From the
An optical biosensor technology based on surface plasmon
resonance was used to determine the kinetic rate constants for
interactions between the CryIA(c) toxin from Bacillus thuringiensis and brush border membrane vesicles purified from susceptible and
resistant larvae of diamondback moth (Plutella xylostella).
CryIA(c) association and dissociation rate constants for vesicles from
susceptible larvae were determined to be 4.5
The Gram-positive, spore-forming bacterium Bacillus
thuringiensis is the most widely used microbial pest control agent
(1-3). Many strains of B. thuringiensis contain
parasporal crystals comprised of one or more insecticidal crystal
proteins. After ingestion by insects, the crystals are solubilized in
the midgut and the proteins are activated by gut proteases. These
activated toxins bind to specific high affinity sites on the midgut
epithelial cell layer in susceptible insects. A breakdown in gut
integrity and eventual larval death follows the initial binding
event
(4, 5, 6) . The toxin inserts into the
membrane of cultured insect cells
(7) or lipid
bilayers
(8, 9, 10) and subsequently forms ion
channels. In larvae of the moth Manduca sexta, a
membrane-bound aminopeptidase N is the receptor for the CryIA(c) toxin
of B. thuringiensis(11, 12) .
Demand for
B. thuringiensis is growing rapidly because of its
environmental compatibility and amenability to genetic engineering
(13). Whether B. thuringiensis toxins are delivered in
conventional sprays or in transgenic plants, the greatest threat to
their continued success is the evolution of resistance in
pests
(14, 15) . Many insects have evolved with a
resistance to B. thuringiensis in response to laboratory
selection; the diamondback moth, Plutella xylostella, is the
first pest to develop resistance to B. thuringiensis in open
field populations (14-16).
Results from P. xylostella and the indian meal moth, Plodia interpunctella, suggest
that reduced binding of toxins to midgut receptors is a primary
mechanism of resistance to B.
thuringiensis(17, 18, 19, 20) . One
resistant strain of P. interpunctella also showed reduced
proteolytic activation of a B. thuringiensis toxin
(21) . In laboratory-selected resistant strains of
Heliothis virescens, reduced binding was not associated with
resistance to B. thuringiensis(22, 23) ,
suggesting that changes in postbinding events like calcium influx and
loss of pH regulation
(7) could also contribute to resistance.
Previous studies comparing the binding of B. thuringiensis toxins to resistant and susceptible strains were performed at
equilibrium using BBMVs
Independent measurement of the rates of
association and dissociation of toxin-receptor complexes can be made
with an optical detection system that exploits a phenomenon called
surface plasmon resonance
(24, 25, 26) . In this
system, one of the reactants (either toxin or receptor) is immobilized
to a hydrophilic dextran layer on the surface of a gold sensor chip in
a microflow cell
(27) . As binding occurs, the increase in total
mass attached to the sensor chip changes the angle of polarized light
reflected from the surface of the chip. These changes are detected by a
diode array. By linking this system to a personal computer, one can
measure complex association and dissociation in real time. We
previously used this approach to determine association and dissociation
rates for the B. thuringiensis toxin CryIA(b) and immobilized
BBMVs from larvae of Choristoneura fumiferana(26) .
To achieve a better understanding of the interactions between
CryIA(c) and its specific binding protein on the brush border surface,
the kinetic binding rates of activated CryIA(c) toxin to BBMVs isolated
from P. xylostella larvae were determined. The aim of this
work was to assess if the CryIA(c) resistance phenotype in a
field-resistant population could be correlated to alterations in toxin
binding rates.
We increased the LC
Nonspecific binding of B. thuringiensis toxins to exposed
plasma membranes has been noted by Ryerse et al.(37) .
To assess the extent of nonspecific binding of Cry toxins to BBMVs from
P. xylostella, we used papain-activated CryIIIA, which does
not bind to the midgut epithelial cells of P. xylostella (19).
CryIIIA was either immobilized to the dextran layer on the CM5 sensor
chip surface (immobilized toxin configuration) or injected over
immobilized P. xylostella BBMVs (immobilized vesicle
configuration). To further examine toxin binding specificity to BBMVs,
homologous/heterologous toxin competition experiments were used.
