From the
PanVera, Madison, Wisconsin 53719 and the
¶Department of Biomolecular Chemistry, University
of Wisconsin, Madison, Wisconsin 53706
Received for publication, February 10, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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The cause of this increase in the assortment of phosphorylated proteins has
not yet been completely explored; however, EGF-induced receptor
internalization and degradation occurred at a slower rate with an oncogenic
EGFR mutant possessing a COOH-terminal truncation at residue 973 rather than
with wild-type EGFR (10,
13). Cells expressing this
mutant develop a transformed phenotype when grown in the presence of low EGF
concentrations (13). Moreover,
data suggest that 973-EGFR and ErbB2 heterodimers form in cells
expressing this mutant. The ErbB2 in these cells is tyrosyl phosphorylated in
the absence of added ligand
(12). Interestingly, the
oncogene product of the avian erythroblastosis virus (v-erbB) is an
NH2- and COOH-terminal truncated version of EGFR (ErbB1)
(14,
15).
Cellular changes in phosphotyrosine levels may occur not only as a result of changes in the catalytic properties of the enzyme, but may also reflect alterations in the regulation of other kinases and/or phosphatases in addition to the possible changes in receptor internalization and dimerization already mentioned. For example, although cellular phosphorylation of caveolin-1 is more pronounced in cells expressing COOH-terminal truncated EGFR (9), whether the mutated EGFR directly phosphorylates this substrate remains unclear. Therefore, to precisely isolate the effects of an EGFR mutation on the tyrosine kinase activity, it was necessary to purify the mutant and compare its activity to that of wild-type enzyme in a biochemical assay.
In this study, an in vitro fluorescence polarization (FP) assay
(described below) has been used quantitatively to compare steady-state kinetic
parameters of wild-type EGFR with those of the ct1022 mutant. FP measures
molecular rotation occurring during the fluorescence lifetime, the period
between excitation and emission of a fluorophore. Relatively small molecules
tumble quickly in solution. When excited by plane-polarized light, these
molecules emit light that is relatively depolarized. In contrast, larger
molecules and complexes tumble more slowly in solution, and emit more highly
polarized light (16). The FP
competitive immunoassay involves the addition of a fluorescently labeled
phosphopeptide tracer (a relatively small molecule) and an antibody (a
relatively large molecule) specific for phosphorylated tyrosine to a kinase
reaction. Initially, the phosphopeptide tracer binds to the antibody. As the
kinase reaction proceeds, however, phosphorylated substrate binds to the
antibody and displaces the tracer. Free fluorescent tracer possesses a lower
polarization value than does antibody-bound tracer; therefore, displacing the
fluorescent tracer decreases the polarization value. Changes in polarization
can readily be monitored with a FP instrument
(Fig. 1)
(17,
18). To determine whether the
steady-state kinetic parameters for wild-type EGFR and for the ct1022 mutant
would vary with the choice of substrate, five different substrates were
employed: two synthetic peptide substrates, two natural substrates (the Shc
adapter protein, and a 321-residue PLC- fragment containing the
complete SH2-SH2-SH3 domains), and a poly(Glu,Tyr) 4:1 copolymer.
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Fig. 2 depicts the basic structural features of EGFR (3). A recently solved crystal structure of EGFR encompassing residues 672998 suggests that for this enzyme, interaction of the COOH-terminal substrate tyrosines with the active site, rather than phosphorylation of the kinase domain "activation loop," is important for regulating cellular processes (19). Additionally, the structure reveals that Leu955 of the Leu955-Val956-Ile957 dimerization motif is closely associated with the kinase domain, and we therefore hypothesize that truncations in the COOH terminus may cause conformational changes that alter substrate specificity.
