R E S U L T S Effect of NO donors on AIIt-mediated liposome aggregation To determine whether NO donors affect AIIt’s functions, we exposed AIIt to GSNO and measured AIIt-mediated liposom
Trang 1Inactivation of annexin II tetramer by S -nitrosoglutathione
Lin Liu1,2, Edward Enright2, Peng Sun1, Shwu Yar Tsai1, Pragna Mehta2, David L Beckman2
and David M Terrian3
1 Department of Physiological Sciences, Oklahoma State University, USA; Departments of 2 Physiology, and 3 Anatomy
and Cell Biology, East Carolina University, USA
We investigated the effect of nitric oxide (NO) donors on the
activities of annexin II tetramer (AIIt), a member of the
Ca2+-dependent phospholipid-binding protein family
Incubation of purified AIIt with S-nitrosoglutathione
(GSNO) led to the inhibition of AIIt-mediated liposome
aggregation This effect was dose-dependent with an IC50of
approximately 100 lM Sodium nitroprusside, another NO
donor also inhibited AIIt-mediated liposome aggregation,
whereas reduced glutathione, nitrate, or nitrite had no
effects GSNO also inhibited AIIt-mediated membrane
fusion, but not the binding of AIIt to the membrane GSNO
only has a modest effect on liposome aggregation mediated
by annexins I, III or IV The binding of AIIt to the mem-brane protected the reactive sites of GSNO on AIIt GSNO did not inhibit AIIt-mediated liposome aggregation in the presence of dithiothreitol Taken together, our results sug-gest that GSNO inactivates AIIt possibly via S-nitrosylation and/or the formation of disulfide bonds
Keywords: annexin; nitric oxide; S-nitrosoglutathione; lipo-some aggregation; membrane fusion
Annexins are a multigene family of Ca2+-dependent
phospholipid-binding proteins and plays roles in many
membrane-associated events including exocytosis,
endocy-tosis, ion transport, inflammation, anticoagulation,
inhi-bition of phospholipases, signal transduction, Ca2+
homeostasis, cell-matrix, cell-cell or cell–virus interaction,
etc [1–7] However, most studies were carried out in vitro
Physiological functions of annexins are still unclear
although progress has been made in the past several years
Annexin VI and VII knock-out mice [8,9], and annexin VI
over expression transgenic mice [10] have been generated
and annexin-related diseases (annexinopathies) have
recently been recognized [11]
Annexins share common structural features, i.e a
con-served core domain of four or eight repeats of
approxi-mately 70 amino acids and a short variable N-terminal
segment The C-terminal core domain contains Ca2+-and
phospholipid-binding sites N-termini of annexins are
regulatory and are subjected to various post-translational
modifications including proteolysis and phosphorylation
Annexin II, a member of this family, exists as a monomer
(p36) or a heterotetramer [(p36)2(p11)2] The latter consists
of two annexin II monomers; each associated with p11
protein, member of S100 family of Ca2+-binding proteins
Annexin II binds to acidic phospholipids or biological membranes and causes them to aggregate and fuse [12–14] The formation of annexin II tetramers (AIIt) markedly reduces the Ca2+requirement for its membrane aggregation activity compared to annexin II monomers [12,14] How-ever, the N-terminal phosphorylation of annexin II tetra-mer by protein kinase C (PKC) or protein tyrosine kinase pp60c–srcinhibits its membrane aggregation activity without affecting its membrane binding activity [15,16] In vitro incubation of annexin II tetramers with plasma membrane vesicles and chromaffin granules results in the formation of
a plasma membrane vesicle-annexin II tetramer-chromaffin granule complex [16] An annexin II bridge between the plasma membrane and secretory granules has been observed
in chromaffin cells and anterior pituitary secretory cells using electron microscopy [17,18] Reconstitution experi-ments have demonstrated that annexin II can enhance secretory activity from permeabilized chromaffin cells [19]
A role of annexin II in regulated exocytosis in pulmonary artery endothelial cells has been documented [20] We have previously shown that annexin II tetramer promotes in vitro fusion of lamellar bodies with liposomes This process is enhanced by arachidonic acid, a lung surfactant secreta-gogue and is inhibited by 4,4¢-diisothiocyanatostilbene-2, 2¢-disulfonic acid (DIDS) and phenothiazines, inhibitors of lung surfactant secretion [14,21] Annexin II also partially restores surfactant secretion from permeabilized type II cells [22] Furthermore, annexin II translocates from cytoplasm
to the plasma membrane of type II cells upon stimulation [23] These results suggest that annexin II is involved in membrane fusion during surfactant secretion
In addition to its well-studied membrane fusion activity
in exocytosis and endocytosis, biological activities of annexin II extend to both intracellular and extracellular compartments Annexin II may regulate the organization of cytoskeleton by binding to F-actin [24] Heterodimer formation between annexin II and DNA polymerase a
Correspondence to L Liu, Department of Physiological Sciences,
Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078,
USA Fax: + 1 405 744 8263, Tel.: + 1 405 744 4526,
E-mail: liulin@okstate.edu
Abbreviations: AIIt, annexin II tetramer; GSH, reduced glutathione;
GSNO, S-nitrosoglutathione; NBD-PtdEtn,
N-(7-nitro-2-1,3-ben-zoxadiazol-4-yl) diacyl PtdEtn; NO, nitric oxide; PtdEtn,
phospha-tidylethanolamine; PtdSer, phosphatidylserine; Rh-PtdEtn,
N-(lissamine rhodamine B sulfonyl) diacyl PtdEtn;
SNP, sodium nitroprusside.
(Received 13 May 2002, revised 11 July 2002, accepted 16 July 2002)
Trang 2indicates a role for annexin II in DNA replication [4].
