(Received for publication, August 29, 1996, and in revised form, December 6, 1996)
From the Venom and Toxin Research Group, Buthus martensi Karsch venom exhibits
nitrergic action in rat anococcygeus muscle (ACM). We have purified a
novel toxin, makatoxin I (MkTx I), which exhibits nitrergic action, to
homogeneity from this venom by a combination of gel-filtration,
cation-exchange chromatography, and reverse-phase chromatography. Its
purity was assessed by capillary electrophoresis and mass spectrometry.
Its molecular weight was found to be 7031.71 ± 2.88 as calculated from electrospray mass spectrographic data. The complete amino acid
sequence was elucidated by sequencing of reduced and
S-pyridylethylated toxin and a carboxyl-terminal peptide,
P55-64, generated by the cleavage of toxin with endoproteinase Lys-C.
The complete sequence of MkTx I is
GRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPDKVPIRISGSCR. This toxin is composed of 64 amino acid residues and contains 8 half-cystine residues. Structurally, MkTx I has high similarity to
Bot I and Bot II when compared with toxins from other scorpion species.
The effects of MkTx I on nitrergic responses were investigated using
the rat isolated ACM mounted in Krebs solution (37 °C, 5% CO2 in O2). MkTx I (2 µg/ml) markedly relaxed
the carbachol-precontracted ACM; the relaxation was inhibited by the
stereoselective inhibitor of nitric oxide synthase,
N Scorpion venoms consist of complex mixtures of several toxins that
exhibit various pharmacological activities including selective actions
at sodium and potassium channels, which are the major molecular targets
of scorpion toxins (1). The scorpion Buthus martensi Karsch
is widely distributed in China (2), but relatively few reports have
been published on the biological properties of its venom
(MKV)1 or toxins. Electrophysiological
studies indicate that MKV contains some neurotoxic compounds that
possibly act on the Na1+ channels in the excitable membrane
of nerve and muscle (3), cultured mouse cardiomyocyte (4), and rat
anterior pituitary cells (5).
In a preliminary study we observed that MKV produced relaxant responses
of the rat precontracted anococcygeus muscle (ACM), suggesting that
some constituent toxin(s) present in MKV could mimic the effects of
nitrergic transmission involving the release of the inhibitory
neurotransmitter nitric oxide (NO).2
Therefore, we were interested in isolating and purifying the bioactive
component(s) present in the venom as well as identifying and
characterizing the pure toxin(s) mediating the relaxant responses of
the rat ACM. In this communication we report the isolation, purification, and complete amino acid sequence of a novel toxin, MkTx
I, isolated from the venom of B. martensi Karsch and provide the first documentation of some novel nitrergic actions mediated by a
scorpion toxin.
The bright white lyophilized crystals
of crude venom of B. martensi Karsch were obtained from the
Huazhen Pharmaceutical Animal Research Institute (Guangzhou, China).
Carbachol (carbamylcholine chloride),
N Capillary electrophoresis was
performed on a BioFocus 3000 capillary electrophoresis system
(Bio-Rad). The sample was injected using pressure mode (5 p.s.i./s) to
a 25 µm × 24-cm coated capillary and run in 0.1 M
phosphate buffer (pH 2.5) under 12.00 kV from + to Samples were
dissolved in deionized water and analyzed using a electrospray mass
spectra system (Perkin-Elmer Sciex API III LC/MS/MS systems). The
ionspray voltage was set to 4000 V. The orifice voltage was set at 75 V, and the interface temperature was set at 60 °C. Mass scan range
was 500-2000 atomic mass units. Nitrogen was used as curtain gas with
a flow rate of 0.6 liters/min and as nebulizer gas with a pressure
setting of 30 p.s.i. A Perkin-Elmer series 200 quaternary pump was
used for solvent delivery.
100 µg of MkTx I
were dissolved in 0.5 ml of buffer solution containing 6 M
guanidinium hydrochloride, 0.13 M Tris, and 0.1 mg/ml EDTA,
adjusted to pH 8.0 with HCl. 1% 2-mercaptoethanol was added in 20 mol/1 mol disulfide bonds in the protein. The solution was then
incubated at room temperature for 3 h. A solution of 10%
4-vinylpyridine was added to give a 3-fold molar ratio to
2-mercaptoethanol and incubated at room temperature for 90 min. After
the reaction, the solution was loaded to a Bio-gel P-2 column (2 × 20 cm), equilibrated, and eluted with 9% formic acid for desalting.
The protein peak was pooled and lyophilized.
Digestion of pyridylethylated MkTx I (100 µg) with endoproteinase Lys-C was performed at 37 °C overnight in
10 mM Tris-HCl, pH 8.8, containing 4 M urea
(substrate:enzyme ratio was 50:1). The reaction was stopped by lowering
the temperature to 4 °C. The mixtures were centrifuged, and the
supernatant was fractionated by reverse-phase chromatography using a
Vydac C8 column (2.1 × 150 mm) on Applied Biosystems 140B solvent
delivery systems and Applied Biosystems 1000s diode array detector.
