13,
1991[Abstract/Free Full Text]
36.
Sigal E,
Grunberger D,
Cashman JR,
Craik CS,
Caughey GH,
Nadel JA:
Arachidonate 15-LOX from human eosinophil-enriched leukocytes: Partial purification and properties.
Biochem Biophys Res Commun
150:376,
1988[Medline]
[Order article via Infotrieve]
37.
Palek J:
Red cell calcium content and transmembrane calcium movement in sickle cell anemia.
J Lab Clin Med
89:1365,
1977[Medline]
[Order article via Infotrieve]
38.
Damonte G,
Guide L,
Sdraffa A,
Benatti U,
Melloni E,
Forteleoni G,
Meloni T,
Carafoli E,
DeFlora E:
Mechanism of pertubation of erythrocyte calcium homeostasis in favism.
Cell Calcium
13:649,
1992[Medline]
[Order article via Infotrieve]
39.
Schilstra MJ,
Veldink GA,
Vliegenthart JFG:
Kinetic analysis of the induction period in lipoxygenase catalysis.
Biochemistry
32:7686,
1993[Medline]
[Order article via Infotrieve]
40.
Nelson MJ,
Chase DB,
Seitz SP:
Photolysis of "purple" lipoxygenase: Implications for the structure of the chromophore.
Biochemistry
34:6159,
1995[Medline]
[Order article via Infotrieve]
41.
Dixon RA,
Diel RE,
Opsa E,
Rands E,
Vickers PJ,
Evans JF,
Gillard JW,
Miller DK:
Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis.
Nature
343:282,
1990[Medline]
[Order article via Infotrieve]
42.
Ford-Hutchinson AW:
FLAP: A novel drug target for inhibiting the synthesis of leukotrienes.
Trends Pharmacol Sci
121:68,
1991
43.
Gawler DJ,
Zhang LJ,
Moran MF:
Mutation-deletion analysis of a Ca(2+)-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras.
Biochem J
307:487,
1995
44. Clark JD, Schievella AR, Nalefski, Li LL: Cytosolic
phospholipase A2. J Lipid Med Cell Signal 12:83, 1995
45.
Boyington JC,
Gaffney BJ,
Amzel LM:
The three-dimensional structure of an arachidonic acid 15-lipoxygenase.
Science
260:1482,
1993[Abstract/Free Full Text]
46.
Minor W,
Steczko J,
Stec B,
Otwinowski Z,
Bolin JT,
Walter R,
Axelrod B:
Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution.
Biochemistry
35:10687,
1996[Medline]
[Order article via Infotrieve]
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X. Tang, B. B. Holmes, K. Nithipatikom, C. J. Hillard, H. Kuhn, and W. B. Campbell
Reticulocyte 15-Lipoxygenase-I Is Important in Acetylcholine-Induced Endothelium-Dependent Vasorelaxation in Rabbit Aorta
Arterioscler Thromb Vasc Biol,
January 1, 2006;
26(1):
78 - 84.
[Abstract]
[Full Text]
[PDF]
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C. Hornig, D. Albert, L. Fischer, M. Hornig, O. Radmark, D. Steinhilber, and O. Werz
1-Oleoyl-2-acetylglycerol Stimulates 5-Lipoxygenase Activity via a Putative (Phospho)lipid Binding Site within the N-terminal C2-like Domain
J. Biol. Chem.,
July 22, 2005;
280(29):
26913 - 26921.
[Abstract]
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M. J. Coffey, G. E. Jarvis, J. M. Gibbins, B. Coles, N. E. Barrett, O. R.E. Wylie, and V. B. O'Donnell
Platelet 12-Lipoxygenase Activation via Glycoprotein VI: Involvement of Multiple Signaling Pathways in Agonist Control of H(P)ETE Synthesis
Circ. Res.,
June 25, 2004;
94(12):
1598 - 1605.