Partitioning of annexin II between nuclei and cytoplasm is
controlled by a nuclear export signal and p11 [25]
Annexin II also exists in extracellular cell surface and acts
as a receptor for cytomegalovirus [26], tenascin C [27], and
tissue plasminogen activator [28] Annexin II tetramer has
been identified as a plasmin reductase [29] and may be
involved in cancer [30]
Nitric oxide (NO) is a membrane-permeable intracellular
and intercellular messenger and plays an important role in
vascular tone, neurotransmission and pulmonary functions
However, it can be toxic when generated in excess Alveolar
epithelium is constantly exposed to NO from two sources:
inhaled air and endogenous production from lung cells
inclu-ding macrophages, endothelial cells, vascular smooth muscle
cells, and epithelial cells NO is generated fromL-arginine by
NO synthase (NOS) Two types of NOS have been described
One is a Ca2+-dependent and constitutive form (cNOS),
which is stimulated by agents that increase intracellular
Ca2+ Another is a Ca2+-independent and inducible form
(iNOS), which is induced by cytokines and/or endotoxins
and is transcriptionally regulated Both types of NOS are
present in alveolar type II cells [31] NO has been shown to
alter lung surfactant metabolism [31] We have previously
shown that NO donors inhibit lung surfactant secretion from
cultured type II cells at high concentrations [32]
There are five cysteine residues in each annexin II
mononer (four in human) and two in the p11 subunit
However, the role of cysteine residues in AIIt’s functions has
not been appreciated We have previously shown that
treatment of AIIt by N-ethylmaleimide resulted in the loss
of its activity [33] NO and its derivatives have been reported
to react with the sulfhydryl groups of several cellular
proteins including calpain [34], protein kinase C [35], low
molecular weight phosphotyrosine protein phosphatase [36]
and glyceraldehyde-3-phosphate dehydrogenase [37], and
inactivate these proteins We reasoned that NO donors
might also inhibit AIIt’s activity, and this could be another
mechanism of NO-mediated inhibition of lung surfactant
secretion, in addition to the nitration of annexin II by
peroxynitrite [38] As nitrosothiols occur naturally in human
airways [39], we chosen S-nitrosoglutathione (GSNO) as a
NO donor In this report, we determined: (a) whether
GSNO influences annexin II’s activities including
mem-brane aggregation, memmem-brane fusion, and memmem-brane
bind-ing; (b) whether the GSNO effect is specific to annexin II;
(c) whether Ca2+and phospholipid alter the GSNO effect
on annexin II; and (d) whether the GSNO effect is due to
the modification of cysteine residues of annexin II
M A T E R I A L S A N D M E T H O D S
Materials
S-Nitrosoglutathione (GSNO) was purchased from Cayman
(Ann Arbor, MI, USA) Dithiothreitol, reduced glutathione
(GSH), sodium nitrate, sodium nitrite and sodium
nitro-prusside (SNP) were from Sigma (St Louis, MO, USA)
Phosphatidylserine (PtdSer), phosphatidylethanolamine
(PtdEtn), N-(7-nitro-2-1,3-benzoxadiazol-4-yl) diacyl
PtdEtn (NBD-PtdEtn) and N-(lissamine rhodamine B
sulfonyl) diacyl PtdEtn (Rh-PtdEtn) were from Avanti Polar
Lipids (Alabaster, AL, USA) DEAE-Sepharose CL 6B,
Sephacryl S-300, Mono S and Mono Q columns were from Amersham Biosciences Corp (Piscataway, NJ, USA) 1,1¢-bis(4-anillino) naphthalene-5, 5¢-disulfonic acid (bis-ANS) was from Molecular Probes (Eugene, OR, USA) Biospin 6 column was from Bio-Rad (Melville, NY, USA) Anti-annexin I, II and IV antibodies were from Zymed (San Francisco, CA, USA) Anti-annexin III antibodies were kindly provided by Dr J D Ernst (University of California San Francisco, USA)
Purification of annexins I–IV Annexins were isolated from bovine lung tissue according
to Khanna et al [40] as previously described in detail [22] The bovine lung tissue (300 g) was powdered in a blender
at slightly above liquid nitrogen temperature One litre of buffer A (10 mMimidazole, pH 7.4, 150 mMNaCl, 1 mM
dithiothreitol, 100 lgÆmL)1soybean trypsin, 1 mMPMSF,
5 lgÆmL)1leupeptin and 2 mMEGTA) was added to the powder Once dissolved, the mixture was centrifuged at
650 g for 10 min Ca2+concentration in the supernatant was then adjusted to 2 mMby the addition of 0.1MCa2+ stock solution The membrane fraction was collected by centrifugation at 24 000 g for 40 min and washed three times in buffer B (10 mM imidazole, pH 7.4, 150 mM
NaCl, 1 mM dithiothreitol and 1 mM Ca2+) The final pellet was resuspended in buffer C (buffer B plus 5 mM
EGTA) and centrifuged at 100 000 g for 1 h The supernatant containing all annexins was dialyzed against buffer D (10 imidazole, pH 7.4, 0.5 mMdithiothreitol and
1 mM EGTA) for 2 days with three changes of buffer D The dialyzate was centrifuged at 100 000 g for 1 h The supernatant (the crude annexin preparation) was loaded
on a DEAE-sepharose column (2.5· 20 cm) and eluted using a linear salt gradient (0–0.3M NaCl in buffer D) Three peaks were resolved: peak A (10–35 mM NaCl) contained annexins I and II; peak B (45–60 mM NaCl) contained annexins III and IV and peak C (160–190 mM
NaCl) contained annexins V and VI Annexins were identified by Western blot using specific antibodies Fraction A was concentrated and applied to a Sephacryl S-300 column (1.5· 150 cm) equilibrated with buffer E (40 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.5 mM dithio-threitol and 1 mM EGTA) Two peaks containing annexin I plus annnexin II monomer, and annexin II tetramer were collected separately The low molecular weight peak was dialyzed against buffer F (25 mM Mes,
pH 6.0 and 0.5 mM dithiothreitol) and applied to an FPLC Mono S column at a flow rate of 1 mLÆmin)1 The column was developed with a gradient of 0–0.4MNaCl in buffer F Annexin I and annexin II monomer were eluted at 0.125M and 0.225M NaCl, respectively The higher molecular weight peak (annexin II tetramer) was also purified by Mono S column chromatography as decribed above Similarly, peak B, from the DEAE column, was concentrated and chromatographed on a Sephacryl S 300 column The major peak containing annexins III and IV was dialyzed against buffer G (40 mMTris, pH 8.5 and 0.5 mM
dithiothreitol) and applied to an FPLC Mono Q column Annexins III and IV were eluted at 0.114M and 0.077M
NaCl when a gradient of 0–0.15MNaCl in buffer G was applied All annexins were homogenous as revealed by SDS/ PAGE and staining with coommassie Brilliant Blue
Trang 3Preparation of liposomes
Liposomes were prepared by the extrusion method [41]
Phospholipids dissolved in chloroform were dried in a test
tube under a stream of nitrogen gas The lipid film was
hydrated with liposome buffer (40 mM Hepes, pH 7.0,
100 mM KCl) by vigorously vortexing The resulting
suspension was passed through a 0.1-lm-filter membrane
three times using an Extruder (Lipex Biomembrane,
Vancouver, Canada)
Preparation of lamellar bodies
Lamellar bodies were isolated from male Sprague–Dawley
rat lung tissue by upward flotation [42] on a discontinuous
sucrose gradient (1.0, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2M)