The amino-terminal amino acid
sequence of MkTx I and the sequence of the peptide P55-64 produced by
endoproteinase Lys-C digestion were determined by using an Applied
Biosystems 477A pulsed liquid-phase sequencer or Procise equipped with
an on-line 120A phenylthiohydantoin-derivative analyzer.
Male Sprague Dawley rats
weighing 280-360 g were stunned and exsanguinated. The paired
anococcygeus muscles were dissected as described previously (6) and set
up in isolated organ baths containing 7 ml of Krebs solution of the
following composition: NaCl (118 mM), KCl(4.8
mM), KH2PO4 (1.2 mM),
CaCl2 (2.5 mM), NaHCO3 (25 mM), MgSO4 (2.4 mM), and
D-(+)-glucose (11 mM). The solution was
maintained at 37 °C and continuously aerated with 5% carbon dioxide
in oxygen. The organ responses against 1 g of tension were recorded by
means of an isometric transducer (model FT 03) on a Grass polygraph.
The preparation was allowed to equilibrate for about 30-40 min with
changes of Krebs solution at 15-min intervals. Motor or relaxant
responses of the muscle were elicited by electrical field stimulation
(20-30 V, 10 Hz for 10 s, 1-ms pulse width) every 2 min.
Motor responses of the ACM to field
stimulation (FS) were first blocked by phentolamine (5 µM), and the tone of the muscle was then raised by the
addition of carbachol (3 µM). Relaxant nitrergic
responses of the ACM to FS (15-20 min) were recorded, and FS was then
stopped. Relaxant responses of the ACM to MkTx I (2 µg/ml) were then
obtained in the absence of FS. Similar experiments were carried out in
the presence of 50 µM L-NAME to produce
blockade of the relaxant nitrergic responses of the ACM to FS. When the relaxant responses to FS were blocked, FS was stopped, and MkTx I (2 µg/ml) was then tested in the presence of L-NAME. Because the relaxant responses to MkTx I were also blocked by
L-NAME, 1 µM sodium nitroprusside was then
added, and the relaxant responses were recorded.
Separation of soluble
venom MKV was initially performed by gel-filtration chromatography in a
Bio-gel P-30 column (2.5 × 115 cm). The lyophilized venom (500 mg) was dissolved in 10 ml of 0.05 M ammonium bicarbonate,
and insoluble material was removed by centrifugation at 3500 × g for 10 min. Supernatant was loaded onto the column.
Protein elution monitored at 280 nm showed 7 fractions, MK1 to MK7
(Fig. 1A). All fractions were pooled,
lyophilized, and tested for nitrergic action. Only MK4 displayed
nitrergic action. This low molecular weight fraction was then submitted to the next purification steps and to an exhaustive study.
MK4 was dissolved in 0.02 M ammonium acetate (pH 6.0) and
loaded onto a POROS® HS column (4.6 × 50 mm)
equilibrated with the same buffer on a BioCAD perfusion chromatography
workstation (Fig. 1B). Proteins were eluted using a linear
gradient formed by mixing 0.02 M ammonium acetate (pH 6.0)
and 1 M ammonium acetate (pH 6.5). Fractions were pooled
according to their absorbance at 280 nm and lyophilized thrice to
remove the ammonium acetate. MK4B, MK4C, and MK4D showed nitrergic
actions, but other fractions did not display any nitrergic actions.
The main fraction MK4C from cation-exchange column was dissolved
in 0.1% trifluoroacetic acid and loaded onto a
POROS® R 2/H column (4.6 × 50 mm), and
fractions were eluted using a linear gradient of 0.1% trifluoroacetic
acid with 80% acetonitrile on a BioCAD workstation, as shown in Fig.
1C, yielding three fractions, MK4C1 to MK4C3. MK4C1 did not
show any nitrergic action, whereas MK4C2 and MK4C3 were found to
display nitrergic activity. MK4C2 is a purified peptide and was named
MkTx I. After three steps of isolation, 7.6 mg of MkTx I was purified
from 500 mg of crude venom. Both capillary electrophoresis and mass
spectrometry indicated that MkTx I is homogeneous (Fig.
2). The molecular weight of MkTx I determined by mass
spectrometry analysis is 7031.71 ± 2.88.
The automatic
Edman degradation of 15 pmol of S-pyridylethylated MkTx I
proceeded with a repetitive yield of 93.7% during the first 56 cycles,
which led to the unequivocal identification of the corresponding
residues labeled a in Fig. 4A. From step 57 onward, the amount of identified phenylthiohydantoin-derivative was
low, although the repetitive yield remained fairly good. To determine
unambiguous sequence at the carboxyl-terminal end, 100 µg of
S-pyridylethylated MkTx I were treated with endoproteinase Lys-C, and the resulting peptide mixtures were separated by
reverse-phase high performance liquid chromatography (Fig.
