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Neither barium nor calcium prevents the inhibition by Bacillus thuringiensis ╬┤-endotoxin of sodium- or potassium gradient-dependent amino acid accumulation by tobacco hornworm midgut brush border membrane vesicles.

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Archives of Insect Biochemistry and Physiology 12:267-277 (1 989)
Neither Barium nor Calcium Prevents the
Inhibition by Bacillus thuringiensis
6-Endotoxin of Sodium- or Potassium GradientDependent Amino Acid Accumulation by
Tobacco Hornworm Midgut Brush Border
Membrane Vesicles
Michael G . Wolfersberger
Department of Biology, Temple University, Philadelphia
Rapid filtration assays were used to determine the effects of barium, calcium
and an insecticidal &endotoxin from Bacillus thuringiensis o n sodium and
potassium ion gradient dependent phenylalanine accumulation by brush border membrane vesicles from the larval midgut of the tobacco hornworm
(Manduca sexta). Neither barium nor calcium had a significant effect on sodium
ion gradient dependent phenylalanine accumulation by the membrane vesicles. Both barium and calcium inhibited potassium ion gradient dependent
phenylalanine accumulation by the membrane vesicles. B. thuringiensis
&endotoxin inhibited both sodium and potassium ion gradient dependent
phenylalanine accumulation by the vesicles. Inhibition of both sodium and
potassium ion gradient dependent phenylalanine accumulation increased similarly with increasing S-endotoxin concentration. Neither barium nor calcium
had any effect on &endotoxin inhibition of either sodium or potassium dependent phenylalanine accumulation by the vesicles.
Key words: phenylalanine, cotransport, Manduca sexta
INTRODUCTION
During sporulation subspecies of Bacillus thuringiensis that contain and express
Cry1 genes produce parasporal proteins that are selectively toxic to lepidopteran insect larvae [11. These &endotoxin proteins usually accumulate to high
concentration and form crystals within the sporulating bacterium [2]. Therefore, they are now commonly refered to as insecticidal crystal proteins.
Received October 23,1989; accepted December 22,1989.
Address reprint requests to M.G. Wolfersberger, Ph.D., Biology Department, Temple University, Philadelphia, PA 19122, (215) 7878879.
0 1989 Alan R. Liss, Inc.
268
Wolfersberger
The insecticidal action of B . thuringiensis ICPs* occurs in the larval midgut.
Ingested parasporal crystals dissolve in the alkaline midgut lumen, and the
ICPs are hydrolyzed by digestive enzymes to serine-protease resistant toxins
with molecular weights between 55,000 and 70,000 [3]. The toxins diffuse through
the peritrophic membrane and bind to specific receptors on the luminal membrane of midgut columnar cells [4]. The binding between toxin and receptor,
within 10 min, becomes an irreversible association [4,5]. This transition from
reversible binding to irreversible association appears to coincide with insertion of at least the hydrophobic N-terminal portion of the toxin protein into
the lipid bilayer of the brush border membrane to form a transmembrane pore
[6]. The osmoregulatory mechanisms of the cells are unable to compensate
for the membrane permeability increase resulting from formation of these new
membrane pores. The cells swell and eventually lyse [7,8].
While there is general agreement on the overall mode of action of B .
thuringiensis ICPs, the nature of the primary membrane lesion resulting from
toxin receptor interaction is controversial. From the results of their studies with
insect cell lines, Knowles and Ellar [7] concluded that the primary membrane
lesion was a pore with a minimum cross sectional area of 78 A2. However,
studies of the effects of B. thuringiensis ICPs on ion gradient driven amino acid
accumulation by BBMV from larval Pieris brassicae midgut lead Sacchi et al. [9]
to the conclusion that the primary membrane lesion was a pore selectively
permeable to potassium ions. The maximum cross-sectional area of a pore with
this permeant ion selectivity is expected to be less than 16 A* [lo].
