Toxkon Vol . 28, No . 3, pp . 329-339, 1990.
0041-0101/90 53.00 + .00 ® 1990 Pcgenwn Pros pk
Printed in Oreat Britain.
AMINO ACID SEQUENCES OF EIGHT PHOSPHOLIPASES A2 FROM THE VENOM OF AUSTRALIAN KING BROWN SNAKE, PSEUDECHIS A USTRALIS GüIKAHISA TAKASAKI, FUJIKO YUTANI
and
TSUYOSHI KAJIYASHIKI
Dt:partment of Cht:alistry, Faculty of Science, Tohoku University, Aobayama, Sendai 980, Japan (Acceptedfor publication 15 .4agust 1989)
C. TAKASAKI, F. YUTANI and T. KAJIYASHIKI . Amino acid sequences of eight phospholipases A2 from the venom of Australian king brown snake, Pseudechis australis. Toxicon 28, 329-339, 1990.-The amino acid sequences of eight phospholipases A2 (Pa-1G, Pa-3, Pa-5, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-15) which had been isolated from the venom of Australian king brown snake (Pseudechis australis) were elucidated . Pa-1G, Pa-3 and Pa-15 showed micro-heterogeneity at the 103rd position and Pa-5 was separated into two components, Pa-Sa ([Pro-18 and Tyr-61]Pa-S) and Pa-Sb ([Ser-18 and Phe61]Pa-5). All the phospholipase A2 molecules except Pa-1Ga and Pa-1Gb which lack the 118th residue, consisted of a single chain of 118 amino acid residues including 14 half-cystine residues and all the common residues among phospholipases A2 from other sources. From comparison studies, Asp-50, Lys58 and Asp-90 seem to be important for the toxicity, and we propose that the domain for the presynaptic toxicity consists of seven hydrophilic residues, i.e. Arg-43, Lys-46, Asp-50, Glu-54, Lys-58, Asp-90 and Glu-94 .
INTRODUCTION
A NUMBER of phospholipases A2 (PLA2, EC 3.1 .1.4) have been purified, and their amino acid sequences determined, from the venoms of various snakes, including those belonging to the three families Elapidae, Crotalidae and Viperidae. All the sequences show a high degree of homology: Some of the basic PLA2s show presynaptic neurotoxicity while other basic PLA2s or acidic PLA2s do not show this type of toxicity (for a review, see EAKER, 1978). The structure-function relationship in PLA2 enzymes have been discussed (for example; DuIrroN and HinsR, 1983; KINI and IWANAGA, 1986; T3AI et al., 1987 ; KoNno et al., 1989). Two PLA2 enzymes, Pa-11 and Pa-13 have been purified from the venom of Australian king brown snake (Pseudechis australis, NI3HIDA et al., 1985a) and sequenced (NISHIDA et al., 19856) . In addition, 13 isoenzymes of PLA2 were isolated from the venom o~ _P. australisal including Pa-1G, an acidic PLA2 with neurotoxicity (TAKASAKI et ., 1990). The present paper describes the complete amino acid sequences of eight PLA2 enzymes (Pa-1G, Pa-3, Pa-5, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-15) from the venom of P. australis. 329
330 Phospholipases A,
C. TAKASAKI et al. MATERIALS AND METHODS
Phospholipases A Pa-1G, Pa-3, Pa-S, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-IS were purified from the venom of king brown snake (Pseudechis oonfmlin), as already described (TAKASAKI et al ., 1990).
Proteinases
Lysyl~ndopeptidase from Achrornobacter lyticus, Staphylococcus aweus V8 proteinase, chymotrypsin and carboxypeptidase Y from yeast were purchased from Wako Pure Chemical industries (Osaka, Japan), Miles Laboratories Inc. (Elkhart, IN, U.S .A.), Worthington Biochemical Corp . (Freehold, NJ, U.S.A .) and Oriental Yeast Co . (Osaka, Japan), respectively.