Preincubation with a toxin homologous to that found on the immobilized
surface should specifically block BBMV binding to the surface, whereas
using a heterologous toxin having its own specific receptor should not
compete, resulting in BBMV binding to the sensor chip surface.
Dissociation rate constants were determined by a
different method than was described previously for BBMVs
(26) .
Because the level of nonspecific binding is lowest in the immobilized
toxin configuration, all dissociation experiments were performed by
immobilizing CryIA(c) to the sensor chip surface and saturating the
surface with BBMVs. The absence of vesicles when the flow is replaced
with buffer allows the determination of kd by plotting the log
of the drop in response against the time interval,
ln(Rt
Binding to BBMVs
from resistant P. xylostella was unexpected because several
studies using B. thuringiensis-resistant P. xylostella and CryIA(b)
(18, 19) or CryIA(c) toxin
(20) demonstrated little or no binding of these toxins to BBMVs.
In particular, the resistant larvae tested here were derived from the
same strain that showed very little binding of radiolabeled CryIA(c) to
BBMVs
(20) . The difference between the present work and that
with radiolabeled toxin is difficult to explain, especially because we
do not yet understand the relationship between results of surface
plasmon resonance kinetic studies compared with results of equilibrium
binding studies. One of the problems with both the equilibrium binding
studies and the present work is that toxin-vesicle interactions are
presumably biphasic. The first phase consists of toxin-receptor
binding, whereas the second phase involves integration of the toxin
into the membrane. Indeed, it has been shown that B. thuringiensis toxins can integrate into lipid bilayers in the absence of a
specific receptor (8-10). Because one cannot discriminate between
the two phases, most binding calculations represent an amalgamation of
the two, with the possible reappearance of free receptor to further
confound the results. To determine the extent of the membrane
integration problem, solubilization of the CryIA(c) receptor could be
achieved by cleaving the glycosylphosphatidylinositol
anchor
(38) , thus stripping it from the vesicle and eliminating
interference from the lipid environment.
A possible explanation for
the contrasting binding results may rest with the additional BBMV
processing required by the surface plasmon resonance technique. BBMVs
are sonicated to enable insertion of the biotin-fatty acid anchor and
to reduce the vesicle size so that the BBMVs are small enough to pass
through the 0.5-µm diameter flow tubing in the apparatus.
Cation-precipitated vesicles used directly in receptor studies normally
range in size from 0.1 to 1 µm and possess different
morphologies
(33, 39) . It is possible that the
sonication step may disrupt the masking of the receptor by one or more
tightly associated proteins in the vesicles prepared from resistant
larvae. It has been shown that the 120-kDa receptor from M. sexta is tightly associated with a number of other membrane
proteins.
CryIC toxin also bound to BBMVs
from either susceptible or resistant larvae but did not compete with
CryIA(c) in heterologous competition experiments, indicating that this
toxin recognizes a different receptor than CryIA(c). Activated CryIE
toxin, which is nontoxic to P. xylostella, showed no net
binding (i.e. the amount of RU remaining bound after toxin
flow is switched to buffer) to immobilized BBMVs in agreement with
previous results, indicating that binding is a prerequisite for
toxicity
(5, 19, 40) . The association and
dissociation rate constants and consequently the calculated affinity
constant for CryIA(c) toxin to BBMVs from both susceptible and
resistant larvae were not significantly different. The high affinity
binding of CryIA(c) to the receptor from resistant larvae supports the
view that toxin binding does not necessarily result in
toxicity
(23, 41, 42) . The
K
One interesting discovery from our immobilized
toxin experiments was the capacity of toxin molecules to multimerize on
an immobilized toxin surface. Although CryIA(c) toxin associates
rapidly with the CryIA(c) toxin immobilized to the hydrogel on the
sensor chip surface, the formed protein complexes dissociate rapidly.
What role these toxin-toxin interactions may play in toxicity is
uncertain at this point, although one could speculate that, like the
role proposed for the receptor
(43) , toxin-toxin binding could
increase the effective concentration of the toxin at the microvillar
membrane surface. It is unknown if the toxin acts in the form of a
monomer or a multimer at the cell surface to create a pore, but the
appearance of clearly defined ion channel substates in planar lipid
bilayers suggests that the toxin may act in an multimeric form
(9) similar to the hexameric
We thank R. Ménard and M.
O'Connor-McCourt for helpful discussions, N. Finson, T.
