From the Division of Medical Oncology, University of
Colorado Health Science Center, Denver, Colorado 80262 and the
¶ Division of Medical Oncology, University of Washington and
Veterans Affairs Medical Center, Seattle, Washington 98108
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ABSTRACT |
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Granulocyte-macrophage colony-stimulating factor
(GM-CSF) regulates differentiation, survival, and proliferation of
myeloid progenitor cells. The biologic actions of GM-CSF are mediated by its binding to the and
subunits of the GM-CSF receptor (GM-CSFR
and
c, respectively). To determine whether identical regions of the
c protein mediate both cell growth and
differentiation, we expressed cDNA constructs encoding the human
wild-type (897 amino acids) and truncated
c (h
c) subunits along
with the wild-type human GM-CSFR
subunit in the murine WT19 cell
line, an FDC-P1-derived cell line that differentiates toward the
monocytic lineage in response to murine GM-CSF. Whereas the WT19 cell
line carrying the C-terminal deleted h
c subunit of 627 amino acids
was still able to grow in human GM-CSF (hGM-CSF), 681 amino acids of
the h
c were necessary for cell differentiation. The addition of
hGM-CSF to WT19 cell lines containing the h
c627 subunit stimulated
the phosphorylation of ERK (extracellular signal-regulated kinase) and
induced the tyrosine-phosphorylation of SHP-2 and STAT5, suggesting that the activation of these molecules is insufficient to mediate the
induction of differentiation. A point mutation of tyrosine 628 to
phenylalanine (Y628F) within h
c681 abolished the ability of hGM-CSF
to induce differentiation. Our results indicate that the signals
required for hGM-CSF-induced differentiation and cell growth are
mediated by different regions of the h
c subunit.
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INTRODUCTION |
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Granulocyte-macrophage colony-stimulating factor (GM-CSF)1 is secreted by activated T cells, endothelial cells, fibroblasts, mast cells, B cells, and macrophages and plays an important role in promoting differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage (CFU-GM) progenitor cells. GM-CSF also enhances the function of mature neutrophils, monocytes, and eosinophils, and stimulates the burst-promoting activity for BFU-E (1-3). In addition, in murine models, alveolar proteinosis, a disease caused by excessive secretion of protein into the alveolar spaces, is associated with the deletion of the GM-CSF gene, suggesting that GM-CSF is essential for alveolar macrophage functions (4, 5).
The biologic actions of GM-CSF are mediated by its binding to a
specific receptor consisting of and
subunits, both of which are
members of the type I cytokine receptor family (6, 7). The
subunit
(GM-CSFR
) binds GM-CSF with low affinity (6). The
subunit does
not bind GM-CSF itself, but it forms a high affinity receptor in
combination with the
subunit (7). The
chain is referred to as
the common
chain (
c) because it is also utilized by interleukin
3 (IL-3) and interleukin 5 (IL-5) (8, 9).
We have recently reported that the cytoplasmic domain of the human
GM-CSFR (hGM-CSFR
) is indispensable for cellular differentiation induced by human GM-CSF (hGM-CSF) (10). In addition, the cytoplasmic domain of the human
c (h
c) has recently been demonstrated to be
essential for hGM-CSF-mediated myeloid cell differentiation (11).
However, the exact mechanism of differentiation by GM-CSF has not been
clearly elucidated. In the present study, we examined the role of
c
in GM-CSF-mediated cell differentiation by using the murine WT19 cell
line, an FDC-P1-derived cell line that uniformly differentiates toward
the monocytic lineage in response to murine GM-CSF (mGM-CSF), but grows
without differentiation in the presence of murine IL-3 (mIL-3) (10,
12). By expressing the wild-type and mutated h
c constructs along
with the wild-type hGM-CSFR
in WT19 cells, we demonstrate the
following results. 1) Mutation of the two prolines in the box 1 region
of h
c, which are necessary for Jak2 activation, results in the loss
of hGM-CSF-induced cell differentiation. 2) The h
c C-terminal
truncation mutants demonstrate that 681 amino acids of the h
c are
sufficient to mediate hGM-CSF-induced differentiation, but the
truncation of the h
c subunit to amino acid 627 abolishes this
differentiation response. In contrast, 559 amino acids of the h
c are
sufficient for hGM-CSF-induced cell growth. 3) Within the
h
c681 subunit, a point mutation of tyrosine 628 to phenylalanine
abolishes the ability of h
c681 to induce differentiation. 4) The
activation of either ERK or STAT5 by hGM-CSF does not correlate with
the ability of hGM-CSF to induce myeloid cell differentiation. These
results demonstrate that hGM-CSF-mediated cell growth and
differentiation require distinct regions of the h
c cytoplasmic
domain.