Crawford and Harvey [ll]showed that addition of barium or calcium ions
to the luminal solution could prevent or reverse the inhibition by €3. thuringiensis
ICPs of the short circuit current across isolated M. sexta larval midgut. Since,
under control conditions, the short circuit current is nearly identical to net
potassium ion transport across isolated M. sexta midgut [12] and at least barium is well known to block a variety of potassium ion channels selectively
[13],the results of Crawford and Harvey appeared to support the conclusions
of Sacchi et al. about the nature of the primary target cell membrane lesion.
However, the experiments of Crawford and Harvey were performed on whole
isolated midguts and used bathing solutions in which potassium was the
only alkali metal cation. Therefore, it was of interest to see if similar results
could be obtained using other alkali metal salts and a cell-free isolated target
membrane system.
The system used in the studies reported herein is essentially the same as
that used by Sacchi et al. [9] except the BBMV are prepared from larval M .
sexta midgut, the amino acid used to monitor membrane ion permeability
changes is phenylalanine, and the toxin is isolated from the HD-73 strain of
B. thuringiensis subsp. kurstaki. Not only all of the individual components of
this system, but also some of their interactions are well characterized. Unlike
strain HD-1 and many other strains of B. thuringiensis, the HD-73 strain produces only one ICP [l].The primary structure of the ICP produced by B .
thuringiensis strain HD-73 is known [14] and its activity as a M . sexta larvicide
*Abbreviations used: BBMV = brush border membrane vesicles; ICP = insecticidal crystal protein.
Toxin Inhibition of Amino Acid Cotransport
269
has been determined by several groups [5,14-161. The polypeptide composition and enrichment of several marker enzymes in larval M. sexta midgut BBMV
are known [17] and the ion selectivity of phenylalanine accumulation by M.
sexfa BBMV has been studied in detail [18]. There have been several quantitative studies of the binding between HD-73 toxin and M. sexta BBMV, and a
good correlation between in vitro binding and in vivo larvicidal activity has
been established [5,15,16].
MATERIALS AND METHODS
Bacteria
Bacillus thuringiensis subspecies kurstaki strain HD-73, kindly provided by
Dr. Howard Dulmage (USDA-ARS, Cotton Insects Research, Brownsville, TX)
was the source of the ICP used in all experiments. Bacteria were grown to
sporulation at 30°C on the modified agar medium of Yousten and Rogoff [19].
Sporulated cultures were flushed from the agar surface with distilled water.
The suspension was centrifuged at 20,OOOg for 20 min at 4°C. The pellet, which
consisted of spores, parasporal crystals, and some cell debris, was washed
three times with distilled water.
Parasporal Crystal Purification
Parasporal crystals were separated from the other components of sporulated
cultures using a two-phase extraction system with dextran sulfate and polyethylene glycol [20]. This procedure has been shown routinely to yield crystals contaminated by less than 1%spores [21]. The purified crystals were
suspended at approximately 10 mg/ml in 1 mM EDTA. The crystal suspension was stored in aliquots frozen at - 80°C.
Toxin Preparation
Toxin was isolated as described by Hofmann and Luthy [22], with slight
modifications. Purified parasporal crystals were suspended at approximately
3 mg/ml in SO mM sodium carbonate buffer (pH 9.5) containing 10 mM
dithiothreitol. The mixture was incubated for 1 h at 37"C, centrifuged for 5
min at approximately lO,OOOg, and the ICP solution was decanted from the
undissolved material. Trypsin (0.1 mg per 1 mg of ICP) was added to the ICP
solution. After incubation for 1h at 37"C, the incubation mixture was applied
to a Sephacryl S-300 column. The column was eluted with 0.15 M NaCl in 50
mM sodium carbonate buffer (pH 9.5). One milliliter fractions were collected
and the absorbance of each fraction at 280 nm was determined. The toxin containing fractions were pooled and dialyzed overnight at 4°C against 100 volumes of 5 mM sodium carbonate buffer (pH 9.5). The dialyzed toxin solution
was freeze-dried and stored desiccated at 4°C. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis [23] of toxin preparations revealed a single
band of approximately 67,000.