Gonsalves, and B. Helvig for rearing insects, A. M. Mes-Masson for
providing the dissected mouse intestines, R. Frutos for providing the
recombinant cryIC and cryIE genes in B.
thuringiensis, and W. J. Moar for providing CryIC toxin.
10
M
s
and 3.2
10
s
, respectively,
resulting in a calculated affinity constant of 7 nM. CryIE
toxin did not kill susceptible or resistant larvae and did not bind to
brush border vesicles. Contrary to expectations based on previous
studies of binding in resistant P. xylostella, the binding
kinetics for CryIA(c) did not differ significantly between susceptible
larvae and those that were resistant to CryIA(c). Determination of the
number of CryIA(c) receptors revealed an approximately 3-fold decrease
in total CryIA(c) receptor numbers for resistant vesicles. These
results suggest that factors other than binding may be altered in our
resistant diamondback moth strain. They also support the view that
binding is not sufficient for toxicity.
(
)
and radioactively
labeled
toxins
(17, 18, 20, 22, 23) . The
calculated affinity constants have thus been based on the net effects
of association and dissociation of toxin-receptor complexes.
Information on the rate of both complex formation and dissociation
would be valuable because dramatic rate alterations may not always be
evident at equilibrium.
Insects
We studied larvae from two Hawaiian
strains of diamondback moth. The susceptible strain (LAB-P) was derived
from individuals collected from Pulehu, Maui
(16) . The
susceptible strain had been reared in the laboratory for >80
generations without exposure to any insecticide. The resistant strain
(NO-QA) was derived from individuals collected from a watercress farm
near Pearl City, Oahu. Repeated exposure to sprays of commercial
formulations of B. thuringiensiskurstaki in the
field produced a 20-fold increase in the LC (concentration
needed to kill 50% of larvae) of the resistant strain relative to the
susceptible strain (16).
of the
resistant strain in the laboratory by selecting it repeatedly with
Dipel
(28, 29) , a commercial formulation of the HD-1
strain of B. thuringiensiskurstaki that contains
spores and insecticidal crystal proteins CryIA(a), CryIA(b), CryIA(c),
CryIIA, and CryIIB
(1, 3, 30) . The concentration
of Dipel ranged from 25.6 to 2560 mg of active ingredient / liter. In
most selected generations, mortality was between 40 and 70%. Larvae
were reared on cabbage, and colonies were maintained as described
previously
(16, 20) .
Insect Bioassays
CryIA(b), CryIA(c), CryIC, and
CryIE protoxins were tested against third instar larvae of the
susceptible and resistant strains using leaf residue
bioassays
(16, 31) . For each toxin, resistant and
susceptible strains were tested simultaneously. Four replicates of an
average of 10 larvae (total of 40) from each strain were tested at l0
and 100 µg of protoxin/ml. Mortality was recorded 2 days after
larvae were placed on treated leaves. For CryIB, mortality at 10 and
100 µg of protoxin/ml was estimated from previously reported probit
regression lines
(32) .
BBMV Purification
Late instar larvae from each
strain were frozen. After approximately 500-800 larvae were
thawed, their guts were removed, placed in ice-cold buffer A (300
mM mannitol, 5 mM EGTA, and 17 mM Tris base,
pH 7.5), and then frozen on dry ice. BBMVs were purified from frozen
tissue by homogenization and selective magnesium precipitation as
described by Wolfersberger et al.(33) . All protein
concentrations were determined by the method of Bradford
(34) .
As a control, BBMVs were prepared from excised small intestines from
6-8-week-old FVB mice.
BBMV Biotinylation and Enzyme
Characterization
Purified BBMVs were biotinylated by
co-sonication with N-biotin-phosphatidylethanolamine in a
ratio of 1:100 (w/w; phosphatidyl N-biotinethanolamine:BBMV
protein) and 0.01% cholesterol (Sigma) as described
elsewhere
(26) . Following sonication, biotinylated BBMVs were
centrifuged at 100,000 g for 1 h, and the pellet was
resuspended in HBS. The resuspended pellet was then sonicated briefly
and re-centrifuged at 100,000
g for 1 h, and the
protein concentration of the final resuspension was determined and
adjusted to a final concentration of 1.0 mg of vesicle protein/ml. The
preparation was aliquoted and stored at -80 °C for up to 6
months. The amount of contaminating non-gut membrane material varied
among the different BBMV preparations. To normalize susceptible and
resistant vesicle preparations for the determination of maximal ligand
binding (R
), the specific activity of the gut
membrane enzyme marker leucine aminopeptidase in washed, freshly
prepared, biotinylated BBMVs was determined by the spectrophotometric
assay of Tuppy et al. (35) using leucine
p-nitroanilide as a substrate. We arbitrarily defined 1 unit
of leucine aminopeptidase enzyme activity as
A
/min = 1.0.