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In this paper, we used an FP competitive immunoassay to measure the amount of phosphorylated substrate produced in kinase reactions. We generated standard curves relating FP values to concentrations of phosphorylated forms of each substrate. In addition, we performed phosphorylation time course assays with EGFR and the ct1022 mutant for each of the five EGFR substrates described above at multiple substrate concentrations. We monitored the reaction progress using FP. We then determined the corresponding amount (pmol) of phosphorylated substrate generated at each time point using the standard curves for each substrate. We plotted the initial velocities resulting from these time course experiments as a function of substrate concentration and fitted the data to the Michaelis-Menten equation. We determined and compared the steady-state kinetic parameters for phosphorylation catalyzed by both wild-type EGFR and the ct1022 mutant for each substrate.
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EXPERIMENTAL PROCEDURES |
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FP AssayWe used PanVera's tyrosine kinase assay kit, green,
for the FP assays. Polarization was monitored using either PanVera's
BeaconTM 2000 fluorescence polarization instrument or the Tecan Ultra
(490485 nm excitation, 525535 nm emission). Black round-bottom
96-well plates (DYNEX Technologies, Inc.) and 384-well plates
(Cliniplate/Labsystems) were used with the Tecan Ultra. For the
[-32P]ATP assay, Redivue [
-32P]ATP was
purchased from Amersham Biosciences, and Schleicher & Schuell Protran
nitrocellulose membranes were used for filtration.
Substrate PeptidesPeptides employed included angiotensin II
and the Y1173 peptide, a 13-residue peptide mimic for the region around the
Y1173 autophosphorylation site (TAENAEYLRVAPQ). The tyrosyl-phosphorylated
version of angiotensin II was purchased from Calbio-chem at 98% purity.
The tyrosyl-phosphorylated version of the Y1173 peptide was custom synthesized
by the University of Wisconsin Biotechnology Center at
99% purity.
Unphosphorylated forms of angiotensin II and the Y1173 peptide were purchased
from the same vendors at
98 and 99% purity, respectively. Aqueous samples
of these peptides were quantified by amino acid analysis at the Medical
College of Wisconsin Protein/Nucleic Acid Facility. The poly(Glu,Tyr) 4:1
copolymer and ATP were purchased from Sigma, and a fragment of PLC-
(residues 530850, containing two SH2 domains and one SH3 domain) was
purchased from Santa Cruz Biotechnology.
FP Assay ConditionsEach experiment (except the
[-32P]ATP time courses) was performed using PanVera's
tyrosine kinase assay kit, green. All reactions and preincubations were
carried out at 30 °C in the following reaction buffer: 20 mM
Hepes (pH 7.4), 5 mM MgCl2, 2 mM
MnCl2, 0.05 mM Na3VO4, and 1
mM dithiothreitol, unless otherwise noted. EGFR was preincubated
for 15 min with the indicated concentration of ATP to autophosphorylate the
kinase. The reaction was initiated with the addition of substrate, and the
decrease in polarization was measured as described. The 15-min preincubation
provided ample time for autophosphorylation
(23). Furthermore, we observed
that any decrease in polarization caused by autophosphorylation leveled off
within the 15-min preincubation (data not shown), so that any observed change
in polarization was because of substrate phosphorylation, not
autophosphorylation. All FP assays were conducted in the presence of antibody
and tracer at 1x concentrations. Assays performed in end-point mode were
quenched by the addition of protein-tyrosine kinase quench buffer (12
mM EDTA, pH 7.5) and antibody and tracer to 1x, for final
concentrations of 6 mM EDTA, 1x antibody, and 1x
tracer.
[-32P]ATP Phosphate Incorporation
AssayThe amount of phosphate incorporated into the substrate was
quantified using radiolabeled ATP
(23,
24). Aliquots of the reaction
mixture (60 µl) were removed and quenched with 700 µl of 10%
trichloroacetic acid. Three microliters (3 µl) of acetylated bovine serum
albumin (10 mg/ml) was added, and the precipitated poly(Glu,Tyr) 4:1 copolymer
or the Shc adapter protein was separated from the radiolabeled ATP by
filtration. The radioactivity incorporated into the product was quantified by
liquid scintillation counting.
Data AnalysisAll data were fitted with PrismTM
software, version 3.0 (GraphPad Software Inc.) using the equations below.