Enzyme digestions
Lysyl~ndopeptidase digestions of reduced and S-carboxymethylated (RCM) proteins were carried out in 50 mM Tris-HCl buffer, pH 9.1 at an enzyme/substrate ratio of 1 :200 (w/w) and 37°C for 4 hr . V8 proteinase digestions were carried out in 50 mM NH,HCO, at an enzyme/substrate ratio of 1 :100 (w/w) and 37°C for I S hr. Chymotryptic digestions were carried out in 50 mM NH,HCO, at an enzyme/substrate ratio of 1 :250 (wJw) and '37°C for 2 hr . The digestions of RCM-proteins or peptides (20 nmole) with carboxypeptidase Y (0.5 hg) were carried ôut in 50 mM citrate buffer, pH 6.0 for 30 min to 6 hr, and aliquots of the digests were taken out at suitable intervals, added with an equal volume of 2 M acetic acid and directly applied to the amino acid analyzer.
Pur~cation of peptides
Peptides were separated with a Pharmacia fast protein liquid chromatography system (FPLC) equipped with a column of Pcp RPC HR 5/5 (Pharmacia, Uppsala, Sweden). Elution was performed with a linear gradient of acetonitrile in 0.1 % trifluoroacetic acid from 0 to 30% in 30 min, then 30 to 40% in 5 min at a flow rate of 1.2 ml/min . The fractionated peptides were manually collected by monitoring the absorbance at 214 nm. If necessary, rechromatography was carried out on the same column with a linear gradient of acetonitrile in 10 mM ammonium formate, pH 6.3 . In some cases, gel filtration with a Sephadex G-50 or a Bio Gel P-2 column or ion exchange chromatography with a DEAE~ellulose DE-52 column were carried out.
Amino acid analysis and Edman degradation
Amino acid analysis and the manual Edman degradation were tamed out as described previously (TAKASAKI and T~uv~, 1982). Automated Edman degradation with a gas-phase sequencer (model 470A ; Applied Hiosystems, Foster, CA, U.S .A .) on RCM-Pa-S, peptides C6 from Pa-3, V3 from Pa-5, C3 and C4 from Pa-10A, C6 from Pa-12A and C4 and CS from Pa-15 was carried out essentially as described by HEWICIC et al. (1981) .
RESULTS
Amino acid sequence of phospholipaseA~ Pa-IG RCM-Pa-1G (about 100 nmoles each) was digested with lysyl-endopeptidase or V8 proteinase, and the digests were separated using the FPLC reversed phase system . RCMPa-1G (130 nmole) was also degraded with 6 mg BrCN in 1 .0 ml of 70% formic acid (v/v) at 37 °C for 24 hr. The reaction mixture was applied to a column (1 .2 ctn x 130 cm) of Sephadex G-25 equilibrated with 5 mM HCI. Peptides B 1, B2 and B3 were eluted at distribution coefficient (Kp) values of 0.80, 0.35 and 0.05 respectively . Peptide B3 (95 nmole) was digested with chymotrypsin . Sequence studies on Pa-IG are summarized in Fig. 1 . Both threonine and alanine residues were detected at the 103rd position, therefore, Pa-1G was a mixture of Pa-1Ga ([Tltr-103]Pa-1G) and Pa-1Gb ([Ala-103]Pa-1G) . They did not separate from each other by reversed phase chromatography at pH 2.0, 5.3 or 6.3.
Amino Acid Sequences of Snake Venom Phospholipases A2
33l
zo 40 NLIOFGNMIQCANKGSRPTRHYMDYGCYCGWGGSGTPVDE ' rB3C1-a ~ B3Ci ~---81-~ ~ B 2V
1
60
BO
LDRCCQTHDDCYGEAEKKGCYPKLTLYSWDCTGNVPICSP ~ L3
L4
--17
-
.~
V2 Y 3-4 _ -~ ________ VT3 _--____----_____ 100
B3 C3
Y4
117
KAECKDFVCACDAEAAKCFAKAÂYNDAMIVNIDTK~RÇ ~1-LS~ ~
L6
T L7--I ~
L6 ~ ~L9~
' :-B3 C4 ~-B3 CS---1~- B 3 C 6 ----1
FIG. I . THE AMINO ACID sDQUENCE OF PHOSPHOLIPASE A= Pa-IG. B, L, V, C and T refer to the peptides derived by cyanogen bromide degradation, lysylendopeptidase, Staphylococcal proteinase, chymotryptic and Cryptic digestions, respectively . Underlines and arrows indicate amino acid residues detected by Edman degradation and carbozypeptidase Y digestion, respectively.