Toxin Purification
The cryIA(b) and
cryIA(c) genes, cloned from the NRD-12 strain of B.
thuringiensis, were expressed in Escherichia coli HB101
(36). The cryIC and cryIE genes were subcloned into a
B. thuringiensis/E. coli shuttle vector and expressed
in a crystal minus mutant of B. thuringiensis. Intracellular
inclusion bodies or crystals were further purified by Renografin
(Squibb) gradients
(36) . All purified protoxins were solubilized
in 40 mM carbonate buffer, pH 10.5, and treated with trypsin
(final concentration, 0.1% (w/v)). The mixture was incubated for 3 h at
ambient temperature, after which the 65,000-dalton trypsin-resistant
protein was purified by ion exchange liquid chromatography using either
Mono Q or Q-Sepharose anion exchangers (Pharmacia Biotech Inc.)
buffered with 40 mM carbonate buffer, pH 10.5. Bound toxins
were eluted using a 50-500 mM NaCl gradient and dialyzed
immediately into distilled water. The activated toxins were freshly
diluted into HBS before use in our binding studies.
Instrumentation and Reagents
The BIAcore system
and CM5 sensor chips were obtained from Pharmacia. Surfactant-free HBS
was used as the running and diluting buffer for all vesicle
experiments. Bovine serum albumin (BSA) (fraction 5, radioimmunoassay
grade) was purchased from Sigma. Rabbit anti-biotin (IgG fraction) was
purchased from Rockland (Gilbertsville, PA). Phosphatidyl
N-biotinethanolamine (lyophilized) was purchased from Avanti
Polar Lipids (Alabaster, AL). The buffers used with the BIAcore machine
contained the following: HBS (10 mM HEPES, pH 7.4, 150
mM NaCl, and 3.4 mM EDTA), HBS-P20 (HBS containing
0.05% BIAcore Surfactant P20), regeneration buffer (50 mM
CAPS, pH 11.0, and 150 mM NaCl), detergent wash (HBS +
0.1% (v/v) Triton X-100), carboxymethylated dextran activation
solutions (A = 0.1 MN-hydroxysuccinimide, B
= 0.1 MN-ethyl-N`-(3-diethylaminopropyl)carbodiimide), coupling
buffer (20 mM ammonium acetate, pH 4.5), and deactivation
solution (1 M ethanolamine, pH 8.5). All protein chemical
immobilizations were done using the standard BIAcore amine coupling
protocol provided with the Pharmacia coupling kit.
Sensor Chip Configuration and Binding
Specificity
We used two sensor chip configurations: immobilized
vesicle and immobilized toxin. In the immobilized vesicle
configuration, 20,000 RU of the rabbit anti-biotin immunoglobulin
dissolved in 20 mM ammonium acetate, pH 5 (equivalent to 20 ng
of protein) was immobilized by standard amine coupling
(25) .
Sonicated vesicles containing the biotin anchor were adjusted to a
protein concentration of 1 mg/ml as described elsewhere
(26) and
injected over the antibody surface. A vesicle level of approximately
1700-1800 RU was reproducibly obtained. The immobilized vesicles
were then washed until a stable baseline was obtained, after which
toxin (freshly prepared in HBS containing 0.01% (w/v) BSA) was
injected. In the immobilized toxin configuration, approximately 5000 RU
of toxin (dissolved as a 0.1 mg/ml stock solution in 20 mM
ammonium acetate, pH 5) was amine-coupled to the dextran. BSA (0.01%
(w/v)) was added to all vesicle preparations immediately prior to
injection over the toxin surface to reduce nonspecific interactions. In
all experiments, a reagent flow rate of 5 µl/min was used.