One-site competition, where mP = polarization expressed in units of milli-P (1
mP = 0.001 P),
![]() | (Eq. 1) |
One-site competition, solving for log picomole of phosphate incorporated is
as follows.
![]() | (Eq. 2) |
![]() | (Eq. 3) |
A single exponential decay, solving for picomole of phosphate incorporated
is as follows.
![]() | (Eq. 4) |
Straight line is the following equation.
![]() | (Eq. 5) |
Michaelis-Menten equation is shown below.
![]() | (Eq. 6) |
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Sigmoidal dose response (variable slope) is shown as,
![]() | (Eq. 7) |
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RESULTS |
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After generating the competition standard curves, we performed FP assays
and calculated the corresponding amounts of phosphorylated product formed from
the transformed one-site competition equation for each substrate
(Equation 2). Using FP, we
measured substrate phosphorylation directly for each substrate at multiple
substrate concentrations. Representative data for both the Y1173 substrate and
the PLC- fragment are shown in Fig.
3B. In the FP assay, product formation is
"coupled" to fluorescent phosphopeptide tracer dissociation from,
and the concomitant binding of non-fluorescent phosphorylated substrate
(product) to, the antibody, resulting in product detection. Bound product
eventually displaces all of the fluorescent phosphopeptide tracer. Therefore,
plots of polarization versus time are not linear over long time
courses. As the assay progresses to completion, the decrease in polarization
gradually plateaus to the polarization value of the free tracer. Although the
equation for a straight line could not describe data for the entire time
course, the equation for a single exponential decay
(Equation 3) described the data
very well, with R2 = 0.98 and 0.99 for the Y1173 peptide
and the PLC-
fragment time courses, respectively. We used
Equation 2 (which converts the
exponential data to logarithms, thus linearizing them) to calculate the extent
of substrate phosphorylation for each individual time point with the
competition standard curves, and plotted the results with respect to time.
These data were linear, and initial velocities were determined from the slopes
of these lines (Equation 5).
Similar experiments were performed at multiple substrate concentrations, and Fig. 3C depicts representative data for the Y1173 peptide substrate. Time courses were prepared involving the preincubation of 0.41.7 nM (0.10.4 units/100 µl) wild-type EGFR or 0.5 nM (0.2 units/100 µl) ct1022 mutant with 2.5 mM ATP, followed by the addition of the Y1173 peptide. Polarization values were transformed into picomole of phosphate transferred/min/pmol of EGFR (Equation 2) as described, and plotted as a function of substrate concentration. Data were fitted to the Michaelis-Menten equation (Equation 6) (26), and the steady-state kinetic parameters were thus determined. The mutant enzyme yielded a 4-fold higher turnover number for the phosphorylation of the Y1173 peptide substrate than did EGFR. The steady-state kinetic parameters determined in this manner for each substrate are described in Table I.
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Because the poly(Glu,Tyr) 4:1 copolymer and the PLC- fragment
contain more than one potential phosphorylation site, the kinetic parameters
for these substrates are apparent values
(27). Full-length PLC-
has four tyrosines that are phosphorylated by EGFR (Tyr-472, Tyr-771, Tyr-783,
and Tyr-1254) (28). Therefore,
the fragment of PLC-
we used as a substrate (residues 538850)
possesses two tyrosines that EGFR can phosphorylate (Tyr-771 and Tyr-783).