The lamellar bodies enriched at the interface between 0.4
and 0.5Msucrose were collected and resuspended in 0.2M
sucrose, 10 mMHepes/Tris buffer (pH 7.4)
Treatment of annexin with NO donors
The following standard procedure was used unless stated
otherwise Annexin II tetramer (5 lg) was mixed in 50 lL
of 40 mMHepes, pH 7.4 with GSNO or other agents After
a 30 min incubation at room temperature, the treated
annexin was then tested for its ability to aggregate and fuse
membrane In some experiments, the unreacted GSNO was
removed by gel filtration using a Biospin 6 chromatography
column according to the manufacture’s instructions In this
case, in order to improve the recovery of annexin protein,
1 mgÆmL)1 of BSA was added to the reaction mixture
before loading on the column
Liposome aggregation and binding assays
Liposome aggregation activity was determined by
monit-oring the changes in turbidity as previously described [14]
Liposomes (PtdSer, 100 lg of lipid) were mixed in 1 mL of
Ca2+-EGTA buffer (40 mMHepes, pH.7.0, 100 mMKCl,
2 mMMgCl2, 1 mMEGTA and various concentrations of
Ca2+) After recording the zero time value (A540nm) of
absorbance at 540 nm, annexin was added to initiate
liposome aggregation and incubation continued for
30 min At the end of incubation, the absorbance at
540 nm (A30540nm) was read again The aggregation activity
was expressed as (A30540nm ) A540nm) For the
time-depend-ence of AIIt-mediated liposome aggregation, the
absorb-ance at 540 nm was read every 2 min At the end of the
liposome aggregation assay, the sample was centrifuged at
100 000 g for 1 h The pellet was analyzed on 10% SDS/
PAGE to determine the amount of AIIt bound to
liposomes The bands were quantitated by densitometry
(GS-710 Calibrated Imaging Densitometer, Bio-Rad,
Hercules, CA) Ca2+-EGTA buffer was prepared according
to the method of Bers [43] and the free Ca2+concentration
was verified using a Ca2+-selective electrophode (Orion
Research, Inc, Boston, MA)
Membrane fusion assay
Membrane fusion between lamellar bodies and liposomes
mediated by AIIt was measured, as described previously
[14], according to the method of Struck et al [44] Fusion was monitored by following the decrease in the efficiency of resonance energy transfer between two fluorescent-labeled phospholipid probes: NBD-PtdEtn (donor) and Rh-PtdEtn (acceptor), due to the dilution of the probes upon membrane fusion Liposomes were composed of PtdSer/ PtdEtn/NBD-PtdEtn/Rh-PtdEtn (24.5 : 74 : 0.75 : 0.75) Labeled liposomes (4 lMin lipid) were mixed with lamellar bodies (20 lgÆmL)1) in 0.5 mL of the assay buffer (40 mM
Hepes, pH 7.0, 100 mMKCl, 2 mM MgCl2, 1 mM EGTA and 2 mM CaCl2) After a 1 min incubation, AIIt was added to initiate the reaction NBD-PtdEtn fluorescence (lambda Ex¼ 450 nm and lambda Em ¼ 530 nm) was monitored as a function of time Fusion was expressed as a percentage of the maximal NBD-PtdEtn fluorescence, which was determined after disrupting the membrane with 0.1% Triton X-100 Because Trition X-100 causes the fluorescence quenching, the maximal fluorescence was corrected by a factor of 1.3 [13]
Other methods Protein concentration was determined by the method of Bradford [45], using bovine plasma gamma globulin as a standard SDS/PAGE was carried out according to Laemmli [46], using a Bio-Rad mini-protean II apparatus
R E S U L T S
Effect of NO donors on AIIt-mediated liposome aggregation
To determine whether NO donors affect AIIt’s functions,
we exposed AIIt to GSNO and measured AIIt-mediated liposome aggregation activity as assessed by monitoring the changes in absorbance at 540 nm Figure 1A shows the time course of AIIt-mediated liposome aggregation in the presence of various concentrations of GSNO Figure 1B depicts a dose-dependence of GSNO inhibition of AIIt-mediated liposome aggregation The concentration effecting 50% inhibition (IC50) was approximately 100 lM Ca2+ (1 mM) and/or GSNO (2 mM) did not cause liposome aggregation in the absence of AIIt under our assay conditions Sodium nitropresside (SNP), another NO donor structurally different from GSNO, also inhibited AIIt-mediated liposome aggregation (Fig 1B) although the IC50 (approximately 2 mM) was higher than that of GSNO To exclude the effect of the unreactive GSNO on liposome aggregation, we removed these small molecules from annexin II protein by gel filtration using a Biospin 6 chromatography column at the end of preincubation and measured its liposome aggregation We observed a similar inhibition to these without column purification (data not shown) Furthermore, GSH, nitrite and nitrate had no effects (Fig 2) GSNO inhibited AIIt-mediated liposome aggregation at all the AIIt concentrations and all the Ca2+ concentrations tested (Figs 3 and 4) At a higher concen-tration of AIIt (10 lg) less inhibition was observed Effect of GSNO on AIIt-mediated membrane fusion Although the mechanisms by which AIIt mediates mem-brane fusion are still unclear, at least three steps are
Trang 4involved: (a) the binding of AIIt to membrane; (b)
membrane aggregation and (c) membrane fusion We have
previously shown that AIIt promotes the fusion of
liposomes with lamellar bodies, the secretory granules of lung alveolar type II cells [14,22] We therefore examined whether GSNO also blocks this process Membrane fusion was monitored by a lipid mixing assay [44] The addition of AIIt caused a rapid fusion of lamellar bodies with liposomes Pre-treatment of AIIt with 0.1 and 1 mMGSNO resulted in 70 ± 4% and 83 ± 10% inhibition of AIIt-mediated membrane fusion, respectively (Fig 5) It was noted that AIIt-mediated membrane fusion was more sensitive to GSNO compared to the membrane aggregation This is probably because GSNO not only affects the membrane aggregation step, but also the membrane fusion step
Fig 1 NO donors inhibit annexin II tetramer (AIIt)–mediated liposome aggregation in a dose-dependent fashion Purified AIIt (5 lg) was incubated in
50 lL of 40 m M Hepes (pH 7.4) buffer containing varying concentrations of S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP) at room temperature for 30 min Liposome aggregation activity was measured by monitoring the turbidity change (A 540nm ) The aggregation assay was carried out in 1 mL of Ca2+-EGTA buffer (40 m M Hepes, pH 7.0, 100 m M KCl, 1 m M EGTA, and 2 m M Ca2+) containing 100 lg phosphatidylserine liposomes AIIt was used to initiate liposome aggregation (A) A representative time course curve of AIIt-mediated liposme aggregation in the presence of various concentrations of GSNO (d) 0 m M (j) 0.001 m M (m ) 0.1 m M ( ) 1 m M (r) 10 m M (B) Dose-dependence of NO donor-mediated inhibition of AIIt-donor-mediated liposome aggregation The activity was expressed as the increase in absorbance at 540 nm after a 30 min incubation over the initial value The results were expressed as percentage control The control was treated the same way as other samples except that
no other reagents were added The data shown are mean ± SE from three experiments (GSNO, d) or mean from two experiments (SNP, j).