Kinetic Rate Analysis
The general rate equation to
describe the interaction between two molecular species can be written
as dR/dt =
kC(R
- R
) -
k
R
, where
R is the response, dR/dt is the rate of
toxin-receptor complex formation, C is the concentration of
injected toxin, R
-
R
is the amount of free remaining
receptor sites at time t (expressed in RU),
k
is the association rate constant
(expressed as M
s
), and
k
is the dissociation rate constant
(expressed as s
)
(25) . By rearranging the
rate equation to dR/dt =
k
CR
-
(k
C +
k
)R
, one
sees that the rate of toxin binding is linearly related to the
response
(25) . When the binding rate of each of the response
curves is plotted against the response (dR/dt versus
R), the association rate k
can be
determined by plotting the slope of the dR/dt versus R curves against toxin concentration. To determine
k
, we used four concentrations of
activated CryIA(c) injected over 1800 RU of BBMVs immobilized by rabbit
anti-biotin IgG.
/Rt
) =
k
(t
-
t
), where Rt
is the response
at an arbitrarily chosen starting time (t
) after
the toxin is replaced by buffer and Rt
and t
are subsequent data points
chosen along the dissociation curve. Ideally, the chosen start point
should be close to the end of the vesicle flow to prevent any
influences by vesicle rebinding. Our arbitrary start point was
generally chosen where the
ln(Rt
/Rt
) versus (t
- t
)
curve initially became linear but before 5-10% of the bound
vesicles had dissociated.
RESULTS
Toxicity to Susceptible and Resistant
Larvae
CryIA(b) and CryIA(c) were extremely toxic to larvae from
the susceptible strain of diamondback moth but did not kill larvae from
the resistant strain (). Two other lepidopteran-specific
toxins, CryIB and CryIC, were highly toxic to both susceptible and
resistant larvae. At the concentrations tested, CryIE caused little or
no mortality to susceptible or resistant larvae. The relative toxicity
of CryIA(b), CryIB, CryIC, and CryIE to the susceptible and resistant
larvae studied here corresponds well with the pattern of toxicity of
the independently derived susceptible and resistant strains of
diamondback moth studied by Ferré et al.(18) .
Cry Binding Characteristics
In the immobilized
toxin configuration, BBMVs from susceptible larvae showed no
significant binding when injected over a surface of immobilized CryIIIA
toxin (Fig. 1). In the immobilized vesicle configuration, CryIIIA
demonstrated limited binding to the P. xylostella BBMVs. The
observed binding was considered to be nonspecific as a result of the
nearly linear (non-saturable) response. The CryIIIA toxin could remain
stably bound to the vesicles for more than 15 min, thereby making it an
ideal candidate for blocking nonspecific CryI toxin binding.
Figure 1:
Nonspecific binding of CryIIIA to
P. xylostella BBMVs. P. xylostella BBMVs isolated
from susceptible larvae were passed over a surface containing 6000 RU
of immobilized CryIIIA (B). On a second surface, approximately
1800 RU of biotin-anchored BBMVs from the same preparation were
immobilized on a surface of immobilized anti-biotin antibodies. A
solution of CryIIIA toxin (1000 nM) was then injected over the
BBMV surface (A). All response curves in this and all
subsequent figures were adjusted for the mass action of the injected
buffer components, and the resonance units were normalized to zero at
the start of injection for comparative
purposes.
To
examine the relationship between toxicity and ligand binding,
immobilized BBMV surfaces prepared from mouse small intestine or from
susceptible larvae were used (Fig. 2). We have found that
CryIA(c) or CryIE could bind, to a limited extent (i.e. approximately 200 RU), to these vesicles.(
)
A preinjection of CryIIIA over the immobilized mouse BBMV
surface followed by an injection of CryIA(c) resulted in a very low
level of bound CryIA(c) toxin (<40 RU). An injection of
trypsin-activated CryIE, which is essentially non-toxic for P.
xylostella (), over a surface of immobilized BBMVs
from susceptible P. xylostella resulted in a small but linear
response. The CryIE toxin complex dissociated completely immediately
after toxin flow was replaced by buffer, suggesting that the observed
increase in RU was nonspecific in nature. By contrast, an injection of
an identical concentration of CryIA(c) over immobilized BBMVs from
susceptible P. xylostella larvae resulted in a high level of
binding that appeared to be nearing saturation. When the toxin flow was
replaced by buffer, the toxin-BBMV complex (approximately 350 RU)
started to slowly dissociate. Surprisingly, CryIA(c) toxin also bound
to BBMVs from resistant P. xylostella larvae, suggesting that
a functional CryIA(c) receptor is present on the surface of the
resistant vesicles.