Steady-state kinetic parameters for angiotensin II and the PLC-
fragment were within experimental error of those previously measured using the
[
-32P]ATP phosphate incorporation assay
(24).2
Verification of Competition Standard Curve Results for the
Poly(Glu,Tyr) 4:1 Copolymer SubstrateAlthough previous control
experiments indicated that the poly(Glu,Tyr) 4:1 copolymer and PLC-
fragment used for generating the competition standard curves had been
completely phosphorylated, we further verified these results by employing an
additional method for generating a standard curve for the poly(Glu,Tyr) 4:1
copolymer. This second approach calls for performing identical reactions and
evaluating them by different methods. We analyzed one reaction with the
traditional kinase assay using [
-32P]ATP, and two other
reactions with the FP technique. Given that conditions (enzyme and substrate
concentrations, temperature, buffer, etc.) were the same for all reactions,
the extent of substrate phosphorylation required to produce a given decrease
in polarization could be calculated. We measured the phosphorylation of the
poly(Glu,Tyr) 4:1 copolymer (150 nM) by the ct1022 mutant (0.5
nM, 0.13 units/100 µl) in three reactions: the FP assay in both
real-time and end-point modes and by the [
-32P]ATP assay
(Fig. 4A) as described
under "Experimental Procedures." Each time course was set up under
identical conditions (10 µM ATP and the buffer and temperature
described), except that the [
-32P]ATP assay reaction
included 7.5 µCi of [
-32P]ATP/100 µl (inset, left
side). Data for the [
-32P]ATP time course were fitted
to the equation for a straight line, with slope = initial velocity = 0.070
± 0.008 pmol of phosphate transferred per min, and intercept =
0.1 ± 0.1 pmol of phosphate transferred. For each time point of
the FP assays, the amount of phosphate transferred to the substrate was
calculated using the equation that describes the [
-32P]ATP
time course: picomole of phosphate incorporated = 0.070 pmol of phosphate
incorporated per min x time 0.1 pmol of phosphate incorporated.
Polarization was plotted as a function of picomole of phosphate incorporated,
and data were fitted to Equation
7, with IC50 real-time assay = 0.27 ±
0.01 pmol of phosphate incorporated, Hill slope = 1.36 ± 0.03,
and IC50 end-point assay = 0.26 ± 0.01 pmol of
phosphate incorporated, Hill slope = 2.5 ± 0.2. These resulting
IC50 values were very similar to each other and to that determined
previously using a competition standard curve
(Fig. 3A) (0.25 pmol
of phosphate incorporated). Therefore, the kinetic parameters determined by
converting polarization to phosphorylated product with either method of
generating a standard curve should be essentially identical. Furthermore, the
inset (right side) presents the same data without a log
scale, described by Equation 3.
For the real-time assay, mPhighest mPlowest =
149.3 ± 0.8 mP, k = 2.77 ± 0.03 pmol of
phosphate1, and mPlowest = 43.9
± 0.2 mP. For the end point assay, mPhighest
mPlowest = 220 ± 9 mP, k = 3.0 ± 0.3 pmol of
phosphate1, and mPlowest = 34 ±
8 mP. A control reaction was performed that also contained 7.5 µCi of
[
-32P]ATP/100 µl, but was analyzed in real time by FP in
the BeaconTM 2000 fluorescence polarization instrument. Results were
within experimental error of those obtained in the absence of
[
-32P]ATP (data not shown).
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Fig. 4B illustrates a representative time course in which the reaction was monitored by FP, and the amount of phosphate incorporated at each time point was calculated from the standard curve. Similar data were collected at multiple substrate concentrations, and steady-state kinetic parameters were measured (Fig. 4C). EGFR (0.50.8 nM, 0.10.2 units/100 µl) or the ct1022 mutant (0.30.5 nM, 0.10.2 units/100 µl) were preincubated with 2.5 mM ATP for 15 min, then substrate was added and polarization was monitored. Polarization values were transformed into picomole of phosphate transferred per min/pmol of EGFR as described, plotted versus time, and initial velocities were described by the slopes of these lines. Initial velocities were then graphed as a function of substrate concentration, and these data were fitted to the Michaelis-Menten equation. The steady-state kinetic parameters thus determined for the poly(Glu,Tyr) 4:1 copolymer as well as the other substrates are shown in Table I, and the turnover number for the phosphorylation of this substrate is 3-fold higher with the ct1022 mutant.
