Chemical modification of arginine residues in α-bungarotoxin

Biochimica et Biophysica A cta, 1159 (1992) 255-261 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

255

BBAPRO 34320

Chemical modification of arginine residues in a-bungarotoxin Shinne-Ren Lin

a

and Chun-Chang Chang b

a Department of Chemistry, Kaohsiung Medical College, Kaohsiung (Taiwan, ROC) and b Department of Biochemistry, Kaohsiung Medical College, Kaohsiung (Taiwan, ROC) (Received 6 May 1992)

Key words: a-Bungarotoxin; Chemical modification; Arginine residue; Nicotinic acetylcholine receptor

The reaction of a-bungarotoxin (a-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of a-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of a-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of a-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in a-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.

Introduction

Up to date, more than 80 highly homologous a-neurotoxins from the venoms of families Elapidae and Hydrophiidae have been sequened. They are structurally grouped into 2 classes, short-chain neurotoxins containing 60-62 amino-acid residues with four disulfide bonds and long-chain neurotoxins of 70-74 residues with five disulfides [1-3]. ot-Bungarotoxin (o~BuTX), a long-chain neurotoxin isolated from the venom of Bungarus multicinctus has been widely used to study the structure-function of nicotinic acetylcholine receptor (AChR) owing to its specific and irreversible binding to the receptor. We have recently demonstrated that the amino-groups in a-BuTX may participate in the multipoint contact between the toxin and AChR, but none of the individual amino groups are essential for the binding [4]. ~-BuTX contains three arginine residues located on loop 2 (Arg-25 and Arg-36) and in the C-terminal tail (Arg-72). It has been assumed that the invariant Arg-36 appears to play an important electrostatic role in the toxin binding to AChR, while the experimental evidence is relatively poor in comparison with that from other functional groups in a-neurotoxins [1-3,5]. In this work, the role

Correspondence to: C.-C. Chang, Department of Biochemistry, Kaohsiung Medical College, Kaohsiung, Taiwan, ROC 80708.

of arginine residues in lethality, affinity to AChR and immunological activity of a-BuTX was studied by selective modification. Materials and Methods

Bungarus multicinctus venom was collected in our laboratory and a-BuTX was isolated as described by Chen and Chang [6] and further purified according to the method used by Kosen et al. [7]. Torpedo californica was purchased from Pacific Biomarine (Venice, CA, USA) and the electric organs were removed and stored in - 70°C. [125I]a-BuTX (291 C i / m m o l ) was purchased from Amersham, UK. TPCK-Trypsin, Streptococcus aureus V8 proteinase and trifluoroacetic acid (TFA) were from Sigma (St. Louis, USA). 1,2-Cyclohexanedione (CHD) was obtained from Aldrich and p-hydroxyphenylglyoxal (HPG) from Pierce (Rockford, IL, USA). The reverse-phase high-performance liquid chromatography (RP-HPLC) column (TSK-gel, ODS-120T, 0.46 × 25 cm) was from Toyo Soda, Japan. All other reagents were of analytical grade. Modification with CHD. Modification was performed essentially according to the method described by Patthy and Smith [8]. a-BuTX (1 ~mol) in 2 ml of 0.2 M sodium borate buffer (pH 9.0) was incubated with 100-fold molar excess of CHD. The reaction was allowed to proceed at 37°C for 1 h and the mixture was