Amino acid sequence
of phospholipase A1
Pa-3
RCM-Pa-3 (about 100 nmoles each) was digested with lysyl-endopeptidase, V8 proteinase or chymotrypsin and the digests were applied to the FPLC reversed phase system . Sequence studies on Pa-3 are summarized in Fig . 2. Pa-3 was a mixture of Pa-3a ([Thr103]Pa-3) and Pa-3b ([Pro-103]Pa-3) and they were partially separated from each other by reversed phase chromatography at pH 6.3. There were no marked düïerences in the enzymatic or lethal activities between Pa-3a and Pa-3b.
20
40
60
BO
NLIQFGNMIOCANKGSRPTRHYMDYGCYCGWGGSGTPVDE
~--
L
C ~ -~~- C 1--~_ C 3
LDRCCKVHDDCYGEAEKKGCYPKLTLYSWDCTGNVPICSP L 3 --~:-L 4 -1 ,
V 2
6
V
3 A_
C6 -
KAECKDFVCACDAEAAKCFAKA~YNDANWNIDTKTRC I K L6 -~; ~ :-
-I :
___
L 7
-_
~ r L B 1F- L 9
___-_ _ ____V 5 1~
t~
-, H L 10 ---I
C 7 -~~ C 8 -I ~- C 9
FIG . 2. THE AMINO ACn) SEQUENCE OF PHOSPHOLIPASE
See legend of Fig. 1 for further details.
A2 Pa-3 .
332
C . TAKASAKI et al.
NLIQFSNMIQCANKGSR~SLDYADYGCYCGWGGSGTPVDE
LDRCCKVHDDCYAEAGKKGCFPKLTLYSWDCTGNVPICNP
100
118
KTECKDFTCACDAEAAKCFAKAPYKKENWNIDTKTRCK V
V 4
6
V 6 -
i~C5~~C6~ ~- C7--
rr
FIG . 3 . TIIE AMINO ACID ~QUFNCE OF PH08PHOLIPASE Az PS-S .
See legend of Fig . 1 for further details .
Amino acid sequence ofphospholipase AZ Pa-S RCM-Pa-S (about 700 nmoles each) was digested with V8 proteinase or chymotrypsin, and the digests were applied to a column (1.S cm x 18 cm) of DEAE-cellulose DE-S2 equilibrated with 0.01 MNH,HCO,. A linear concentration gradient elution with NH,HC03 was carried out from 0.01 M to 0.6 M over 1 liter. An expected chymotryptic peptide (C1, positions 1-5) was not obtained, probably because of its low solubility . Sequence studies on Pa-5 are summarized in Fig. 3. Pa-5 was a mixture of Pa-Sa (35%) and Pa-Sb (6S%) which were eluted at 27% and 29% acetonitrile, respectively, by reversed phase chromatography at pH 5 .3. Pa-Sa and Pa-Sb were separately reduced, S-carboxymethylated and digested with V8 proteinase, and the digests were separated with reversed phase chromatography at pH 5.3. The amino acid compositions suggested that Pa-Sa and Pa-Sb are [Pro-18 and Tyr-61]Pa-5 and [Ser-18 and Phe-61]Pa-5, respectively . There were no marked differences in the activities of Pa-Sa and Pa-Sb. 20
40
NLIQFKSIIECANRGSRRWLDYADYGCYCGWGGSGTPVDE
~~
i-L I L 2 Li F-L2T1 -~ h Ti~ ~ ~C 1W
C 2
T3 -
~~ C 3 ---; - C4-~~-
C 5
60
80
LDRCCKVHDECYGEAVKQGCFPKLTVYSWKCTENVPICDS .-i ~- L 3 -~F-- L 4 V 3
0
~~ L 5
-~I---- L 6
V 4
,-
100
IIB
RSKCKDFVCACDAAAAKCFAKAPYNKDNYNIDTKTRCQ
--iiL7a7
L B --~H L 9 ~H L10-IF-- L 11
-,H L12--î
V 5 I~C7 ---IF-CB~~ C9 FIG . 4 . THE AMINO ACID SEQUENCE OF PHOÔPHOLIPASE A z
See legend of Fig . 1 for further details .