Insects
Second and third day fifth instar Manduca sexta larvae, weighing 5.2 + 0.6 g,
were used in all experiments. Second or third instar larvae as well as larval
270
Wolfersberger
diet were purchased from Carolina Biological Supply Company (Burlington,
NC). The larvae were reared to experimental size at 27°C with constant light.
Midgut Isolation and BBMV Preparation
Larvae were chilled on ice for 15-20 min. Chilled larvae were transected immediately behind the fourth pair of abdominal appendages and again immediately behind the first pair of thoracic appendages. The integument was cut open
along the dorsal midline and was spread apart, exposing the midgut. The
tracheoles attaching the midgut to the integument were severed. Each excised
midgut was rinsed with an ice-cold solution of 0.3 M mannitol, 5 mM EGTA,
and 17 mM Tris-HC1, pH 7.5 (homogenization buffer). Each midgut was cut
along one of the six longitudinal muscles and opened to form a flat sheet. The
midgut contents were rinsed away with ice-cold homogenization buffer. The
peritrophic membrane and the Malpighian tubules were removed using forceps. The isolated midguts were rinsed with ice-cold homogenization buffer,
gently blotted, weighted, and used immediately for BBMV preparation.
Brush border membrane vesicles were prepared by the differential magnesium precipitation method of Biber et al. [24] as modified and described by
Wolfersberger et al. [25]. Characterization of BBMV produced from larval M .
sexfa midgut by differential magnesium precipitation is described in detail elsewhere [17]. The vast majority of BBMV prepared by differential precipitation
methods are right-side-out; the membrane in the vesicles has the same orientation as in the intact epithelium [26].
Transport Experiments
Uptake experiments were preformed at room temperature (23 2 2°C) using
a rapid filtration technique [25]. Unless otherwise noted, BBMV pellets were
resuspended in 100 mM mannitol, 10 mM Tris-Hepes (pH 8) at a protein concentration of 5 to 7 mg/ml using a syringe equipped with a 22 gauge x 3.8 cm
needle. Incubations were started by mixing aliquots of BBMV suspensions with
equal volume aliquots of solutions containing radioactive phenylalanine and
other components as reported in Table 1 and the figure legends. Some incubations of 15 s or longer were performed by withdrawing, at selected times,
20 pl aliquots from a single incubation mixture, diluting each aliquot with 2
ml of an ice-cold solution of 0.15 M NaCl in 1 mM Tris-Hepes (pH 7), and
immediately filtering the dilute suspension through a prewetted cellulose nitrate
filter (0.65 p.,m pore size, Sartorius No. 11305, Hayward, CA). All other incubations were started by mixing a 10 p1 drop of BBMV suspension and a 10 pl
drop of labeled phenylalanine solution. They were stopped by injecting 2 ml
of ice-cold 0.15 M NaCl in 1 mM Tris-Hepes (pH 7) into the incubation mixture and immediately filtering the dilute mixture. All filters were washed twice
with 4 ml if ice-cold 0.15 M NaCl in 1 mM Tris-Hepes (pH 7), put into a vial
with 10 ml of liquid scintillation cocktail (ScintiVerse BD, Fisher Scientific,
Pittsburgh, PA), and counted in a liquid scintillation spectrometer (Model
2000CA, Packard Instrument, Downers Grove, IL). Aliquots of radioactive
phenylalanine solutions used in each experiment were spotted on filters and
counted. These standard counts were used to convert sample cpm into mols
of phenylalanine [181.