256 desalted by gel filtration on a Sephadex G-50 column (1.8 x 90 cm) equilibrated with 10% acetic acid. In order to modify more arginine residues, the reaction was carried out in 0.2 M sodium borate buffer (pH 9.0) containing 6 M urea with 1000-fold molar excess of CHD according to the procedure described by Wolfenstein-Todel and Santom6 [9]. Modification with p-hydroxyphenylglyoxal. This was performed according to the procedure described by Yamasaki et al. [10] and Kharrat et al. [11]. a-BuTX (1.5/zmol) in 2 ml 0.1 M NaHCO 3 buffer (pH 9.0) was incubated with 100-fold molar excess of HPG. The reaction was allowed to proceed at room temperature for 1 h and the mixture was desalted by passage through a Sephadex G-25 column (2.0 x 45 cm) equilibrated with 0.05 M sodium acetate (pH 5.0). The modified derivatives were separated on a CM-52 column (1.5 X 28 cm) equilibrated with 0.05 M sodium acetate buffer (pH 5.0) and eluted with a linear gradient of 0-0.15 M NaC1 in the same buffer. The three main fractions (Fig. 3) were further purified on a SP-Sephadex C-25 column (1.0 x 28 cm) with a linear gradient from 0.03 M (pH 5.0) to 0.15 M (pH 6.8) of ammonium acetate buffer. Localization of the incorporated groups. The derivatives were reduced and carboxymethylated (RCM) by the procedure described by Crestfield et al. [12], followed by proteolytic digestion. RCM-proteins (1 mg) in 1 ml of 0.1 M ammonium bicarbonate (pH 8.0) were digested with trypsin for 6 h or with S. aureus V8 proteinase for 8 h in 0.05 M sodium phosphate buffer (pH 7.8) at 37°C, substrate/enzyme ratio of 50:1 (w/w). The hydrolysates were separated by RP-HPLC on Toyo Soda ODS-120T column (0.46 x 25 cm) equilibrated with 0.1% TFA and eluted with gradients of acetonitrile in 0.1% TFA as shown in the figure legends. The peptides were lyophilized for amino-acid analysis. Assay for lethal toxicity. Mice weighing 16-18 g were injected intraperitoneally with 0.2-ml samples of serial 2-fold dilutions. Four mice of both sexes were used for each dilution and the LDs0 values were calculated according to the 50% end-point method of Reed and Muench [13].

Preparation of AChR-rich membrane fragments. These were prepared from T. californica electric tissues according to the published procedure of ContiTronconi et al. [14] with a slight modification as previously described [4]. The specific activity of the preparation (expressed as nmol of a-BuTX binding/mg protein), measured by the [125I]a-BuTX binding assay, was 0.6 nmol/mg protein. Receptor binding assay. The binding activity of aBuTX and its modified derivatives was determined by modifying the centrifugation assay of Conti-Tronconi et al. [14] as previously described [4]. A constant amount

of AChR-rich membrane fragments (80 /zg) and [125I]a-BuTX (3.44 nM) were incubated with increasing concentrations of a-BuTX or its derivatives at room temperature for 2 h, followed by continuous incubation at 4°C for 16 h. The amount of bound toxin was calculated from the different radioactivity between the total radioactivity input and supernatant. The mean values of duplicate determinations were plotted and the molar concentration of the native or modified toxin able to inhibit 50% of the specific binding of the labeled a-BuTX was defined as ICs0 [15,16]. Both ICs0 values from duplicate determinations fell within 10% of the mean value. Immunological methods. Rabbit anti-a-BuTX sera were prepared according to the procedure previously described [6]. Antigenicity was determined by competitive ELISA as previously described [4]. Results

Reaction of a-BuTX with CHD The reaction product of a-BuTX with 100-fold molar excess of CHD in 0.2 M borate buffer (pH 9.0) was purified by CM-52 chromatography. The major fraction proved to be homogeneous by polyacrylamide gel electrophoresis was subject to amino-acid analysis. As presented in Table I, only one of the three arginine residues in a-BuTX was modified. No more arginine reacted, even with 1000-fold molar excess of CHD, in TABLE I

Amino-acid compositions of a-bungarotoxin and its derivatives modified with CHD or HPG All values are expressed as molar ratios based on leucine = 2.0. C H D and H I through H 3 were prepared by reaction with C H D and HPG, respectively. A m i n o acid

Native

Asp Thr Ser Glu Pro Gly Ala 1/2Cys-Cys Val Met Ile leu Tyr Phe His Lys Arg Trp

4.1 6.7 5.7 5.1 7.7 4.0 5.2 9.8 5.1 0.8 1.8 2.0 1.8 0.9 1.8 5.9 2.9 1.0

n.d., not determined.