rr
Pa-9C.
333
Amino Acid Sequences of Snake Venom Phospholipases A, 20
40
60
80
NLI(~FSNMIDCANKGSRPSLHYADYGCYCGWGGSGTPVDE
LORCCKVHDDCYDQAGKKGCFPKLTLYSWDCTGNVPICNP }-- L 7
irL4
/
L5
1~
I IB
KSKCKDFVCACDAAAAKCFAKAPYNKANWNIDTKTRCK ~-L s~k ~a---- L e "r- L s -F-- L I o -+--- L I I ~-L I z -I ' F-cs ~- cs~--cz -~ FIG . 5 . THE Aanxo ACW 3EQUENCE OF PHOSPHOLIPASE AZ Pa-10A. See legend of Fig. 1 for further details.
Amino acid sequence ofphospholipase A? Pa-9C
RCM-Pa-9C (about 100 nmoles each) was digested with lysyl-endopeptidase, V8 proteinase or chymotrypsin and the digests were separated with the FPLC reversed phase system . Peptide L2 (40 nmole) was digested with trypsin in 0.2 M NH4 HC0 3 at an enzyme/substrate ratio of 1 :100 and 37°C for 16 hr, and the digests were applied to a column (1 .2 cm x 136 cm) of Sephadex G-50 equilibrated with 0 .02 M NH4HC0, and peptides L2T1, L2T2 and L2T3 were eluted at KD values of 0.85, 1 .08 and 0.60, respectively . Sequence studies on Pa-9C are summarized in Fig. 4. Amino acid sequence ofphospholipase A? Pa-10A, Pa-12A, Pa-12C and Pa-15
RCM-Pa-lOA, PCM-Pa-12A, RCM-Pa-12C or RCM-Pa-15 (about 100 nmoles each) was separately digested with lysyl-endopeptidase, V8 proteinase or chymotrypsin . 20
40
NLI~FGNMIQCANKGSRPSLNYADYGCYCGWGGSGTPVDE L 1
--1f------- L 2 V 1
~ C 1 -~ C 2
v-
C 3 -ij-C 4~- C 5 60
80
LDRCC(~VHDNCYE(~AGKKGCFPKLTLYSWKCTGNVPTCNS J-_- L__ 3 ~F--- L 4 -V ~ t 7 __L 4 --~F _ C6 I(D
11B
KTGCKSFVCACDAAAAKCFAKAPYKKENYNIDTKKRCK -~L6 --~
L9 --'~ f-LB-IFL9~r
L10--~L11-~
'~- Y 4 C 7 -H C B
.r- C 9 -
FIG. 6. THE AMINO ACID SEQUENCE OF PHOSPHOLIPASEA= Pa-12A. See legend of Fig. 1 for further details.
33 4
C. TAKASAKI et al .
TABLE
a) b) c) d) e) f) h) i) i) k) 1) m) n) o) P) 4) s) tl u) v) w) x)
1.