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EXPERIMENTAL PROCEDURES |
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Cells and Cell Culture-- The mouse IL-3 (mIL-3)-dependent WT19 cell line derived from the FDC-P1 cell line was a generous gift from Dr. Larry Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle, WA) and was described previously (10, 12). The cells were cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 10% WEHI-3B conditioned medium containing mIL-3.
Reagents-- Recombinant hGM-CSF was purchased from Immunex Corp. (Seattle, WA). Recombinant mGM-CSF and mIL-3 were obtained from Genzyme (Cambridge, MA). PD98059, a specific inhibitor of ERK kinase (MEK) was purchased from Alexis Corp. (San Diego, CA).
Plasmids--
The expression plasmid pEFBOS-hGM-CSFR
containing the wild-type hGM-CSFR
cDNA was a generous gift of
Dr. N. A. Nicola (Walter and Eliza Hall Institute for Medical
Research, Victoria, Australia). The wild-type human GM-CSFR
chain
(h
c) cDNA was removed from the plasmid pKH97 (provided by Dr. A. Miyajima, DNAX Research Institute, Palo Alto, CA), and the 2.9-kilobase
HindIII-NotI insert was ligated into pCEP4
(Invitrogen, Carlsbad, CA), which contains a hygromycin selection
marker giving the plasmid pCEP4-h
cwt.
Establishment of Stable Tranfectants--
20 µg of
pEFBOS-hGM-CSFR was introduced into WT19 cells in combination with 1 µg of pSV2-Neo plasmid (14) by electroporation using Bio-Rad Gene
Pulsar (Richmond, CA), and transfectants were isolated using G418 (0.4 mg/ml). A clone termed "WT19 pEFBOS-hGM-CSFR
" expressing
hGM-CSFR
was used for the transfection of pCEP4-h
c wild-type or
mutant constructs. Resistant clones expressing h
c were then isolated
using hygromycin selection (0.4 mg/ml).
Antibodies--
The polyclonal and monoclonal anti-hc
antibodies, anti-ERK2 polyclonal antibody (specific to both ERK1 and 2)
and the anti-Stat5b polyclonal antibody were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). The anti-STAT5b antibody did not
cross-react with Stat5a (data not shown). The anti-Stat5a polyclonal
antibody was obtained from R & D Systems (Minneapolis, MN) and does not cross-react with STAT5b (data not shown). The anti-phosphotyrosine monoclonal antibody (4G10) was purchased from Upstate Biotechnology (Lake Placid, NY). The phospho-specific anti-ERK polyclonal antibody was obtained from New England Biolabs (Beverly, MA). The
anti-hGM-CSFR
monoclonal antibody was a generous gift of Dr. A. F. Lopez (Institute of Medical and Veterinary Science, Adelaide,
Australia). The anti-F4/80 rat monoclonal antibody was purified from
the culture supernatant of HB198 rat hybridoma cell line obtained from
ATCC.
Immunoblotting-- Cells were lysed in PLC lysis buffer (50 mM HEPES, pH 7.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM NaPPi, 1 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) at 108 cells/ml. The lysates were separated on SDS-polyacrylamide gels and then electrotransferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked for 2 h in 2% bovine serum albumin-TBST (20 mM Tris-HCl, pH 7.6, 0.15 M sodium chloride, 0.1% Tween 20), incubated with primary antibodies in TBST for 1 h, washed three times with TBST, and incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or rabbit immunoglobulin (Amersham Pharmacia Biotech) diluted 1:10,000 in TBST. After three washes in TBST, the blot was developed with the enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Immunoprecipitation-- The cell lysates were incubated with the indicated antibody for 2 h at 4 °C, followed by protein A-Sepharose beads (Amersham Pharmacia Biotech) for an additional 1 h at 4 °C. The beads were washed 3 times in PLC lysis buffer, suspended in SDS-sample buffer, and heated at 95 °C for 5 min. The eluted proteins were applied to an SDS-polyacrylamide gel and detected by Western blotting.
MTS Cell Proliferation Assay-- 5,000 cells were incubated in 100 µl of RPMI 1640 containing 10% fetal bovine serum and various concentrations of hGM-CSF for 14 h at 37 °C in a humidified 5% CO2 atmosphere. 20 µl of freshly prepared combined 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulhophenyl)-2H-tetrazolium, inner salt/phenazine methosulfate (MTS/PMS) solution (Promega, Madison, WI) were added to each sample. After an additional 4 h of incubation at 37 °C, the conversion of MTS into the aqueous soluble formazan was measured at an absorbance of 490 nm.