(4) (7) (6) (5) (8) (4) (5) (10) (5) (1) (2) (2) (2) (1) (2) (6) (3) (1)

Modified derivative CHD

HI

H2

H3

4.2 6.8 5.7 5.1 8.0 4.1 5.1 9.8 5.1 1.1 1.9 2.0 1.8 0.9 1.9 6.1 1.9 n.d.

3.8 6.9 5.8 5.0 7.8 4.1 5.1 9.7 4.8 0.8 1.8 2.0 1.9 1.1 1.8 6.1 1.1 n.d.

3.9 6.9 5.7 4.9 7.9 4.2 5.1 9.6 4.8 0.9 1.9 2.0 1.9 1.2 1.8 6.0 2.2 n.d.

4.0 7.1 5.8 5.0 7.9 4.2 5.1 9.7 4.9 1.0 1.9 2.0 1.8 0.9 1.8 5.9 2.2 n.d.

257 100

0.2

40

80

o o

v ~

tM

0

0.3

,

,

O '1" O

,

B

11 ~o

60

.m m

-= 4 0 .J

4

9

_ - -

-

"-

2J

20

20

3

0.1

20

0

40

60

I

0

I

30

Time (min)

Time

Fig. 1. HPLC chromatogram of the tryptic digests of native (A) and CHD-modified e-BuTX (B). The tryptic digests were separated on a TSK gel ODS-120 T column (0.46x25 cm, Toyo Soda) at a rate of 1.0 ml/min with a linear gradient of acetonitrile in 0.1% TFA as shown by a dotted line. The absorbance was monitored at 215 nm.

I

60

90

(rain)

Fig. 2. Change of lethal toxicity of a-BuTX by reaction with HPG at various pH values, tr-BuTX (1 mg) in 1 ml of 0.1 M NaHCO 3 buffer was allowed to react with 100-fold molar excess of HPG at 37°C at pH 8.0 (o e), pH 9.0 ( • • ), pH 9.5 ( o o ) and pH 10.0 (/x ,x ), respectively. After suitable intervals of time, aliquots were taken for the determination of lethality.

the presence of 6 M urea. The tryptic-digest peptides from RCM-a-BuTX and its CHD-derivative were separated by RP-HPLC. Peptide mapping (Fig. 1) and amino-acid analysis of the isolated peptides (Table II) indicated that Arg-72 was modified in the reaction.

mined at 340 nm based on a molar extinction coefficient of 1.83-104 M -~ cm -~ [10]. Changes of the lethal toxicity of a-BuTX by reaction with HPG at varying pH values are shown in Fig. 2. The toxicity decreased rapidly in the reactions performed at pH 9.0 or higher and was lost pronouncely after 60 min. For the preparation of arginine-modified derivatives, otBuTX was allowed to react with HPG in 0.1 M

Reaction with HPG HPG has been reported to be an arginine-specific reagent and the extent of modification can be deter-

TABLE II

Amino-acid compositions of tryptic fragments of RCM- a-bungarotoxin The peptides obtained from Fig. 1 were analyzed.

Amino acid

Peak 1

CM-Cys Asp Thr Ser Glu Pro Gly Ala Val Met

2

3

0.9 (1) 1.1 (1)

1.0(1) 0.9 (1) 1.0 (1)

0.8(1) 2.7 (3) 0.9(1)

4

0.8 (1) 1.1 (1)

1.8 (2)

5

1.6 (2) 1.0 (1) 1.8 (2) 1.1 (1) 1.9(2) 1.1 (1)

1.0 (1)

6

2.6 (3) 1.9 (2) 1.8 (2) 1.0 (1) 2.0(2) 2.9 (3)

1.0 (1)

7

8 *

1.7 (2)

1.5 (2)

0.8 (1) 0.9(1) 0.9(1) 0.9 (1) 1.0 (1) 1.8(2) 1.5 (2)