Toxin Inhibition of Amino Acid Cotransport
271
TABLE 1. Effects of Barium, Calcium, and B. thuringiensis Toxin on Ion Gradient Dependent
Phenylalanine Accumulation by Brush Border Membrane Vesicles From Larval M . sextu Midgut*
Accumulation
(nmolimg)
Uptake solution (mM)
Vesicles
100 KC1
100 NaCl
100 KCl
15 BaC1,
100 KCI
100 NaCl
15BaCl,
100 KCl
15 CaC1,
100 NaCl
15 CaCI,
100 KC1
100 NaCl
100 KCI
15 BaC1,
100 KCl
100 NaCl
15 BaC1,
100 KCI
15 CaC12
100 NaCl
15 CaClz
Control
Control
2.75
2.84
Control
BaClz
1.60 ? 0.14
1.80 ? 0.36
Control
2.83
Control
1.96 ? 0.13
Control
Toxin
Toxin
2.90 2 0.22
0.46 f 0.14
0.57 ? 0.10
Toxin
Toxin
0.23 2 0.07
0.29 ? 0.05
+ BaCI2
2
?
?
0.26
0.16
0.14
Toxin
0.65 2 0.16
Toxin
0.34 f 0.11
Toxin
0.66 & 0.07
*All uptake solutions contained 100 mM mannitol, 10 mM Tris-HEPES (pH 8), and 0.5 mM phenylalanine in addition to the indicated salts. BBMV were resuspended at approximately 7 mg/ml
in 100 mM mannitol, 10 mM Tris-HEPES (pH 8), with or without 9 pmol/mg toxin and/or 15 mM
BaC12 as indicated. Toxin and barium treated BBMV were preincubated at 23°C for at least 30
min before use in uptake experiments. The reported accumulation is the difference between
vesicular phenylalanine uptake at 90 s and at 60 min (mean ? SD; n = 3).
Protein Determinations
The protein concentration of toxin solutions and BBMV preparations was
determined by the method of Bradford [27] using a Bio-Rad (Richmond, CA)
kit. The protein concentration of parasporal crystal suspensions was determined by the method of Lowry et al. [28]. Bovine serum albumin served as
standard for both protein determinations.
Statistics
Statistical analyses were performed using Student’s t-test. A P-value of
< 0.01 was considered to represent a statistically significant difference.
Reagents
L-[ ri11g2,6-~H]-Phenylalanine,60 Ci/mmol, was purchased from DupontNEN (Boston, MA). Dithiothreitol, L-phenylalanine, and trypsin (type 111) were
from Sigma (St. Louis, MO). Dextran sulphate (sodium salt) and Sephacryl
S-300 (superfine) were from Pharmacia (Uppsala, Sweden). Polyethylene glycol 6000 was from Serva (Heidelberg, FRG). All other chemicals were analytical grade products from either Fisher or Mallinckrodt (St. Louis, MO).
272
Wolfersberger
RESULTS
The time courses of L-phenylalanine uptake by larval M. sexta midgut BBMV
in the presence of an initial NaCl gradient with and without B. thuringiensis
toxin as well a s in the absence of a NaCl gradient are shown in Figure 1. In
the absence of a salt gradient L-phenylalanine uptake is equilibrative. During
the first 90 s of incubation the phenylalanine concentration within the vesicles increases until it is the same as that outside the vesicles; thereafter it remains
constant. In the presence of an initial NaCl gradient there is a transient accumulation of L-phenylalanine within the vesicles to approximately three times
its equilibrum concentration. This transient accumulation of L-phenylalanine
is dependent on sodium ion-amino acid cotransport 1181.It is suppressed greatly
by B. thuringiensis toxin.
The dependence of maximum ion gradient dependent L-phenylalanine accumulation on toxin concentration is shown in Figure 2. Both NaCl and KC1 gradient dependent accumulation of L-phenylalanine decreases to the same extent
with increasing toxin concentration. A plot of the reciprocal of inhibition of
ion gradient dependent phenylalanine accumulation vs the reciprocal of toxin
concentration (Fig. 2, inset) yields a straight line (linear regression correlation
coefficient = 0.997) which extrapolates to complete suppression of accululation
at infinite toxin concentration.