Fig 2 Sodium nitroprusside and S-nitrosoglutathione inhibit
AIIt-mediated liposome aggregation, whereas reduced glutathione, nitrate or
nitrite has little effect AIIt (5 lg) was incubated with sodium
nitro-prusside (SNP, 2 m M ), S-nitrosoglutathione (GSNO, 2 m M ), reduced
glutathione (GSH, 2 m M ), nitrate (2 m M ) or nitrite (2 m M ) in 50 lL of
40 m M Hepes buffer (pH 7.4) for 30 min The treated AIIt was tested
for its ability to mediate liposome aggregation The results were
expressed as a percentage of the control The control was treated the
same way as other samples except that no other reagents were added.
The data shown are mean ± SE from three experiments wP < 0.05
vs control.
Fig 3 A dose-dependence of AIIt-mediated liposome aggregation in the presence or absence of GSNO Various concentrations of AIIt (0–10 lg) were incubated with or without 2 m M GSNO in 50 lL of
40 m M Hepes buffer (pH 7.4) for 30 min Liposome aggregation was determined and expressed as the increase in absorbance at 540 nm after a 30 min incubation over the initial value.
Trang 5Effect of GSNO on the binding of AIIt to membrane
As the binding of AIIt to membrane would be the
first step of AIIt-mediated membrane fusion, we also
investigated whether GSNO inhibits the binding of AIIt
to liposomes We treated AIIt with GSNO for 30 min and mixed the samples with liposomes and 1 mM Ca2+ After a 30 min incubation, we pelleted liposomes by centrifugation and analyzed AIIt associated with lipo-somes on 10% SDS/PAGE As shown in Fig 6A, GSNO had no effect on the amount of AIIt associated with liposomes In the absence of Ca2+, little AIIt was bound to liposomes The results indicate that the modification of annexin II by GSNO does not affect the binding of AIIt to membrane When different amounts of AIIt were treated with GSNO, no inhibition were observed for the binding of AIIt to liposmes at all the AIIt concentrations tested (Fig 6B)
Effect of GSNO on liposome aggregation mediated
by annexins I, III and IV Annexins are a large gene family In mammals, so far, 12 members have been identified and in other organisms more than 60 and over 200 isoforms [47] Annexins share common structural features and some biochemical pro-perties All annexins bind to phospholipids in the presence of Ca2+ Some annexins (I, II, III, IV and VII) are able to mediate liposome aggregation although their Ca2+sensitivities differ [22] We therefore examined whether GSNO inhibits liposome aggregation mediated
by various annexins As shown in Fig 7, GSNO only has a modest effect on liposome aggregation mediated by other annexins
Effect of Ca2+and phospholipid on GSNO inhibition
Ca2+causes protein conformational changes in annexin II [48] and may alter the environment of reactive sites of annexin II by GSNO We tested whether such changes influence the GSNO inhibition of the activity of annexin II AIIt (5 lg) was preincubated in 50 lL of buffer containing 1 mM Ca2+ for 30 min to induce protein conformational changes The mixture was then added to 1 mL of the assay buffer containing 100 lg liposome for measuring liposome aggregation As shown
in Fig 8 (two bars with minus liposome during the preincubation), a similar inhibition was observed when AIIt was pretreated with EGTA or Ca2+, suggesting that the conformational changes caused by Ca2+had no effect
on the GSNO inhibition
After binding to the membrane, some residues in AIIt may be hidden due to the polymerization of AIIt on the membrane or a protein conformational change, and are
no longer accessible to GSNO To test this possibility, AIIt (5 lg) was preincubated with 50 lg liposomes in
50 lL of buffer containing 1 mM Ca2+ to allow AIIt binding to liposomes and then treated with 1 mMGSNO
At the end of preincubation, the mixture was added to
1 mL of assay buffer containing 50 lg liposomes for measuring liposome aggregation As expected, GSNO still inhibited AIIt-mediated liposome aggregation when no
Ca2+existed and thus AIIt did not bind to the membrane during the preincubation However, in the presence of
Ca2+and liposomes during the preincubation, AIIt was bound to the membrane before the addition of GSNO In this case, no significant inhibition was observed (Fig 8)
Fig 4 Ca 2+ -dependence of AIIt-mediated liposome aggregation in the
presence or absence of GSNO AIIt (5 lg) was incubated with or
without 2 m M GSNO in 50 lL of 40 m M Hepes buffer (pH 7.4) for
30 min Liposome aggregation was measured in various
concentra-tions of Ca2+-EGTA buffer and expressed as the increase in
absorb-ance at 540 nm after a 30 min incubation over the initial value.
Fig 5 GSNO inhibits AIIt-mediated fusion of lamellar bodies with
liposomes AIIt (5 lg) was incubated with 0.1 m M or 1 m M GSNO in
50 lL of 40 m M Hepes (pH 7.4) for 30 min and AIIt- mediated
membrane fusion was measured Lipid (4 l M ) in labeled liposomes
(PtdSer/PtdEtn/NBD-PtdEtn/Rh-PtdEtn, 24.5 : 74 : 0.75 : 0.75)
were mixed with 20 lgÆmL)1lamellar bodies in 0.5 mL Ca2+-EGTA
buffer (1 m M free Ca2+) After a stable baseline was established,
AIIt was added to initiate the reaction Fusion was monitored by
following the increase in NBD-PtdEtn fluorescence (Ex ¼ 450 nm,
Em ¼ 530 nm) The data shown are a representative from three
experiments.