1.0 (1) 1.1 (1) 1.1 (1) 1.0 (1) 1.2(1) 2.0(2) 1.6 (2)

1.9 (2) 1.7 (2) 2.1 (2)

1.2(1) 1.2(1)

9

10 *

1.5 (2) 0.8 (1) 0.8 (1) 0.7 (l) 0.8(1) 0.9 (1) 0.9(1) 1.7 (2) 1.6 (2)

0.8 (1)

11 *

1.8 (2) 0.9 (1)

1.9 (2) 1.1 (1)

1.6(2)

2.0(2)

0.9(1)

1.1 (1)

0.8 (1)

0.8 (1)

1.0 (1)

1.0 (1)

2.0 (2)

2.9 (3) 0.9(1)

1.0 (1)

1.0 (1)

1.0 (1)

1.0 (1)

Phe

Lys His Arg 1.0 (1) Corresponding peptide 71-74

1.0 (3) 0.9(1) 0.9(1) 65-74

1.0 (1) 0.9(1)

2.0 (2)

1.0 (1)

1.0 (1) 65-70

* The peptides containing the Trp-28 residue.

52-64

52-70

39-51

39-51

2.6 (3) 1.0 (1) 3.6 (4) 1.9(2) 0.9(1) 2.8 (3) 1.0(1) 1.9(2) 1.6 (2) 1.7 (2) 0.9(1) 0.9 (1)

Ile

Leu Tyr

12

26-~38

39-51

0.9(1) 1.0 (1)

1.1 (1)

0.9(1)

1.0 (1)

26-36

27-36

0.9(1) 1.1 (1) 1-25

258

O.3

H1 H2

O

0D 0.2 N

<

o,,/ I.i

0

I

I

I

i

I

I

40

60

80

100

120

140

Fraction

Number

Fig. 3. Separation of HPG-modified a-BuTX derivatives on a CM-52 column. The reaction products of a-BuTX with 100-fold molar excess of HPG were applied on a CM-52 column (1.5×28 cm) equilibrated with 0.03 M sodium acetate buffer (pH 5.0) and eluted by a linear gradient from 0-0.15 M NaCl in the same buffer solution. 5-ml fractions were collected and the protein fractions were pooled as indicated by bars and lyophilized.

NaHCO 3 buffer (pH 9.0) for 1 h. About 5 protein fractions were obtained from the reaction products by a CM-52 chomatography (Fig. 3). The three major fractions indicated by bars and marked as H1, H2 and H 3 were collected separately and further purified by SP-Sephadex C-25 chomatography (data not shown).

Spectral determination of the extent of modification based on the molar absorption coefficient and an amino-acid analysis (Table I) indicated that two HPG groups were incorporated into fraction Hi, while fractions H 2 and H 3 were monoderivatives. In order to locate the modified arginine residues, the RCM-derivatives of native and modified a-BuTX were digested with S. aureus V8 proteinase and the resulting peptides were separated by RP-HPLC . As shown in Fig. 4, either a-BuTX or the modified derivatives revealed five totally different peptides marked as A through G. The probable sequences of peptides assigned from their amino-acid compositions (Table III) indicated that the five peptides (A, B, E, F and G) are corresponding to the sequences of t~-BuTX fragments, 3141, 57-74, 42-56, 1-20 and 21-30, respectively. However, peptides A a n d / o r B in control t~-BuTX were shifted to the respective C a n d / o r D in the chromatographic profiles of the modified derivatives (Fig. 4). Amino-acid analysis indicated that the peptides C and D are corresponding to the peptides A and B, except that one arginine had disappeared (Table III). These results revealed that the fraction H l is both Arg-36 and -72 modified derivative, while fractions H 2 and H 3 are monoderivatives modified at Arg-72 and -36, respectively.