GST Fusion Protein and Affinity Purification--
GST-MycN262 (a
generous gift from Dr. E. M. Blackwood, University of California,
San Diego, La Jolla, CA) was described previously (15). GST-MycN262
fusion protein was induced with 0.2 mM
isopropyl--D-thiogalactopyranoside and purified over
glutathione-Sepharose according to the manufacturer recommendations
(Amersham Pharmacia Biotech).
Immune Complex Kinase Assay--
The cell lysates (1 × 107 cells/sample) were incubated with 0.4 µg of
polyclonal anti-ERK2 antibody for 2 h at 4 °C, followed by
protein A-Sepharose beads (Amersham Pharmacia Biotech) for an
additional 1 h. The beads were washed 3 times in PLC lysis buffer
and then once in kinase buffer (20 mM HEPES, pH7.9, 10 mM MgCl2, 0.2 mM EDTA, 0.5 mM sodium vanadate, 10 mM -glycerophosphate, and 1 mM dithiothreitol). The kinase reaction was initiated
by the addition of 40 µl of kinase buffer with 20 µM
ATP, 5 µCi of [
-32P]ATP, and 5 µg of GST-MycN262
and was allowed to proceed for 15 min at 30 °C. The reaction was
terminated by the addition of 2× SDS sample buffer. Samples were
boiled and resolved by SDS-PAGE, and the fixed gel was exposed to an
x-ray film and the PhosphorImager (Molecular Dynamics).
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RESULTS |
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Expression of Wild-type and Mutant hc in WT19 Cells--
To
study the role of h
c in hGM-CSF-mediated differentiation, we
constructed a series of cytoplasmic deletion and point mutants of the
h
c subunit (Fig. 1A). These
mutant h
c subunits were transfected into WT19 cells expressing the
wild-type hGM-CSFR
subunit (Fig. 1, B and C).
The amino acid length of each h
c mutant represents the number of
amino acids from the N terminus including the leader sequence. As
evaluated by Western blotting and flow-cytometry, the expression level
of each mutant h
c subunit in these clonal cell lines (Fig.
2, A and B) was
found to be similar. The mutations introduced into the h
c
cytoplasmic domain did not affect the high affinity ligand binding as
reported by others (16) (data not shown). At least three clones were
isolated expressing each h
c mutant and were analyzed in the
following experiments (see below).
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Induction of Monocytic Differentiation through hc in WT19
Cells--
Parental WT19 cells growing in mIL-3 demonstrated a
myeloblastic morphology including rounded nuclei, fine chromatin, and basophilic cytoplasm. In response to mGM-CSF, the cells demonstrated monocytic characteristics: an indented nucleus with fine stranded appearance, increased cytoplasm containing a variable number of vacuoles, and larger total cell size. WT-19 cells treated with mGM-CSF
demonstrated significantly increased surface expression of F4/80, a
marker of monocyte differentiation (17). As previously reported (10),
hGM-CSF treatment of WT19
wt cells expressing hGM-CSFR
and the
wild-type h
c subunits induced monocytic differentiation of WT19
cells, as measured by changes in morphology and the surface expression
of F4/80 (Fig. 3, A and
B).
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Proliferation and Differentiation Signals Are Mediated by Different
Regions of hc--
GM-CSF regulates both cell differentiation and
proliferation of myeloid progenitor cells. To investigate whether
differentiation and proliferation signals are generated by different or
identical regions of the h
c subunit, the growth of each of the above
mutants was examined at low concentrations (1-40 pg/ml) of hGM-CSF
(Fig. 5). The growth response to hGM-CSF
of WT19 cells containing h
c796 was similar to
wt cells. Cells
containing the h
c627, h
c681, and h
c681Y628F transfectants grew
at a reduced rate when compared with
wt cells incubated with
comparable concentrations of hGM-CSF. Similar results were obtained
with WT19 cells containing h
c559 and 592 (data not shown). However,
PA cells did not show any growth response to hGM-CSF (data not
shown). Therefore, hGM-CSF-mediated signals for proliferation and
differentiation are controlled by different regions of the h
c
subunit.