Trang 6In an additional experiment, GSNO (1 mM) was directly
added to liposome aggregation assay medium before the
addition of AIIt Under these conditions, a 48% inhibition
was observed However, if GSNO was added 5 min or
10 min after the addition of AIIt, less inhibition (26% or
17%) was seen (data not shown) Presumably, this is due
to the binding of AIIt to liposomes These results indicate
that the reactive sites on AIIt were protected by the
binding of AIIt to the membrane
Fig 6 GSNO does not affect the binding of AIIt to liposomes (A) AIIt was incubated with GSNO (1 m M ) for 30 min At the end of incubation, AIIt was mixed with liposomes in the presence of 1 m M EGTA or Ca2+ After a 30 min incubation, liposomes were sedimented by centrifugation and AIIt associated with liposomes was analyzed by 10% SDS/PAGE The data shown are a representative from three experiments (B) A dose dependence of AIIt binding to liposomes in the presence or absence of GSNO The conditions were the same as in the figure legend of Fig 3 At the end of the aggregation assay, liposomes were sedimented by centrifugation AIIt associated with liposomes were analyzed by SDS/PAGE and quantitated by densitometry The results were expressed as the percentage of the maximal binding (i.e 10 lg AIIt without GSNO).
Fig 7 A dose-dependence of liposome aggregation mediated by
ann-exin I, III and IV in the presence or absence of GSNO Various amounts
of annexins I, III and IV (0–10 lg) were incubated with or without
2 m M GSNO for 30 min Liposome aggregation was determined and
expressed as the increase in absorbance at 540 nm after a 30 min
incubation over the initial value For the comparison, the results were
expressed as percentages of the maximal activity (i.e 10 lg of annexins
without GSNO treatment) The latter values were 0.28, 0.32, and 0.23
for annexins I, III, and IV, respectively (d) AI (s) AI + GSNO (.)
AIII (,) AIII + GSNO (j) AIV (h) AIV + GSNO.
Fig 8 Ca2+-induced protein conformational change in AIIt has no effect on GSNO inhibition of AIIt-mediated liposome aggregation However, GSNO does not inhibit AIIt-mediated liposome aggregation once AIIt binds to membrane For the first two bars, AIIt (5 lg) was incubated with or without 1 m M GSNO in 50 lL of 40 m M Hepes (pH 7.4) containing 1 m M EGTA or 1 m M Ca2+for 30 min The samples were added to 1 mL of the assay buffer containing 100 lg liposome for aggregation activity determination For the last two bars, AIIt (5 lg) was preincubated with 50 lg liposome in the presence or absence of 1 m M GSNO in 50 lL of 40 m M Hepes (pH 7.4) containing
1 m M EGTA or Ca 2+ for 30 min The samples were then added to
1 mL of assay buffer containing 50 lg liposome for aggregation activity determination The zero time absorbance was recorded sepa-rately using 1 mL of the assay buffer containing 100 lg liposomes The results were expressed as percentage control (i.e activity of GSNO-treated AIIt/activity of unGSNO-treated AIIt · 100%) The data shown are mean ± SE from three experiments.
Trang 7Effect of dithiothreitol on GSNO inhibition
of AIIt-mediated liposome aggregation
To evaluate whether the GSNO inhibition of AIIt-mediated
liposome aggregation is involved in the formation of
disulfide bonds, we incubated purified AIIt (5 lg) with
GSNO (1 mM) in the presence of the reducing agent,
dithiothreitol As shown in Fig 9, when dithiothreitol
(0.5 mM) was included in the incubation medium, the
inhibition of AIIt-mediated liposome aggregation by
GSNO was no longer observed, suggesting that the
inactivation of AIIt may be due to the formation of
disulfide bridges However, no intermolecular disulfide
bonds between annexin molecules were formed, because
when GSNO-treated AIIt was resolved on nonreduced
SDS/PAGE, no extra-bands were seen (data not shown)
However, we cannot rule out the possibility of disulfide
bond formation between AIIt and glutathione because of
the resolution of SDS/PAGE
Conformational changes
To detect possible conformational changes of AIIt treated
with GSNO, we used the hydrophobic fluorescent probe,
bis-ANS This dye binds to hydrophobic sites of proteins
and causes an increase of intensity in fluorescence with a
concomitant shift to the lower wavelength [49] As expected,
the addition of AIIt to the bis-ANS aqueous solution, the
fluorescence increased and maximal emission wavelength
was shifted from 510 nm to 490 nm (data not shown)
Those changes are less, compared to annexin I [50]
GSNO-treated AIIt had a similar increase of fluorescence and wavelength shifts GSNO itself had no effect on either fluorescence or maximum wavelength The results suggest that GSNO does not cause a major conformational change
of AIIt as detected by the fluorescent dye, bis-ANS However, it is possible that the method used here may not
be able to detect small conformational changes
D I S C U S S I O N
Annexins are subjected to various post-translational modi-fications Although the phosphorylation of tyrosine or serine/threonine residues in annexin has been extensively studied, the relationship between other residues and annex-in’s activity attracted less attention When AIIt was treated with N-ethylmaleimide, a sulfhydryl agent, AIIt’s activity was reduced [33] However, modification of annexin by reactive nitrogen species has not been reported Our recent study has shown that AIIt can be nitrated by peroxynitrite
to form nitrotyrosine and such modification inhibited AIIt-mediated liposome aggregation [38] In the present study,
we, for the first time, showed that NO donors, GSNO and SNP, also inhibit annexin II’s activities including membrane aggregation and fusion This modification was abolished in the presence of dithiothreitol Although physiological significance of this in vitro observation remains to be determined, it might imply a new post-translational modi-fication and possibly a regulatory mechanism for annexin II
in cells Recently, Fas-induced caspase-3 de-nitrosylation was observed in lymphocyte cells, but the factors responsible for the de-nitrosylation was not identified [51] Because NO inhibits surfactant secretion from alveoar type II cells [32] and AIIt is a criticial component for the secretion of lung surfactant in type II cells [14,22], NO inhibition of AIIt’s activity may provide an alternative mechanism
of NO-mediated reduction of lung surfactant secretion Annexins including annexin II have also shown to be associated with oxidative stress [52–56], NO modification may also have implications in this process as well as other biological activities of annexin II
NO and its derivatives can attack protein targets involved
in many physiological processes and thus modifies their functions Interaction of NO with the heme or nonheme iron of proteins leads to activation of soluble guanylyl cyclase [57] and inactivation of cyclooxygenase [58] or mitochondrial complexes I and II [59] NO also regulates protein functions by covalent attachment of the NO group
to cysteine residues in proteins via S-nitrosylation, which may involve other nitrogen species such as NO+ Increasing amounts of evidence demonstrate that this post-transla-tional modification may represent an important cellular regulatory mechanism [34–37] Depending on different proteins, S-nitrosylation may be followed by secondary modification For example, for glyceraldehyde-3-phosphate dehydrogenase, S-nitrosylation of four active site cysteines
in the tetramer ultimately results in S-ADP-ribosylation and inactivation [37] If vicinal thiols in the protein are S-nitrosylated, a more stable disulfide may be formed One of the examples is the N-methyl-D-asparate receptor [60] Our present study has shown that the GSNO inhibition
of annexin II-mediated liposome aggregation is no longer observed in the presence of dithiothreitol, suggesting that, most likely, the modification of annexin II’s activity by
Fig 9 Effect of ditiothreitol on the inhibition of AIIt-mediated liposome
aggregation caused by GSNO AIIt (5 lg) was incubated with 1 m M
GSNO in the presence of the reducing agent, dithiothreitol (0, 0.1, 0.5,
1.0 m M ) After a 30 min incubation, liposome aggregation activity was
determined The results were expressed as a percentage of the control.