Biological activity of the modified toxins We have prepared two monoderivatives modified at Arg-36 or -72, and one of the derivatives modified at

TABLE Ili

Amino-acid composition of proteolytic peptides from RCM-a-BuTX and HPG-a-BuTX derivatives The peptides obtained from Fig. 4 were analyzed and peak nomenclature is described in the figure legends. Amino acid

Peak A

CM-Cys Asp Thr Ser Glu Pro Gly Ala Val Met lie leu Tyr Phe His Lys Arg Corresponding peptide Arg-modified

0.96 (1)

1.63 (2) 1.00 (1) 1.33 (1) 0.95 (1) 1.65 (2)

B 2.93 (3) 1.97 (2) 2.14 (2) 1.02 (1) 1.34 (1) 2.93 (3) 1.01 (1) 1.00 (1)

C 0.94(1)

1.50 (2) 1.00 (1) 1.31 (1) 0.97 (1) 1.56 (2)

D 2.91 (3) 1.99 (2) 2.01 (2) 1.01 (1) 1.32 (1) 2.98 (3) 1.21 (1)

E

F

1.95 (2)

1.89 (2)

0.78 (1) 0.82 (1) 1.26 (1) 1.89 (2) 1.00 (1) 1.72 (2)

3.78 (4) 1.68 (2) 1.00 (1) 2.76 (3) 1.18 (1) 1.75 (2) 1.84 (2)

1.00 (1)

G 1.82 (2) 1.74 (2)

0.97 (1) 1.78 (2) 0.86 (1) 0.61 (1) 0.95 (1) 1.26 (1) 0.89 (1) 31-41

1.00 (1) 1.01 (1)

0.91 (1) 0.98 (1) 1.96 (2) 1.01 (1) 57-74

1.32 (1) 0.19 (0) 31-41 Arg-36

1.05 (1) 2.21 (2) 0.29 (0) 57-74 Arg-72

1.28 (1) 1.73 (2)

42-55

1.12 (1) 0.96 (1) 1-20

21-30

259 TABLE IV cI-BuTX

B

E

Lethality and binding affinity to A C h R o f a - B u T X and its derivatives 0.5

CHD and fractions H 1 through H3 were obtained from reaction with CHD and HPG, respectively. HI

Derivative

D

Activity

F

Lethal potency b

Binding potency

LD50 (p,g/g)

Relative lethality (%)

iC50 c (nM)

Relative activity (%)

0.14 0.27 0.98 0.27 0.37

100 50 14 50 38

204 398 1513 425 630

100 51 13 48 32

0.5

0

u~

D E

H2

F

a-BuTX CHD (Arg-72) a H I (Arg-36,72) H 2 (Arg-72) H 3 (Arg-36)

0.5

0

0

H3

20

B

30

E

40

F

50

60

a The modifed arginine residue(s). b Measured i.p. with mice weighing 16-18 g. c Calculated from the molar concentration to induce the 50% inhibition of [125Ila-BuTX binding to the AChR in duplicate determinations from Fig. 5.

Time(min)

Fig. 4. HPLC chromatogram of the proteolytic digests of native and HPG-modified a-BuTX with S. aureus V8 proteinase. Chomatography was performed under the same conditions as in Fig. 1, except that elution was performed with three stages of linear gradients of acetonitrile: 0 - 2 5 % for 50 min, 25-35% for 30 min and 35-60% for 20 min, respectively. Peaks eluted at the same retention time were referred to with the same letters.

both positions. No derivative modified at Arg-25 was obtained, indicating that it may be located in the interior region of the molecule. The relative binding affinity of the modified derivatives to AChR was measured by their ability to com-

pete with [125I]a-BuTX (Fig. 5). A parallel relationship between the binding affinity and lethality was observed (Table IV). A monoderivative at Arg-72 showed about 50% residual activities, but the activities were decreased to approx, one-third of the toxin when Arg-36 was modified, indicating that the conservative Arg-36 is more closely related to the toxin-AChR binding. Approx. 13% of the residual activities were observed when both Arg-36 and -72 were modified. These re-

100

1.0

\

'13 C 0

m

x

L

50

d

\

0.5

3

mi

\\

o

:,.

i

B

i

C

100

D

m

50

d.