The effects of barium, calcium, and B. thuringiensis toxin on maximum
potassium and sodium gradient dependent L-phenylalanine accumulation
by BBMV prepared from larval M. sexta midgut are summarized in Table 1.
5
4
3
&
m9
i
2
1
1
2
3
4
5
'
6
+I
60
time (min)
Fig. 1 . Time courses for the uptake of L-phenylalanine by brush border membrane vesicles
(e)
from larval Manduca sexta rnidgut. L-phenylalanine (0.5 mM) uptake by control vesicles
and by vesicles preincubated with 9 pmol/rng Bacillus thuringiensis toxin
).(
in the presence of
an initial NaCl gradient (50 m M outside and 0 mM inside). L-phenylalanine (0.5 mM) uptake by
control vesicles in the presence of NaCl but in the absence of a salt gradient (0).
Toxin Inhibition of Amino Acid Cotransport
6t
C
273
0
0.8.
._
c
-0
i
"0
0.6-
8
.-c
1/
Ctoxl
> 0.4 0
9
~
0.2
-
3
6
[tox]
9
pmol /mg
Fig. 2. Effect of Bacillus thuringiensis toxin on maximum transient L-phenylalanine accumulation by brush border membrane vesicles from larval M. sexta midgut. Vesicles were suspended
in 100 mM mannitol, 10 mM Tris-Hepes, pH 8; toxin was added to give the indicated final concentrations; and the mixtures were incubated for 30 min at 23°C. Preincubated vesicles were
mixed with an equal volume of a medium composed of 100 m M mannitol, 10 mM Tris-Hepes,
pH 8,O.S mM L-3H-phenylalanine,100 mM KCI (O),
or 100 mM NaCl ( 0 )Vesicle
.
phenylalanine
uptake after 90 s and 60 min of incubation was determined. Accumulation i s the difference
between uptake at 90 s and 60 min. Relative accumulation i s accumulation by the toxin treated
vesicles/accumulation by untreated vesicles. Each point represents the mean k SD of three
determinations. The inset i s a plot of the reciprocal of relative accumulation vs the reciprocal
of toxin concentration. A, relative accumulation in the absence of toxin; A, , relative accumulation in the presence of toxin.
The difference between L-phenylalanine uptake by larval M. sexta midgut
BBMV after 90 s and 60 min of incubation was the same in the presence of
an initial gradient of either KCI or NaCI. There was also no difference between
the maximum potassium gradient dependent and the maximum sodium
gradient dependent accumulation of phenylalanine by B . thuringiensis toxin
treated BBMV. However, maximum phenylalanine accumulation by the toxin
treated BBMV was only 18%of maximum phenylalanine accumulation by the
untreated BBMV.
Neither barium nor calcium had a significant effect on maximum sodium
gradient dependent L-phenylalanine accumulation by either control or toxin
treated larval M. sexta midgut BBMV. However, maximum potassium gradient dependent phenylalanine accumulation by control BBMV was reduced 29%
by calcium and 38% by barium. Inclusion of barium and calcium in the incubation medium resulted in approximately the same percent reduction of maximum potassium gradient dependent phenylalanine accumulation by toxin
treated BBMV. The reduction of maximum potassium gradient dependent phenylalanine accumulation by control vesicles in the presence of either barium
274
Wolfersberger
or calcium was statistically significant but the difference between barium and
calcium inhibition was not. The reduction in the presence of either barium or
calcium of maximum potassium gradient dependent phenylalanine accumulation by toxin treated BBMV was not statistically significant. The barium dependent reduction of maximum potassium gradient dependent phenylalanine
accumulation by both control and toxin treated BBMV was the same regardless of whether the BBMV were preincubated with BaC12 or BaC12 was present only during the uptake measurements.