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Activation of the MAP Kinase Pathway Is Not Sufficient to Stimulate
hGM-CSF-induced Differentiation--
To determine which
hGM-CSF-induced signals are important for growth and/or differentiation
of WT19 cells, the signaling pathways activated by hGM-CSF were
examined. Shc and SHP-2, when phosphorylated on tyrosines by GM-CSF
stimulation, bind to the SH2-containing Grb2 protein (24, 25) and
mediate the activation of the Ras-ERK pathway. hGM-CSF-induced
tyrosine-phosphorylation of Shc, and SHP-2 was equivalent in
WT19wt,
681,
681Y628F and
627 cell lines,
suggesting that these proteins are similarly regulated in cells that
are or are not capable of undergoing hGM-CSF-induced differentiation
(Fig. 6, A and B).
In contrast, cells that contain the box 1 proline mutant (h
cPA) were
incapable of both Shc and SHP-2 phosphorylation (Fig. 6, A
and B).
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Tyrosine Phosphorylation of STAT5 Proteins Is Not Sufficient to
Mediate hGM-CSF-induced Differentiation--
An important substrate of
the Jak2 proteins whose tyrosine phosphorylation is induced by the
addition of hGM-CSF is STAT5 (31). To determine whether STAT5
activation is sufficient to induce the differentiated phenotype,
tyrosine phosphorylation of STAT5 proteins by hGM-CSF in the mutant
hc cell lines was examined. Stat5 consists of two proteins, Stat5a
and Stat5b, encoded by separate genes, both of which have been shown to
be activated by GM-CSF (31). To investigate the hGM-CSF-mediated
activation of Stat5 proteins, immunoprecipitations were performed using
specific antibodies against Stat5a or 5b from hGM-CSF-stimulated cell
lysates followed by immunoblotting with the anti-phosphotyrosine
antibody. Both Stat5a and 5b were phosphorylated on tyrosine after
hGM-CSF treatment in WT19
wt and 681 cells, which respond to
hGM-CSF with differentiation, and in WT19
627 and
681Y628F
cells, which do not differentiate (Fig.
8). These results suggest that the activation of Stat5 proteins is not sufficient to induce
differentiation of WT19 cells.
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DISCUSSION |
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By expressing truncated forms of hc in WT19 cells, we
demonstrated that hGM-CSF-induced morphological differentiation and the
surface expression of F4/80 requires h
c subunits of 832, 796, or 681 amino acids in length (Fig. 3, Table I). In contrast, WT19 cells
expressing h
c subunit containing 627 amino acids or less did not
differentiate but were capable of growth in the presence of hGM-CSF
(Fig. 3). Mutation of the two prolines in the box 1 region blocked both
growth and differentiation. Thus, these results suggest that cell
growth and differentiation are mediated by different regions of the
receptor and that the signals generated by the box 1 region of the
receptor are necessary for both cellular events.
Similar results to those described above have been generated using M1
and WEHI-3B D+ cells as a target cell population for transfection (11).
In this study, wild-type hc and h
c783, but not h
c541 or 461, could mediate hGM-CSF-induced morphological change and differentiation
marker induction, whereas induction of cell differentiation by
h
c626 required a 50- to 100-fold higher concentration of hGM-CSF (M1
cells). However, in the WT19 cell system, neither h
c627 nor
h
c681Y628F could induce cell differentiation even at a very high
concentration (1 µg/ml) of hGM-CSF (data not shown). The results
derived from M1 cells suggest that the region between amino acids 627 and 783 is necessary for the full differentiation signal, whereas our
results more narrowly delineate the crucial region for differentiation
to lie between 628 and 681.
The results described here demonstrate that tyrosine 628 is highly
phosphorylated after hGM-CSF treatment in vivo (Fig. 4). In
the hc681 construct, this phosphotyrosine is essential for differentiation. Tyr-627 and its surrounding amino acids are conserved between human and mouse
c (SLEYLCL), suggesting that in both species
this tyrosine may play an important role in differentiation. In the
macrophage colony stimulating factor signaling, both Tyr-807 and
Tyr-721 of macrophage colony stimulating factor receptor were shown to
be necessary for monocytic differentiation and for the tyrosine
phosphorylation of phospholipase C-
(PLC
) (12, 22). The
involvement of PLC
in M-CSF-mediated cell differentiation was
further demonstrated by the inhibition of differentiation using
U-73122, a compound that acts as a specific inhibitor of PLC
(22).
The surrounding amino acid sequence of h
c Tyr-628 (YLCL) is
homologous to the optimal binding site for the N-terminal SH2 domain of
PLC
in a phosphopeptide binding assay (32). However, PLC
may not
be involved in GM-CSF-induced differentiation as suggested by the
following data. First, tyrosine phosphorylation of PLC
was not
induced by addition of hGM-CSF to WT19 cells (data not shown) or
another myeloid cell line treated with this hormone (33), although
M-CSF induced significant tyrosine phosphorylation of PLC
. Second,
U-73122 did not inhibit the differentiation of WT19 cells by mGM-CSF at
1 µM, a concentration 10 times higher than that capable
of inhibiting the M-CSF-mediated differentiation (22) (data not
shown).