The control was treated as the same way as other samples except that
no GSNO and dithiothreitol was added The data shown are
mean ± SE from three experiments.
Trang 8GSNO is through S-transnitrosylation and the formation of
disulfide bond(s) As no dimers or polymers in
GSNO-treated AIIt were observed on nonreduced SDS/PAGE, the
disulfide bonds could be formed either within the AIIt
molecules or between annexin II thiol and GSNO [61]
UV and fluorescence studies of annexin II revealed a
Ca2+-induced conformational change in which the
aroma-tic amino acids, tyrosine and tryptophan, expose more to
the aqueous phase [48] For annexin V, Ca2+ causes
conformational changes in domain III that leads to the
formation of an additional Ca2+-binding site and exposure
of Trp187 to the solvent [62,63] These conformational
changes appear not to affect the GSNO reaction with
annexin II, as a similar inhibition of AIIt-mediated
lipo-some aggregation by GSNO was observed whether AIIt
was pretreated with Ca2+ or not prior to the liposome
aggregation assay Probably, because Ca2+only induced a
modest conformational change circular dichroism studies
failed to detect major changes in secondary structure of
Ca2+-bound annexin II [48]
The present study indicated that GSNO no longer inhibits
AIIt-mediated liposome aggregation once the protein binds
to the membrane This is consistent with the finding that
some annexins are accessible to quenchers in the solution
more than in the membrane-bound state [64] There are
several possibilities: (a) after the binding of annexin II, the
reactive sites were hidden by membrane; (b) the binding of
AIIt to membrane causes a conformational change [65], such
changes may bury the reactive sites of GSNO more deeply in
the protein matrix therefore rendering them inaccessible to
GSNO; (c) annexins V and XII has been shown to form
trimers or hexamers on membrane [66,67] We have
previ-ously shown that AIIt can self-associate in the presence of
Ca2+[23] Therefore, it is possible that AIIt forms polymers
on membrane, thus hiding the reactive sites
Nitrosothiols occur naturally in human plasma mainly as
the nitrosothiol of human serum albumin [68]
S-nitroso-glutathione has been identified on normal airways [39] and
in neutrophils [69] S-nitrothiol concentrations in inflamed
and transplanted lungs were much higher than normal
subjects The half-life of GSNO in the lavage fluid is
approximately 3 h, much longer than NO [39] Therefore,
GSNO may contribute to physiological and pathological
processes in the lung and GSNO regulation of annexin II
activity may be physiologically relevant
A C K N O W L E D G E M E N T S
This work was supported by US Public Health Service Grant NHLBI
HL-52146, OCAST HR01-093 and OAES (to L L.) We thank Ms.
Dierra Davis and Ms Krista J Schone for secretarial assistance.
R E F E R E N C E S
1 Creutz, C.E (1992) The annexins and exocytosis Science 258,
924–931.
2 Gerke, V & Moss, S.E (1997) Annexins and membrane
dynamics Biochim Biophys Acta 1357, 129–154.
3 Raynal, P & Pollard, H.B (1994) Annexins: the problem of
assessing the biological role for a gene family of multifunctional
calcium-and phospholipid- binding proteins Biochim Biophys.
Acta 1197, 63–93.
4 Waisman, D.M (1995) Annexin II tetramer: structure and
func-tion Mol Cell Biochem 149/150, 301–322.
5 Bandorowicz-Pikula, J., Buchet, R & Pikula, S (2001) Annexins
as nucleotide-binding proteins: facts and speculations Bioessays
23, 170–178.
6 Kourie, J.I & Wood, H.B (2000) Biophysical and molecular properties of annexin-formed channels Prog Biophys Mol Biol.
73, 91–134.
7 Gerke, V & Moss, S.E (2002) Annexins: from structure to function Physiol Rev 82, 331–371.
8 Hawkins, T.E., Roes, J., Rees, D., Monkhouse, J & Moss, S.E (1999) Immunological development and cardiovascular function are normal in annexin VI null mutant mice Mol Cell Biol 19, 8028–8032.
9 Srivastava, M., Atwater, I., Glasman, M., Leighton, X., Goping, G., Caohuy, H., Miller, G., Pichel, J., Westphal, H., Mears, D., Rojas, E & Pollard, H.B (1999) Defects in inositol 1,4,5-tripho-sphate receptor expression, Ca(2+) signaling, and insulin secre-tion in the anx7 (+/-) knockout mouse Proc Natl Acad Sci USA
96, 13783–13788.
10 Gunteski-Hamblin, A.M., Song, G., Walsh, R.A., Frenzke, M., Boivin, G.P., Dorn, G.W., Kaetzel, M.A., Horseman, N.D & Dedman, J.R (1996) Annexin VI overexpression targeted to heart alters cardiomyocyte function in transgenic mice Am J Physiol.