0

2.0

;s Log ~To xi n~(nM)

I

Fig. 5. Competition experiments of a-BuTX and its modified derivatives with [125I]a-BuTX for binding to AChR. The experimental procedures were described in Materials and Methods. ( o o), a-BuTX; (e e), CHD (Arg-72); (zx zx), H 2 (Arg-72); ([] [] ), H 3 (Arg-36); ( × × ), H i (Arg-36 and -72) modified derivatives. B and B o denote the bound radioactivity in the presence and absence of competitors, respectively. The non-specific binding was substracted and the results shown are the average of duplicate determinations.

i

9

7

-Log

5

,,i

9

i

i

7

5

EToxin~(M)

Fig. 6. Competitive ELISA of anti-a-BuTX sera with a-BuTX and its modified derivatives. Antisera were diluted 10000-fold for use. In all cases, the continuous curve corresponds to the inhibition observed in the presence of a-BuTX. The derivatives are: (A), CHD (Arg-72); (B), H 2 (Arg-72); (C), H 3 (Arg-36); (D), H x (Arg-36 and -72). B and B 0 represent the absorbance at 492 nm in the presence and absence of competitors, respectively. The non-specific binding was substracted and the results shown are the average of duplicate determinations.

260 suits indicate that these residues may be involved in the AChR-binding, but no individual arginine residue can presently be determined as absolutely essential.

Antigenic activity of the modified toxins As shown in Fig. 6, most of the monoderivative modified at Arg-36 or -72 retained its antigenic activity as measured by a competitive ELISA. Subsequently, the activity was decreased to half of that of a - B u T X when the both residues were modified.

Discussion By chemical modification and comparison of the sequence data, the contribution of individual aminoacid residues in snake c~-neurotoxins to the AChR-binding has been investigated [1-3,5,17]. It has been assumed that the conserved Arg-37 (corresponding to Arg-36 in a - B u T X ) plays an important electrostatic role in neurotoxicity. However, there is little experimental evidence, one from cobrotoxin [18] and the other from a-cobratoxin, a long-chain neurotoxin from Naja naja siamensis [19]. These results have excluded the arginine residues or any particular one of them as absolutely essential for toxicity, but one or more of them may be involved in the AChR-binding. In this study, among the three arginine residues (Arg-25, -36 and -72) in a-BuTX, only one at position 72 was modified in borate buffer (pH 9.0), even with 1000-fold molar excess of C H D in the presence of 6 M urea. Apart from Arg-72, Arg-36 as well as both were modified in sodium bicarbonate buffer (pH 9.0). These results indicate that Arg-72 in the C-terminal tail of a - B u T X is the most accessible to modification, Arg-36 is less reactive and Arg-25 is probably located in the interior region of the molecule. We have suggested that Lys-26 in a - B u T X is the least reactive to modification with trinitrobenzene sulfonate [4] and Tyr-24 was resistant to nitration reported by Chen et al. [20]. It has been showed that the four invariant disulfide bonds and Tyr-24, which is buried in the molecule, form a central core to stabilize the active conformation of ct-neurotoxins [21]. Thus, Arg-25 and Lys-26 located adjacent to the core seem to be in inaccessible states to modification. The central loop (loop II) of the long-chain neurotoxins containing most of the highly conserved residues, including Lys-26, Trp-28, Arg-36 and Lys-38 (numeration based on a - B u T X sequence), has been proposed to be an essential region for the AChR-binding [13,5,22]. However, Lys-26 in a - B u T X has been suggested to be located in the interior region of the molecule [4] and Trp-28 is not required for the toxin lethality [23]. Morever, about one-third to one-half of the lethality and binding affinity for A C h R of the toxin was still retained when Arg-36 and Lys-38 [4] were

modified, respectively, indicating that the cationic groups of Arg-36 and Lys-38 may be involved in the binding, but neither is absolutely essential. Endo et al. [24] demonstrated that the binding affinity to A C h R reduced greatly after cleavage of the C-terminal four to five residues by carboxypeptidase P, suggesting that Lys-70 and Arg-72 are directly involved in the binding. However, the results from the derivatization of Arg-72 in the present work and of Lys-70 in the previous study [4] show no pronounced decrease in lethality and binding activity. The antigenicity of a - B u T X was still retained essentially intact after only one arginine at position 36 or 72 was modified. Thus, the observed loss of biological activity is more likely due to the disappearance of an electrostatic interaction between the toxin and A C h R than to a conformational change of the toxin. Further experiments by using of monoclonal and anti-peptide antibodes would be required to determine precisely the location and structure of the epitopes which are closely related to the AChR-binding sites of a-BuTX. This work is in progress.