[Abstract]
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M. Walther, R. Wiesner, and H. Kuhn
Investigations into Calcium-dependent Membrane Association of 15-Lipoxygenase-1: MECHANISTIC ROLES OF SURFACE-EXPOSED HYDROPHOBIC AMINO ACIDS AND CALCIUM
J. Biol. Chem.,
January 30, 2004;
279(5):
3717 - 3725.
[Abstract]
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X. Tang, N. Spitzbarth, H. Kuhn, P. Chaitidis, and W. B. Campbell
Interleukin-13 Upregulates Vasodilatory 15-Lipoxygenase Eicosanoids in Rabbit Aorta
Arterioscler Thromb Vasc Biol,
October 1, 2003;
23(10):
1768 - 1774.
[Abstract]
[Full Text]
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D. Zhu, M. Medhora, W. B. Campbell, N. Spitzbarth, J. E. Baker, and E. R. Jacobs
Chronic Hypoxia Activates Lung 15-Lipoxygenase, Which Catalyzes Production of 15-HETE and Enhances Constriction in Neonatal Rabbit Pulmonary Arteries
Circ. Res.,
May 16, 2003;
92(9):
992 - 1000.
[Abstract]
[Full Text]
[PDF]
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M. Walther, M. Anton, M. Wiedmann, R. Fletterick, and H. Kuhn
The N-terminal Domain of the Reticulocyte-type 15-Lipoxygenase Is Not Essential for Enzymatic Activity but Contains Determinants for Membrane Binding
J. Biol. Chem.,
July 19, 2002;
277(30):
27360 - 27366.
[Abstract]
[Full Text]
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S. Yokota, T. Oda, and H. D. Fahimi
The Role of 15-lipoxygenase in Disruption of the Peroxisomal Membrane and in Programmed Degradation of Peroxisomes in Normal Rat Liver
J. Histochem. Cytochem.,
May 1, 2001;
49(5):
613 - 622.
[Abstract]
[Full Text]
|
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P. Christmas, J. W. Fox, S. R. Ursino, and R. J. Soberman
Differential Localization of 5- and 15-Lipoxygenases to the Nuclear Envelope in RAW Macrophages
J. Biol. Chem.,
September 3, 1999;
274(36):
25594 - 25598.
[Abstract]
[Full Text]
[PDF]
|
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A. R. Brash
Lipoxygenases: Occurrence, Functions, Catalysis, and Acquisition of Substrate
J. Biol. Chem.,
August 20, 1999;
274(34):
23679 - 23682.
[Full Text]
[PDF]
|
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|
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M. A. Giembycz and M. A. Lindsay
Pharmacology of the Eosinophil
Pharmacol. Rev.,
June 1, 1999;
51(2):
213 - 340.
[Abstract]
[Full Text]
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K. Schnurr, A. Borchert, and H. Kuhn
Inverse regulation of lipid-peroxidizing and hydroperoxyl lipid-reducing enzymes by interleukins 4 and 13
FASEB J,
January 1, 1999;
13(1):
143 - 154.
[Abstract]
[Full Text]
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T. Hammarberg, P. Provost, B. Persson, and O. Radmark
The N-terminal Domain of 5-Lipoxygenase Binds Calcium and Mediates Calcium Stimulation of Enzyme Activity
J. Biol. Chem.,
December 1, 2000;
275(49):
38787 - 38793.
[Abstract]
[Full Text]
[PDF]
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X.-S. Chen and C. D. Funk
The N-terminal "beta -Barrel" Domain of 5-Lipoxygenase Is Essential for Nuclear Membrane Translocation
J. Biol. Chem.,
January 5, 2001;
276(1):
811 - 818.
[Abstract]
[Full Text]
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Y. I. Miller, M.-K. Chang, C. D. Funk, J. R. Feramisco, and J. L. Witztum
12/15-Lipoxygenase Translocation Enhances Site-specific Actin Polymerization in Macrophages Phagocytosing Apoptotic Cells
J. Biol. Chem.,
May 25, 2001;
276(22):
19431 - 19439.
[Abstract]
[Full Text]
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Abstract
Introduction
Abstract