270, H1091–H1100.
11 Rand, J.H (2000) The annexinopathies: a new category of dis-eases Biochim Biophys Acta 1498, 169–173.
12 Drust, D.S & Creutz, C.E (1988) Aggregation of chromaffin granules by calpactin at micromolar levels of calcium Nature 331, 88–91.
13 Blackwood, R.A & Ernst, J.D (1990) Characterization of Ca 2+ -dependent phospholipid binding, vesicle aggregation and mem-brane fusion by annexins Biochem J 266, 195–200.
14 Liu, L., Fisher, A.B & Zimmerman, U.J.P (1995) Lung annexin
II promotes fusion of isolated lamellar bodies with liposomes Biochim Biophys Acta 1259, 166–172.
15 Johnstone, S.A., Hubaishy, I & Waisman, D.M (1992) Phos-phorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein J Biol Chem 267, 25976–25981.
16 Hubaishy, I., Jones, P.G., Bjorge, J., Bellagamba, C., Fitzpatrick, S., Fujita, D.J.X & Waisman, D.M (1995) Modulation of annexin II tetramer by tyrosine phosphorylation Biochemistry
34, 14527–14534.
17 Nakata, T., Sobue, K & Hirokawa, N (1990) Conformational change and localization of calpactin I complex involved in exo-cytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry J Cell Biol 110, 13–25.
18 Senda, T., Okabe, T., Matsuda, M & Fujita, H (1994) Quick-freeze, deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin
II Cell Tissue Res 277, 51–60.
19 Ali, S.M., Geisow, M.J & Burgoyne, R.D (1989) A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells Nature 340, 313–315.
20 Konig, J., Prenen, J., Nilius, B & Gerke, V (1998) The annexin II-p11 complex is involved in regulated exocytosis in bovine pulmonary artery endothelial cells J Biol Chem 273, 19679–19684.
21 Liu, L., Tao, J.Q., Li, H.L & Zimmerman, U.J.P (1997) Inhibi-tion of lung surfactant secreInhibi-tion from alveolar type II cells and annexin II tetramer-mediated membrane fusion by phenothia-zines Arch Biochem Biophys 342, 332–328.
22 Liu, L., Wang, M., Fisher, A.B & Zimmerman, U.J.P (1996) Involvement of annexin II in exocytosis of lamellar bodies from alveolar epithelial type II cells Am J Physiol 270, L668– L676.
23 Liu, L (1999) Calcium-dependent self-association of annexin II:
a possible implication in exocytosis Cell Signal 11, 317–324.
Trang 924 Filipenko, N.R & Waisman, D.M (2001) The C-terminus of
annexin II mediates binding to F-actin J Biol Chem 276, 5310–
5315.
25 Eberhard, D.A., Karns, L.R., Vandenberg, S.R & Creutz, C.E.
(2001) Control of the nuclear-cytoplasmic partitioning of annexin
II by a nuclear export signal and by p11 binding J Cell Sci 114,
3155–3166.
26 Wright, J.F., Kurosky, A & Wasi, S (1994) An endothelial
cell-surface form of annexin II binds human cytomegalovirus.
Biochem Biophys Res Commun 198, 983–989.
27 Chung, C.Y & Erickson, H.P (1994) Cell surface annexin II is a
high affinity receptor for the alternatively spliced segment of
tenascin-C J Cell Biol 126, 539–548.
28 Tsao, F.H., Chen, X & Vu, V.X (1994) Immunocharacterization
and developmental regulation of rabbit lung calcium-dependent
phospholipid-binding proteins Biochim Biophys Acta 1213, 91–
99.
29 Kwon, M., Caplan, J.F., Filipenko, N.R., Choi, K.S., Fitzpatrick,
S.L., Zhang, L & Waisman, D.M (2002) Identification of annexin
II heterotetramer as a plasmin reductase J Biol Chem 277, 10903–
10911.
30 Bastian, B.C (1997) Annexins in cancer and autoimmune diseases.
Cell Mol Life Sci 53, 554–556.
31 Hallman, M & Bry, K (1996) Nitric oxide and lung surfactant.
Semin Perinatol 20, 173–185.
32 Beckman, D.L., Mehta, P., Enright, E & Liu, L (1998) Nitric
oxide donor, spermine NONOate, modulate lung surfactant
secretion from cultured alveolar type II cells FASEB J 12, A491.
33 Singh, T.K & Liu, L (2000) Modification of cysteine residues by
N-ethylmaleimide inhibits annexin II tetramer mediated liposome
aggregation Arch Biochem Biophys 381, 235–240.
34 Michetti, M., Salamino, F., Melloni, E & Pontremoli, S (1995)
Reversible inactivation of calpain isoforms by nitric oxide.
Biochem Biophys Res Commun 207, 1009–1014.
35 Gopalakrishna, R., Chen, Z.H & Gundimeda, U (1993) Nitric
oxide and nitric oxide-generating agents induce a reversible
inactivation of protein kinase C activity and phorbol ester binding.
J Biol Chem 268, 27180–27185.
36 Caselli, A., Camici, G., Manao, G., Moneti, G., Pazzagli, L.,
Cappugi, G & Ramponi, G (1994) Nitric oxide causes
inactiva-tion of the low molecular weight phosphotyrosine protein
phos-phatase J Biol Chem 269, 24878–24882.
37 Mohr, S., Stamler, J.S & Brune, B (1996) Post-translational
modification of glyceraldehyde-3-phosphate dehydrogenase by
S-nitrosylation and subsequent NADH attachment J Biol Chem.
271, 4209–4214.
38 Rowan, W.H., Sun, P & Liu, L (2002) Nitration of annexin II
tetramer Biochemistry 41, 1409–1420.
39 Gaston, B., Reilly, J., Drazen, J.M., Fackler, J., Ramdev, P.,
Arnelle, D., Mullins, M.E., Sugarbaker, D.J., Chee, C., Singel,
D.J., Loscalzo, J & Stamler, J.S (1993) Endogenous nitrogen
oxides and bronchodilator S-nitrosothiols in human airways.
Proc Natl Acad Sci USA 90, 10957–10961.
40 Khanna, N.C., Helwig, E.D., Ikebuchi, N.W., Fitzpatrick, S.,
Bajwa, R.X & Waisman, D.M (1990) Purification and
char-acterization of annexin proteins from bovine lung Biochemistry
29, 4852–4862.