3). Peptides P1-28, P33-54, and P55-64, but not
P29-32, were identified by mass spectrometry analysis. All three
values obtained matched the calculated molecular masses deduced from
the sequences of respective peptides (Table I). The
automatic Edman degradation of P55-64 allowed the identification of
the carboxyl-terminal part amino acid residues. Thus, the amino acid
sequence of MkTx I was established as shown in Fig.
4A, which is comparable to mass spectrometric
analysis, with a calculated molecular weight of 7038.83.
Mass spectrometry analysis of MkTx I and peptides from Lys-C
digest
All cysteine residues are pyridylethylated. Department
of Anatomy,
Bioprocessing Technology Centre,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-nitro-L-arginine methyl ester (50 µM). Thus, MkTx I is the first
-toxin that can mediate
nitrergic responses in the rat isolated ACM.
Drugs and Chemicals
-nitro-L-arginine methyl ester
(L-NAME), and phentolamine hydrochloride were obtained from
Sigma. 2-mercaptoethanol and 4-vinylpyridine were
obtained from Merck. Bio-gel P-2 and Bio-gel P-30 were obtained from
Bio-Rad, POROS 20 HQ and POROS® R 2/H 4.6/5.0 were
obtained from PerSeptive Biosystem (Cambridge, MA). Endoproteinase
Lys-C was purchased from Wako Pure Chemicals (Osaka, Japan). Reagents
used in the automatic protein sequenator were obtained from Applied
Biosystems (Foster City, CA).
at 15 °C
for 40 min. Migration was monitored at 200 nm.
Isolation and Purification of MkTx I
Fig. 1.
Separation and purification of MkTx I. Horizontal bars indicate pooled fractions. A,
gel-filtration chromatogram of MKV on Bio-gel P-30. The column
(2.5 × 115 cm) was equilibrated and eluted with 0.05 M ammonium bicarbonate. Flow rate, 27.6 ml/h; fraction
size, 9.2 ml. 500 mg of MKV were dissolved in 10 ml of ammonium
bicarbonate and centrifuged at 3500 × g for 10 min.
Supernatant was loaded onto the column. B, cation-exchange
chromatogram of MK4 on POROS® HS column. The fraction MK4
from Bio-gel P-30 was separated in a POROS HS column, equilibrated, and
run with ammonium acetate. A linear gradient was formed by mixing 0.02 M ammonium acetate (pH 6.0) with 1 M ammonium
acetate (pH 6.5). Fraction size, 2.5 ml; flow rate, 5 ml/min.
C, reverse-phase chromatogram of MK4C on POROS®
R 2/H column. The fractions of MK4C from cation-exchange separation were obtained using a reverse-phase column (R 2/H 4.6/50) on a BioCAD
workstation with a linear gradient of 0.1% trifluoroacetic acid with
80% acetonitrile. Fraction size, 2.5 ml; flow rate, 5 ml/min.
[View Larger Version of this Image (21K GIF file)]
Fig. 2.
Homogeneity of MkTx I. Ionspray mass
spectra of makatoxin I. Ionspray voltage, 4000 V; orifice voltage, 75 V; mass scan range, 500-2000 atomic mass units. Inset,
capillary electrophoresis of makatoxin I. Sample was injected at 5 p.s.i./s to a 25 µm × 24-cm coated capillary and run in 0.1 M phosphate buffer (pH 2.5) under 12.00 kV from + to at 15 °C for 40 min.
[View Larger Version of this Image (12K GIF file)]
Fig. 4.
A, amino acid sequence determination of
MkTx I. a, results from Edman degradation of
S-pyridylethylated MkTx I. b, results from Edman
degradation of the peptide P55-64 from Lys-C proteinase digestion.
c, complete amino acid sequence of MkTx I. A dash
indicates an unidentified residue. B, amino acid sequence
comparison of MkTx I with other scorpion toxins. Amino acid sequences
are arranged with cysteine residues (C) in register with
those of MkTx I, leaving gaps when necessary. The jutting
(J) and blunt (B) loops are identified by
dashed lines. The residues conserved to MkTx I are shown in bold. Percentages of similarity are listed to the
right of the figure. Sources: Bot I and Bot II from B. occitanus tunetanus (7, 8), Lqq IV and Lqq III from L. quinquestriatus quinquestriatus (9, 10), Lqh IT from L. quinquestriatus hebraeus (IT-insect toxin) (11), Bom III from
B. occitanus mardochei (12), and Css II from C. suffusus suffusus (13).
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
Reverse-phase high performance liquid
chromatography fractionation of peptides released by digestion of
pyridylethylated MkTx I with endoproteinase Lys-C. Solvent A,
0.1% trifluoroacetic acid; solvent B, 70% acetonitrile in 0.085%
trifluoroacetic acid; flow rate, 0.2 ml/min. Elution was done with a
linear gradient from 5 to 65% solvent B at 50 min, followed by a
linear gradient to 100% solvent B at 55 min.