Figure 2:
Specificity of CryI toxin binding to
BBMVs. BBMVs from either P. xylostella larvae or mouse
intestine were immobilized to an anti-biotin surface. A 1000
nM solution of CryIIIA was preinjected to reduce nonspecific
binding over the BBMV surface for 400 s followed by a 1000 nM
solution of a trypsin-activated lepidopteran-specific toxin. The
curves are labeled with the injected toxin followed by the
type of BBMV. R, BBMVs from resistant P. xylostella;
S, BBMVs from susceptible P. xylostella; M,
BBMVs from mouse small intestine.
In light of the CryIE results presented above,
we determined whether CryI trypsin-activated toxins can bind to
themselves to form dimers or other multimeric forms by passing CryIA(c)
toxin over an immobilized homologous toxin surface. As shown in
Fig. 3
, approximately 500 RU of toxin remained bound after the
injection. When the toxin flow was replaced by buffer, the toxin
complexes dissociated, rapidly dropping to 50% of the total bound after
120 s. The gentler slope of the observed biphasic dissociation curve is
due to rebinding of the toxin to freed immobilized binding sites.
Figure 3:
Binding of CryIA(c) to immobilized
homologous toxin. Approximately 6000 RU of trypsin-activated CryIA(c)
were immobilized and washed with HBS to remove unbound toxin until a
stable baseline was reached. A 400-s injection of a 1000 nM
CryIA(c) toxin stock solution was passed over the toxin surface. After
the end of toxin injection, the flow was switched to HBS for an
extended period.
To
further characterize the specificity of toxin binding to BBMVs from
resistant P. xylostella, the vesicles were preincubated with
either a homologous or heterologous toxin (CryIA(c) or CryIC,
respectively) before injection over an immobilized CryIA(c) surface
(Fig. 4). Preincubation of BBMVs from resistant larvae with
CryIC, a heterologous protein highly toxic to both susceptible and
resistant P. xylostella larvae, resulted in net binding to the
CryIA(c) surface. The binding curve is similar to that generated when
the vesicles were preincubated with BSA. However, little or no net
capture of BBMVs occurred at the end of the vesicle injection when
preincubated with the homologous toxin. The specific blocking of BBMV
binding to an immobilized CryIA(c) surface suggests that BBMVs from
resistant P. xylostella larvae possess a receptor that
specifically binds CryIA(c) toxin.
Figure 4:
Homologous/heterologous competition of
CryI toxins for BBMVs purified from resistant P. xylostella larvae. Vesicles from resistant insects were preincubated for 30
min on ice with 5 mM of BSA, CryIC, or CryIA(c). The BBMVs
were then injected over a surface of immobilized CryIA(c)
(approximately 6000 RU).
Determination of Kinetic Rate Constants
Typical
CryIA(c) response curves using immobilized BBMVs from susceptible
P. xylostella larvae are shown in Fig. 5A. The
four different toxin concentration curves showed that the rate of
increase in RU, which represents toxin binding to the vesicles,
generally decreased with time. The association rate can be calculated
by determining the slopes from the linear part of the curve produced by
plotting the binding rates (dR/dt) of the four
separate toxin concentrations used against the response
(R).(
)
By plotting these four slopes
against the toxin concentration, the k
can be calculated from the slope of the fitted line
(Fig. 5B) and was determined to be 4.4
10
M
s
. A similar
binding response was observed with CryIA(c) using immobilized BBMVs
from resistant larvae (Fig. 6A). The plot of the four
dR/dt versus R slopes against toxin concentration produced a
k
(3.6
10
M
s
) for BBMVs
from resistant larvae (Fig. 5B) that was not
significantly different than that determined for BBMVs from susceptible
larvae.
Figure 5:
Association rate kinetics of CryIA(c) with
immobilized BBMVs from susceptible P. xylostella larvae.
A, typical response curve showing injections of four different
CryIA(c) concentrations. The data obtained in A were
transformed into binding rate versus response curves (not
shown) as described under ``Materials and Methods.''