Acknowledgements This work was supported by grant NSC 81-0412-B037-27 from the National Science Council, Taiwan, ROC. We also thank Drs. L.Y. Chuang and K.W. Kuo for the purification of AChR-rich membranes from electric organ of Torpedo californica.

References 1 Karlsson, E. (1979) in Handbook Experimental Pharmacology (Lee, C.Y., ed.) Vol. 52, pp. 159-212, Springer, Berlin. 2 Dufton, M.J. and Hider, R.C. (1983) CRC Crit. Rev. Biochem. 14, 113-171. 3 Lentz, T.L. and Wilson, P.T. (1988) Int. Rev. Neurobiol. 29, 117-160. 4 Lin, S.R. and Chang, C.C. (1991) Toxicon 29, 937-950. 5 Love, R.A. and Stroud, R.M. (1986) Prot. Eng. 1, 37-46. 6 Chen, H.M. and Chang, C.C. (1982) J. Chin. Biochem. Soc. 11, 32-48. 7 Kosen, P.A., Finer-Moore, J., McCarthy, M.P. and Basus, V.J. (1988) Biochemistry27, 2775-2781. 8 Patthy, L. and Smith, E.L. (1975) J. Biol. Chem. 250, 565-569. 9 Wolfenstein-Todel, C. and Santom6, J.A. (1983) Int. J. Pept. Protein Res. 22, 611-616. 10 Yamasaki, R.B., Vega, A. and Feeny, R.E. (1980) Anal. Biochem. 109, 32-40. 11 Kharrat, R., Darbon, H., Rochat, H. and Granier, C. (1989) Eur. J. Biochem. 181,381-390. 12 Crestfield, A.M., Moore, S. and Stein, W.H. (1963) J. Biol. Chem. 238, 622-627. 13 Reed, L.J. and Muench, H. (1938) Am. J. Hyg. 27, 493-497. 14 Conti-Tronconi, B.M., Tang, F., Walgrave, S. and Gallagher, W. (1990) Biochemistry29, 1046-1054. 15 Jover, E., Martin-Moutot, N., Couraud, F. and Rochat, H. (1978) Biochem. Biophys. Res. Commun. 85, 377-382.

261 16 Kharrat, R., Darbon, H., Granier, C. and Rochat, H. (1990) Toxicon 28, 509-523. 17 Endo, T. and Tamiya, N. (1987) Pharmacol. Ther. 34, 403-451. 18 Yang, C.C., Chang, C.C. and Liou, I.F. (1974) Biochim. Biophys. Acta 365, 1-14. 19 Martin, B.M., Chibber, B.A. and Maelicke, A. (1983) J. Biol. Chem. 258, 8714-8722. 20 Chen, Y.H., Tai, J.C., Huang, W.J., Lai, M.Z., Hung, M.C., Lai, M.D. and Yang, J.T. (1982) Biochemistry 21, 2592-2600.

21 Yang, C.C. (1987) in Pharmacology (Rand, M.J. and Raper, C. eds.) pp. 871-874, Elsevier, Amsterdam. 22 Lentz, T.L (1991) Biochemistry 30, 10949-10957. 23 Chang, C.C., Kawata, Y., Sakiyama, F. and Hayashi, K. (1990) Eur. J. Biochem. 193, 567-572. 24 Endo, T., Oya, M., Tamiya, N. and Hayashi, K. (1987) Biochem° istry 26, 4592-4598.