Similarly, a standard curve for the 46-kDa isoform of the Shc adapter protein was generated by running two identical reactions (0.5 µM Shc, 10 µM ATP, and 5.1 nM ct1022 mutant at 30 °C): one analyzed by FP in real time using the BeaconTM 2000 fluorescence polarization instrument, and the other by the radioactive assay as described. An initial velocity of 0.025 ± 0.004 pmol/min was measured (data not shown) and standard curves similar to those for the poly(Glu,Tyr) 4:1 copolymer were generated, with k = 1.88 ± 0.03 pmol1 from the direct fit using a single exponential decay, and IC50 = 0.46 ± 0.03 pmol of phosphate incorporated (data not shown). Steady-state kinetic parameters for catalysis of phosphorylation by wild-type and mutant enzyme were determined by fitting plots of initial velocity versus substrate concentration to the Michaelis-Menten equation (Equation 6), and Table I summarizes the kinetic parameters so determined for the Shc adapter protein as well as all of the other substrates.
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DISCUSSION |
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The FP assay has the advantages of being nonradioactive and amenable to
high throughput screening. Furthermore, we have shown that this particular FP
assay can be performed at relatively high ATP concentrations (2.5
mM), which would be more difficult with the traditional radioactive
assay. In the [-32P]ATP assay, sensitivity is directly
proportional to the concentration of radioactive ATP and inversely
proportional to the concentration of unlabeled ATP. Therefore, the amount of
radioactive ATP needed to conduct assays at physiological ATP concentrations,
which are often in the millimolar range, would be quite high. Some examples of
intracellular ATP concentrations include 3 mM for yeast
(29), 1 mM in
smooth muscle cells (30), and
2.6 mM in human astrocytoma cells
(31). We found that the
Km values for ATP were in the micromolar range
for certain substrates. For example, for the poly(Glu,Tyr) 4:1 copolymer
(substrate concentration = 2 ng/µl (60 nM), using the FP assay
at room temperature, as described under "Experimental
Procedures"), the Km(ATP,EGFR (wild-type))
= 6 ± 2 µM and the
Km(ATP,ct1022 mutant) = 1.9 ± 0.8
µM, data not shown. However, we observed that the
Km for ATP with the PLC-
fragment
(wild-type EGFR, substrate concentration = 0.2 µM, using the FP
assay at 30 °C, as described under "Experimental Procedures")
is much greater than 50 µM (data not shown). Therefore, the
amount of [
-32P]ATP required in the
[
-32P]ATP filtration assay (if it were conducted near or
above the Km for ATP) would increase
dramatically. Furthermore, for the Shc adapter protein, while we found that
Km(ATP,ct1022) was less than 10 µM
(substrate concentration = 0.5 µM, using the FP assay at 30
°C), the Km(ATP,EGFR (wild-type)) was greater
than 100 µM (substrate concentration = 1.5 µM,
using the FP assay at 30 °C). Consequently, FP may be an easier method for
analyzing the kinetics of the wild-type enzyme for this natural substrate.
Moreover, we have demonstrated that this assay is accurate in real time (Fig. 4A). The comparison of the real time versus the end point assay for the poly(Glu,Tyr) 4:1 copolymer suggests that, for this particular FP assay, the time required for phosphorylated product to come to equilibrium with the antibody and tracer is so brief that the results are unaffected. Thus, the assay may be performed directly in an instrument measuring FP, rather than by quenching individual time points with EDTA and waiting for antibody and tracer to come to equilibrium. Therefore, during screening experiments, control reactions run in the absence of inhibitor could be monitored to determine the appropriate time to quench the reaction with EDTA.
It is important to keep in mind that the relationship of polarization to phosphorylation of substrate is not linear (Figs. 3, A and B, and 4, A and B), and that the assay is more sensitive to phosphorylation before all of the tracer is displaced. In measuring initial rates to determine kinetic parameters, continuous assays are always preferable to single time point assays (32), and this is also true for the FP technique. A standard curve, however, takes into account the "leveling off" of polarization as tracer is displaced.