41 Hope, M.J., Bally, M.B., Webb, G & Cullis, P.R (1985)
Pro-duction of large unilamellar vesicles by a rapid extrusion
proce-dure, characterization of size distribution, trapped volume and
ability to maintain a membrane potential Biochim Biophys Acta
812, 55–65.
42 Chander, A., Dodia, C.R., Gil, J & Fisher, A.B (1983) Isolation
of lamellar bodies from rat granular pneumocytes in primary
culture Biochim Biophys Acta 753, 119–129.
43 Bers, D.M (1982) A simple method for the accurate determination
of free [Ca] in Ca-EGTA solutions Am J Physiol 242, C404– C408.
44 Struck, D.K., Hoekstra, D & Pagano, R.E (1981) Use of resonance energy transfer to monitor membrane fusion Bio-chemistry 20, 4093–4099.
45 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.
46 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680– 685.
47 Morgan, R.O & Fernandez, M.P (1997) Annexin gene structures and molecular evolutionary genetics Cell Mol Life Sci 53, 508– 515.
48 Gerke, V & Weber, K (1985) Calcium-dependent conformational changes in the 36-kDa subunit of intestinal protein I related to the cellular 36-kDa target of Rous sarcoma virus tyrosine kinase.
J Biol Chem 260, 1688–1695.
49 Rosen, C.G & Weber, G (1969) Dimer formation from 1-amino-8-naphthalenesulfonate catalyzed by bovine serum albumin A new fluorescent molecule with exceptional binding properties Biochemistry 8, 3915–3920.
50 Liu, L & Zimmerman, U.J (1995) An intramolecular disulfide bond is essential for annexin I-mediated liposome aggregation Biochem Mol Biol Int 35, 345–350.
51 Mannick, J.B., Hausladen, A., Liu, L., Hess, D.T., Zeng, M., Miao, Q.X., Kane, L.S., Gow, A.J & Stamler, J.S (1999) Fas-induced caspase denitrosylation Science 284, 651–654.
52 Ammendola, R., Fiore, F., Esposito, F., Caserta, G., Mesuraca, M., Russo, T & Cimino, F (1995) Differentially expressed mRNAs as a consequence of oxidative stress in intact cells FEBS Lett 371, 209–213.
53 Li, Y.D., Patel, J.M., Zhang, J & Block, E.R (1997) Over-expression of plasma membrane annexin II in NO 2 -exposed pulmonary artery endothelial cells Free Radic Biol Med 23, 120–126.
54 Hoyal, C.R., Thomas, A.P & Forman, H.J (1996) Hydroper-oxide-induced increases in intracellular calcium due to annexin VI translocation and inactivation of plasma membrane Ca2+ -ATPase J Biol Chem 271, 29205–29210.
55 Polla, B.S., Jacquier-Sarlin, M.R., Kantengwa, S., Mariethoz, E., Hennet, T., Russo-Marie, F & Cossarizza, A (1996) TNF alpha alters mitochondrial membrane potential in L929 but not in TNF alpha-resistant L929.12 cells: relationship with the expression of stress proteins, annexin 1 and superoxide dismutase activity Free Radic Res 25, 125–131.
56 Sacre, S.M & Moss, S.E (2002) Intracellular localization of endothelial cell annexins is differentially regulated by oxidative stress Exp Cell Res 274, 254–263.
57 Ignarro, L.J (1989) Heme-dependent activation of soluble guanylate cyclase by nitric oxide: regulation of enzyme activity by porphyrins and metalloporphyrins Semin Hematol 26, 63–76.
58 Kanner, J., Harel, S & Granit, R (1992) Nitric oxide, an inhibitor
of lipid oxidation by lipoxygenase, cyclooxygenase and hemoglobin Lipids 27, 46–49.
59 Drapier, J.C & Hibbs, J.B.J (1988) Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in 1-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells J Immunol 140, 2829–2838.
60 Lei, S.Z., Pan, Z.H., Aggarwal, S.K., Chen, H.S., Hartman, J., Sucher, N.J & Lipton, S.A (1992) Effect of nitric oxide produc-tion on the redox modulatory site of the NMDA receptor-channel complex Neuron 8, 1087–1099.
Trang 1061 Xian, M., Chen, X., Liu, Z., Wang, K & Wang, P.G (2000)
Inhibition of papain by S-nitrosothiols: formation of mixed
disulfides J Biol Chem 275, 20467–20473.
62 Concha, N.O., Head, J.F., Kaetzel, M.A., Dedman, J.R &
Seaton, B.A (1993) Rat annexin V crystal structure: Ca2+
-induced conformational changes Science 261, 1321–1324.
63 Meers, P & Mealy, T (1993) Relationship between annexin V
tryptophan exposure, calcium, and phospholipid binding.
Biochemistry 32, 5411–5418.
64 Hofmann, A., Escherich, A., Lewit-Bentley, A., Benz, J.,
Raguenes-Nicol, C., Russo-Marie, F., Gerke, V., Moroder, L &
Huber, R (1998) Interactions of benzodiazepine derivatives with
annexins J Biol Chem 273, 2885–2894.
65 Pigault, C., Follenius- Wund, A Lux, B & Gerard, D (1990) A
fluorescence spectroscopy study of the calpactin I complex and its
subunits p11 and p36: calcium-dependent conformation changes.
Biochim Biophys Acta 1037, 106–114.
66 Concha, N.O., Head, J.F., Kaetzel, M.A., Dedman, J.R & Seaton, B.A (1992) Annexin V forms calcium-dependent trimeric units on phospholipid vesicles FEBS Lett 314, 159–162.
67 Luecke, H., Chang, B.T., Mailliard, W.S., Schlaepfer, D.D & Haigler, H.T (1995) Crystal structure of the annexin XII hexamer and implications for bilayer insertion Nature 378, 512–515.
68 Stamler, J.S., Jaraki, O., Osborne, J., Simon, D.I., Keaney, J., Vita, J., Singel, D., Valeri, C.R & Loscalzo, J (1992) Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin Proc Natl Acad Sci USA 89, 7674– 7677.
69 Clancy, R.M., Levartovsky, D., Leszczynska-Piziak, J., Yegu-din, J & Abramson, S.B (1994) Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as
a bioactive intermediary Proc Natl Acad Sci USA 91, 3680– 3684.