DISCUSSION
The effects of barium, calcium, and the insecticidal protein toxin from B.
thuringiensis subsp. kurstuki strain HD-73 on both sodium and potassium gradient
dependent phenylalanine accumulation by larval M . sextu midgut BBMV have
been determined. The insecticidal bacterial toxin inhibited, in a dose dependent manner, not only potassium gradient dependent but also sodium gradient dependent accumulation of L-phenylalanine by the midgut BBMV. Fifty
percent inhibition of ion gradient dependent L-phenylalanine accumulation
occurred at a toxin concentration of 3.24 ? 0.35 pmol/mg of BBMV protein.
Differential magnesium precipitation produces midgut BBMV from M . sexta
larvae in a yield of approximately 30% [17] and one obtains approximately 0.6
mg of BBMV from a 5g larva. Therefore, a toxin concentration of 3.24 pmol/mg
BBMV corresponds to a toxin concentration of approximately 1.3 pmol/g
larva. This toxin concentration is of the same order of magnitude as the toxin
concentration in the midgut of a M . sexfu larva that has received an LDS0dose
of toxin [161. The maximum inhibition of ion gradient dependent L-phenylalanine
accumulation observed in these studies was less than 90%. However, a double reciprocal plot of ion gradient dependent L-phenylalanine accumulation
vs. toxin concentration resulted in a straight line which extrapolated to 100%
inhibition of accumulation. This theoretically satisfying result is unlikely to
occur actually because the concentration of receptor sites for HD-73 toxin on
M . sexta BBMV is less than 10 pmol/mg [5,16].
Whereas preincubation with toxins from B. fhuringiensis subsp. kursfuki strain
HD-1 resulted in a dose dependent increase in only the potassium permeability of larval P. brussicue midgut BBMV [9], preincubation with the toxin from
strain HD-73 resulted in similar dose dependent increases in both the potassium and sodium permeability of M . sexta BBMV. Since neither lithium nor
rubidium can substitute for sodium or potassium in L-phenylalanine cotransport
across the brush border membrane of larval M. sexta midgut [MI, one can not
use this system to monitor changes in membrane permeability to these alkali
metal ions.
Numerous factors other than physical size are important in determining
the permeant ion selectivity of a transmembrane pore [101. However, the minimum cross-sectional area of a transmembrane pore permeable to both sodium
and potassium ions must be approximately 17 A2, whereas a transmembrane
pore selectively permeable to potassium ions can have a minimum cross sectional area of less tha 9 A2 [29]. It therefore appears that the minimum size of
the pore formed by interaction of HD-73 toxin with M . sextu BBMV is greater
Toxin Inhibition of Amino Acid Cotransport
275
than that of the pore formed by interaction of HD-1 toxins with P. brussicue
BBMV [9], but it may be considerably smaller than that of the pore formed by
interaction of HD-1 toxins with the membrane of CF1 cells [7].
Both M . sexta and P. brussicue BBMV have been shown to contain more than
one type of specific high affinity binding site for B. thuringiensis CryIA toxin
[5,15]. The membrane of CF1 cells also contains at least one glycoprotein capable of binding CryIA toxins [30]. Since there is extensive amino acid sequence
identity among all CryIA toxins [l],it seems likely that the toxin binding membrane proteins are more important than the toxin itself in determining the
nature of the transmembrane pore formed by toxin membrane interaction.
Neither barium nor calcium had a significant affect on sodium ion gradient
dependent accumulation of L-phenylalanine by BBMV prepared from midguts of M . sexta larvae. These results are consistent with neither of these divalent cations affecting the ion permeability of the BBMV or competing with
sodium for the sodium-phenylalanine cotransporter. Both barium and calcium
inhibited, to a similar extent, potassium ion gradient dependent accumulation of L-phenylalanine by M . sexta BBMV. The extent of calcium or barium
inhibition of potassium gradient dependent L-phenylalanine accumulation was
more than twice what one might expect if these ions competed equally with
potassium for the potassium-phenylalanine cotransporter. However, barium
and calcium frequently bind much more tightly than potassium to sites
accessible to all three ions [lo]. Barium and calcium inhibition of potassium
dependent but not sodium dependent L-phenylalanine accumulation alerts
one to the possibility that these two processes may proceed via different
cotransporters or at least a cotransporter with different affinities or binding
sites for sodium and potassium.