Comparison of Kinetic Parameters for Reactions Catalyzed by the ct1022 Mutant to Those for Wild-type EGFROur data demonstrate that the steady-state kinetic parameters for the ct1022 mutant differ from those of the wild-type enzyme, and these differences depend on the identity of the substrate (Table I). The increases in turnover number for certain substrates along with the studies of internalization and possible heterodimer formation with ErbB2 may explain the broader spectrum of proteins phosphorylated when COOH-terminal truncated mutant varieties of EGFR are expressed (913). Although the reason for the increase in the turnover number for the Y1173 peptide and the poly(Glu,Tyr) 4:1 copolymer is unknown, more rapid product release rather than an increase in the rate of the catalytic step may contribute, as product release has been found to be rate-limiting for the catalysis of phosphorylation of a peptide substrate by ErbB2 (33, 34). More deeply truncated EGFR mutants, on the other hand, have been reported to be less active than wild-type EGFR in vitro with peptide substrates (35).
In addition to the investigations performed with purified proteins,
differences in the amounts of specific phosphorylated proteins formed in the
presence of COOH-terminal truncated versions of EGFR compared with those
formed in the presence of wild-type EGFR have been explored in transfected
cells. The results suggest that phosphorylation of both PLC- and
Ras-GTPase activating protein-associated protein p62 was dramatically reduced
in EGFR mutants truncated at residues 1011 or 973; however, the truncated EGF
receptors could still induce phosphorylation of the Shc adapter protein
(10,
3638).
Furthermore, caveolin-1 phosphorylation occurred in cells expressing truncated
EGF receptors, but not in cells expressing wild-type or kinase-inactive EGFR
(9). As mentioned earlier,
changes in phosphotyrosine levels observed in vivo may be because of
changes in the regulation of other kinases and/or phosphatases, and not due
simply to an alteration in the catalytic properties of the enzyme in question.
However, EGF-dependent cell growth did not occur in cells expressing a ct1022
kinase negative double mutation, but did occur in cells expressing an EGFR
mutant truncated at residue 973, suggesting the phenotypic importance of
kinase activity for COOH-terminal truncation mutants
(13).
Interestingly, we observed that the ct1022 mutant phosphorylated the
PLC- fragment in vitro as efficiently as did purified
wild-type EGFR. Only one of the five tyrosine autophosphorylation sites
remains in the ct1022 mutant (Tyr-992), and this phosphorylation site has been
identified as the critical site for PLC-
binding
(39).
We hypothesize that the lower turnover number for the ct1022 mutant (compared with the wild-type enzyme) for phosphorylating the Shc adapter protein may be because of slower product dissociation, because, as noted earlier, product dissociation is rate-limiting for ErbB2 (33). Furthermore, the lower Km might suggest a tighter association between the ct1022 mutant and Shc than between wild-type EGFR and Shc. In the absence of data describing the individual rate constants, we cannot determine whether the Km equals the dissociation constant for enzyme and substrate. However, we expect that the Shc adapter protein as well as GrbB2 associates with EGFR in vivo and thus participates in the initiation of the mitogen-activated protein kinase cascade (1).
In sum, when compared with wild-type EGFR, several substrates are more efficiently phosphorylated by the ct1022 mutant, whereas other substrates are equally well phosphorylated or even less efficiently phosphorylated by the mutant receptor. These data reveal that COOH-terminal sequences may participate in determining the substrate specificity of EGFR, in addition to serving as docking sites for various effector molecules.
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FOOTNOTES |
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|| Current address: 5 Oakwood Circle, Madison, WI 53719.
To whom correspondence should be addressed: PanVera, 501 Charmany Dr.,
Madison, WI 53719. Tel.: 608-204-5000; Fax: 608-204-5200; E-mail:
jane_beebe{at}panvera.com.
1 The abbreviations used are: EGF, epidermal growth factor; CR, cysteine-rich
domain; ct1022 mutant, EGFR mutant possessing a truncation in the COOH
terminus after residue 1022; EGFR, EGF receptor; FP, fluorescence
polarization; mP, millipolarization unit (1 polarization unit = 1000 mP
units); Y1173 peptide, 13-residue peptide mimic for the region around the
major autophosphorylation tyrosine (tyrosine 1173) and the Shc binding site;
v-erbB, avian erythroblastosis virus; PLC-, phospholipase
C-
.
2 P. J. Bertics, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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