B, slopes of the four curves plotted against toxin
concentration. The slope of the fitted linear curve represents the
association rate constant.
Figure 6:
Association rate kinetics of CryIA(c) with
immobilized BBMVs from resistant P. xylostella larvae.
A, typical response curve showing injections of four different
CryIA(c) concentrations. The data obtained in A were
transformed into binding rate versus response curves (not
shown) as described under ``Materials and Methods.''
B, slopes of the four curves plotted against toxin
concentration. The slope of the fitted linear curve represents the
association rate constant.
Results from a typical experiment used to determine
dissociation rate constants using BBMVs from both susceptible and
resistant insects are shown in Fig. 7. A 600-s BBMV injection
from either susceptible or resistant insects sufficient to saturate an
immobilized CryIA(c) surface was replaced by a continuous flow of
buffer (Fig. 7). The dissociation of the vesicle from the toxin
surface is manifested by the slow drop in RU with time after the buffer
switch. The slope of the transformed dissociation curve
(Fig. 8A) calculated from the susceptible larval BBMV
curve in Fig. 7represents the dissociation rate constant and was
calculated to be 3.94 10
s
. This k
is not
significantly different from that calculated for the resistant larval
BBMVs (Fig. 8B) (2.57
10
s
). Because the affinity constant
(K
) is a ratio of the dissociation and
association rate constants, the K
of
CryIA(c) for its receptor can be determined. As summarized in
, CryIA(c) showed similar high affinity binding to BBMV
preparations from susceptible and resistant larvae.
Figure 7:
Dissociation curves of CryIA(c) with
susceptible and resistant P. xylostella BBMVs. Approximately
6000 RU of activated CryIA(c) toxin were immobilized, and BBMVs from
either susceptible or resistant P. xylostella larvae were
injected until the surfaces were saturated (usually 600 s). Typical
curves showing vesicle saturation and dissociation (after HBS buffer
replaced vesicle flow) are shown. The area of the dissociation segment
of the curve used to calculate k was selected by choosing an
arbitrary starting point (t = 0) a few minutes after
the start of buffer flow but before 5% of the total amount of bound
vesicle was dissociated.
Figure 8:
Calculation of dissociation rate constant.
The dissociation rate constant was calculated from the slope of the
fitted line (solid black line) in
ln(Rt/Rt) versus (t - t
) plots for BBMVs purified from
either susceptible larvae (A) or from resistant larvae
(B). The small dots represent the data points (log of
the drop in response) calculated at 1-s intervals using data from the
segment of the dissociation curve described in Fig.
7.
In the
immobilized vesicle configuration experiments, even though the same
number of BBMV resonance units was immobilized between the two
preparations, BBMVs from resistant larvae tended to bind slightly
higher levels of toxin. By assaying the levels of a gut-specific
membrane marker and normalizing the maximal binding of the toxin to
immobilized BBMVs, a comparative estimate of receptor levels
(R) was determined. The results presented in
revealed only a 3-fold decrease in relative number of
receptors in resistant BBMVs.
DISCUSSION
We determined the kinetic binding characteristics of the
CryIA(c) toxin from B. thuringiensis to BBMVs purified from
both susceptible and CryIA(c)-resistant larvae of P.
xylostella. The results presented here show that CryIA(c) can
specifically recognize and bind to BBMVs prepared from resistant as
well as susceptible diamondback moth larvae. Although the kinetic
parameters were similar for the two larval types, a lower number of
receptors in the resistant BBMVs were observed. However, the minor
difference in receptor numbers does not seem sufficient to account for
the major difference in CryIA(c) susceptibility.
(
)
determined by our optical technique
correlated well (3-4-fold higher) with that calculated at
equilibrium for BBMVs from susceptible larvae using radiolabeled
toxins
(20) .
-toxin pore
(44) or the
heptameric aerolysin channel
(45) . Because the concept of
surface multimerization may have important implications in the toxin
mode of action, further experiments are planned to determine if the
multimeric binding is specific and how CryIA(c) toxin interacts with
heterologous toxins.
Table: 1635000358p4in
Estimated from probit analysis of previously
reported data (32).(119)
Table: Summary of CryIA(c) kinetic rate constants
and calculated affinity constants
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.