The effects of barium and calcium on the small amount of ion gradient dependent L-phenylalanine accumulation detectable in toxin treated BBMV were parallel to their effects on ion gradient dependent phenylalanine accumulation
by untreated BBMV. Neither barium nor calcium inhibited, or stimulated, sodium
dependent phenylalanine accumulation by toxin treated BBMV. Addition of
calcium or barium to the uptake mixtures resulted in 26-37% inhibtion of potassium dependent phenylalanine accumulation by toxin treated BBMV. This level
of inhibition is similar to that obtained with untreated BBMV. However, potassium dependent phenylalanine uptake by toxin treated BBMV is so small that
the differences between accumulation in the presence and in the absence of
calcium or barium are not statistically significant.
Addition of B . thuringiensis ICP to the lumen side bathing solution results
in a large decrease in the short circuit current of an isolated larval M. sextu
midgut [31]. Addition of 4 mM to 6 mM BaC12 or CaCI2 to the lumen side
solution has no significant effect on the short circuit current of an isolated
larval M. sextu midgut bathed in buffered 32 mM KC1 solution [ll].However,
addition of BaC12 or CaC12 to the lumen side bathing solution can prevent or
reverse the effect of ICPs on the short circuit current [ll]. If the mechanism of
barium and calcium protection of short circuit current from inhibition by ICPs
is divalent cation blockage of ICP dependent transmembrane pores in the apical membranes of the midgut columnar cells, one would expect that barium
or calcium would restore potassium dependent phenylalanine accumulation
276
Wolfersberger
by toxin treated M. sextu midgut BBMV to the levels seen with untreated BBMV.
This is clearly not the result obtained.
ICPs from B . thuringiensis subsp. kurstuki strain HD-1 were used in the short
circuit current studies I l l ] , whereas purified toxin from strain HD-73 was used
in the studies with BBMV reported herein. However, because of their great
structural homology and similar efficacy in the absence of barium and calcium, it is difficult to attribute major differences in results of these two studies
to the ICPs. In both studies the external surfaces of the cell membranes were
bathed by simple solutions of similar defined composition with a similar ratio
of barium or calcium to potassium. The major difference between the system
used by Crawford and Harvey [ll]and that used in these BBMV studies is
that the former system contains a piece of intact midgut epithelium composed
of several types of living cells and the latter system contains solution filled
vesicles of the apical membrane of midgut columnar cells. The primary effect
of the toxin in both systems is presumably to participate in forming a pore in
the columnar cell apical membrane. The BBMV are severely limited in their
options for responding to the formation of this transmembrane pore. However, the living cells appear to have barium or calcium dependent mechanisms
for either blocking the pore or compensating for the effects of the pore on the
short circuit current.
ACKNOWLEDGMENTS
The assistance of Ms. Veronica F. Fernandes in bacterial culture and parasporal
crystal isolation is gratefully acknowledged. I thank Mr. Brian B. Hennigan
for assistance in the preparation of illustrations and Dr. William R. Harvey for
valuable comments on the manuscript. This work was supported by research
grants from the United States Department of Agriculture (87-CRCR-1-2487)
and the National Institutes of Health (AI-22444).
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acid, prevent, inhibition, endotoxin, sodium, midgut, amin, thuringiensis, membranes, bacillus, neither, vesicle, potassium, tobacco, gradient, dependence, barium, brush, border, calcium, hornworm, accumulation
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