[View Larger Version of this Image (15K GIF file)]
Peptide
Calculateda
Observedb
MkTx
I
7038.83
7031.71
± 2.88
P1-28
3539.42
3538.47
± 2.12
P33-54
2821.81
2821.64
± 1.53
P55-64
1190.30
1192.99 ± 1.42
a
Molecular masses are calculated from the amino acid
sequences of these peptides.
b
Molecular masses are detected by electrospray mass
spectrometry.
The complete amino acid sequence of MkTx I was compared with known
toxins from other scorpions: a striking degree of similarity was found
with these toxins, for which the total primary structure is known. It
is 81% similar to that of Bot II (7) and 78% similar to that of Bot I
(8), both of which were isolated from North African scorpion B. occitanus tunetanus venom. From 55-77% similarity was found when
compared with other toxins of Lqq IV (9) and Lqq III (10) from
Leiurus quinquestriatus quinquestriatus venom, Lqh IT
(11) from L. quinquestriatus hebraeus venom, and Bom III
(12) from B. occitanus mardochei venom. The least similar one was Css II (13) from Centruroides suffusus suffusus,
which showed only 25% similarity (Fig. 4B).
Experiments were also performed
to determine whether MkTx I could mediate nitrergic responses in the
rat isolated ACM because the venom (MKV) from which it was purified was
observed to possess this pharmacological activity. After blockade of
the adrenergic responses of the ACM by phentolamine (5 µM), FS of the carbachol-precontracted ACM produced
relaxant (inhibitory) responses of the muscle. When FS was stopped, the
addition of MkTx I (2 µg/ml) produced a rapid and marked 39.5 ± 3.4% (n = 4) decrease (relaxation) of the muscle tone,
which then progressively returned to 82.1 ± 4.6%
(n = 4) of the initial (contracted) muscle tone.
Pretreatment with 50 µM L-NAME produced
blockade of the nitrergic responses to FS as well as the relaxant
responses to 2 µg/ml MkTx I; the relaxant responses to 1 µM sodium nitroprusside were not blocked (Fig. 5).
MkTx I is structurally homologous to other scorpion toxins so far
described: (a) it consists of one single chain of 64 residues cross-linked by 4 disulfide bridges; and (b) it lacks
methionine, phenylalanine, and histidine. MkTx I belongs to the
-type scorpion toxin group; it has a short J-loop (from Cys-16 to
Cys-22) and a long B-loop (From Cys-36 to Cys-46) (14). Furthermore, it displayed a very high similarity with toxins of group 3 (Fig. 4B) according to the classification of Rochat et
al. (15). In this group, MkTx I has highest homology with Bot II
from North African scorpion B. occitanus tunetanus venom.
However, it differed significantly from those of
-type neurotoxins
isolated from the venoms of North American and South American
scorpions. For example, Css II showed only 25% similarity with MkTx I. Moreover, MkTx I has 5 aspartic acid and 5 asparagine residues with 1 glutamic acid and 1 glutamine residue, in contrast to
-type scorpion
toxins that contain more glutamic acid and glutamine residues than
aspartic acid and asparagine residues (16, 17).
The marked relaxant responses of the carbachol-precontracted ACM to MkTx I, like the nitrergic (relaxant) responses to FS, were effectively blocked by 50 µM L-NAME, a stereoselective inhibitor of the enzyme nitric oxide synthase involved in the synthesis of NO from L-arginine and oxygen (18, 19). The relaxant responses of the ACM produced by MkTx I are therefore also likely to be NO-mediated because there is now strong evidence that implicates NO as the inhibitory neurotransmitter involved in nitrergic transmission (20) and, consequently, in mediating the relaxant responses of the ACM (21, 22). Unlike the relaxant responses of the ACM to FS and MkTx I, the relaxant responses of the ACM to sodium nitroprusside, an agent that directly releases its NO via metabolism (23), were not blocked by L-NAME. This provides further evidence for the involvement of endogenous NO released prejunctionally in mediating the relaxant responses of the ACM to MkTx I.
In conclusion, a novel toxin MkTx I with nitrergic actions has been purified from the venom of the scorpion B. martensi Karsch, and the novel nitrergic actions of MKV in the rat isolated anococcygeus muscle can be attributed, at least in part, to the actions of the pure toxin MkTx I present in the venom.
We thank Shaolian Zhou (Center for Natural Product Research, Institute of Molecular and Cellular Biology, Singapore) for mass spectrographic analysis.