Although the single tyrosine mutation in the 681 hc containing cell
line had significant biologic effects, this mutation had no significant
effect on differentiation in the wild-type receptor. The wild-type
h
c contains four tyrosines C-terminal to Tyr-628. One of them
(Tyr-882) has a similar amino acid sequence (YLSL) to Tyr-628 (YLCL).
Two of them (Tyr-766 and Tyr-842) have a leucine at the +3 amino acid
(YVEL and YCFL, respectively). The amino acid at +3 position of the
tyrosine is important in dictating the binding affinity for many SH2
domain-containing proteins (32). It is possible that a common signaling
molecule binds to a number of tyrosines in the h
c-mediating cell
differentiation so that, in the wild-type receptor, mutation of a
single tyrosine is not sufficient to abolish cell differentiation.
The role of ERKs in differentiation appears to be complex and may
depend on the cell type and the stimulus involved. The ERK pathway has
been suggested to be essential for the neurite outgrowth of PC12 cells
(27, 34). Also, interleukin 6 (IL-6) can induce PC12 differentiation
when the cells are pretreated with nerve growth factor (35). A mutation
of a specific tyrosine in gp130, the IL-6 receptor signaling subunit
involved in ERK activation, abolishes the induction of cell
differentiation by IL-6 (23). Our results suggest that the ERK pathway
may not be involved in hGM-CSF-mediated differentiation of WT19 cells.
1) Although Tyr-628 of hc was shown to be a binding site for SHP-2
SH2 domains (36), it was not necessary for hGM-CSF-induced tyrosine
phosphorylation of SHP-2 or ERK activation (Fig. 6) (36). 2) PD98059, a
specific inhibitor of MEK, blocked activation, but this compound had no significant effect on differentiation. 3) The expression of a constitutively active mutant of MEK in WT19 cells did not induce differentiation.2 Thus,
activation of ERK does not appear to be sufficient or necessary for
hGM-CSF-induced differentiation of WT19 cells.
Activation of Stat proteins by several cytokines has been suggested to
be involved in cell differentiation (37-42). In WT19 cells transfected
with hGM-CSFR and wild-type h
c, hGM-CSF induced tyrosine
phosphorylation of both isoforms of Stat5, but not other Stat proteins
(Stat1, Stat3, and Stat6 examined). Tyr-628 was not necessary for the
hGM-CSF-mediated tyrosine phosphorylation of either Stat5a or 5b. In
addition, both h
c628 and h
c681Y628F could induce tyrosine
phosphorylation of Stat5a and 5b in response to hGM-CSF equivalent to
that seen in WT19 cells containing the wild-type receptor (Fig. 8).
These results suggest that Stat5 activation is not sufficient for the
induction of cellular differentiation. In a recent report, using
chimeric receptors of erythropoietin and IL-3, a delayed activation of
Stat5 was suggested to be important for the erythroid differentiation
induction of BaF/3 cells (43). However, we did not see a delay in the
time course of Stat5 activation in WT19 transfectants which
differentiate in response to hGM-CSF in contrast to those cell lines
which are capable of only growth (data not shown). Thus, none of the
signals examined (STAT5 activation along with SHP-2 and Shc
phosphorylation) are sufficient to modulate differentiation.
In conclusion, our data suggest that cell differentiation and cell
growth induced by hGM-CSF are mediated by different regions of the
hc subunit. Attempts are underway to define the novel signals
generated by amino acids 627-681 of the h
c which control the
differentiated phenotype.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK44741 (to A. S. K.) and CA45672 (to M. B. L.) and a Veterans Affairs Merit Award (to M. B. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by direct grants from the University of Alabama and the University of Colorado.
To whom correspondence should be addressed: Division of
Medical Oncology, University of Colorado Health Science Center, 4200 East Ninth Ave., Denver, CO 80262. Tel.: 303-315-8802; Fax:
303-315-8825.
1 The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; GM-CSFR, GM-CSF receptor; hGM-CSF, human GM-CSF; mGM-CSF, murine GM-CSF; ERK, extracellular signal-regulated kinase; IL, interleukin; MEK, ERK kinase; PCR, polymerase chain reaction; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulhophenyl)-2H-tetrazolium, inner salt; PMS, phenazine methosulfate; PLC, phospholipase C; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.
2 T. Matsuguchi and A.Kraft, unpublished data.
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REFERENCES |
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