COMPARISON OF THE AMINO ACID SEQUENCE OF VARIOUS PHOBPHOLIPASES SNAKES
Paeudechis auatralia
Pa-1Ga Pa-1Gb Pa-3a Pa-3b Pa-Sa Pa-Sb Pa-9C~ Pa-10A Pa-11 Pa-12A Pa-12C Pa-13 Pa-15a Pa-15b Notechis s . acutatus Notexin Enhvdrinâ échiatosa Myotoxin Laticauda c0lubrina LcPLA-II~~ Laticauda semifaaciata LsPLA III LePLA I IPV LsPLA I Na a niaricollie basic PLA Na a m . moesambica CM-I Na a na a atra acidic PLA Hemachatua haemachatua DE-I
h) i) k) 1) m) n) o) P) 41 r) s) t) u) v) w) x)
FROM THE VENOM OF ELAPIll
NLIyhGNMÎQCANKGSRP~Y~Y~YCGW~S~~~VDEÎ~D~tCC~ 0 NLIQFGNMIQCANKGSRp~y~YGCYCGWGGSGTPVDELDRCC NLIQFGNMIQCANKGSR YGCYCGWGGSGTPVDELDRC~~ NLIQFGNMIQCANRGSRP~Y YGCYCGWGGSGTPVDELDRCC/vH NLIQ MIQCANRGSRPSLDYADYGCYCGWGGSGTPVDELDRCCQVHDN NLIQ MIQCANRGS SLDYADYGCYCGWGGSGTPVDELDRCCQVHDN NLIQ~IICAI~GS~DYADYGCYCGWGGSGTPVDELDRC~~ NLIQ MIQCANKGSRPSI~SIADYGCYCGWGGSGTPVDELDRC NLIQFGNMIQCANKGSRPSLDYADYGCYCGWGGSGTPVDELDRCCQVHDN NLIQFGNMIQCANRGSRPSL/YADYGCYCGWGGSGTPVDELDRCCQVHDN NLIQFGNMIQCANKGSRPSLDYADYGCYCGWGGSGTPVDELDRCCQ7HDN ~IQCANKGSR DYGCY~TPVDELDRCC NLVQFSYLIQCANHGKRPTWHYMDYGCYCGAGGSGTPVDELDRCCKIHDD NLVQFSYVITCANHNRRSSLDYADYGCYCGAGGSGTPVDELDRCCKIHDD NLIQFSELIQCANRGKRATYYYMDYGCYCGKGGSGTPVDDLDRCCKTHDD NLVQFTYLIQCANSGKRASYHYADYGCYCGAGGSGTPVDELDRCCKIHDN NLVQFSYLIQCANTGKRASYHYADYGCYCGAGGSGTPVDELORCCKIHDN NLVQFSNLIQCNVKGSRASYHYADYGCYCGAGGSGTpVDELDRCCKIHDN NLYQFHNMIHCTVP-SRPWWHFADYGCYCGRGGRGTPVDDLDRCCQVHDN NLYQFKNMIHCTVP-SRPWWHFADYGCYCGRGGKGTAVDDLDRCCQVHDN NLYQFRNMIQCTVP-SRSWWDFADYGCYCGRGGSGTpVDDLDRCCQVHDN NLYQFKNMIKCTVP-SRSWWHFANYGCYCGRGGSGTPVDDLDRCCQTHDN
1 0
9 a) b) c) d) e) f)
AZ
1 1
1 1 _9*
0 _ ~1l~RK-G~KLTLYSFI" C7GN -~K~V~A~L~A~ÂÂK~~A CY(~i/!tK-G KLTLYS =CI~VCACD CFA K-G KLTLYS C~yCACD CFAK I CRK-G KLTLYS CACD CFARAP I CKKCFAKAPYKRE CKGRK-G KLTLYS CD IDT GKK-GCFPRLTLYSf~CTGNV CD KCFAKAPYKRE ID CKGCFPKLI~1[SWKCT~IV FVCACDAAAAKCFAKAPI~/NYNI C,ÂGKK-GCFPKLTLYSW/CTGN FVCACDAAAAKCFAKAP ~IDT CKCYEQAGRK-GCFPKLTLYSWRCTGNVPTCNSRPG-CRSFVCACDAAAAKCFARAPYRKENYNIDTKKRCKCYEQAGRK-GCFPKLTLYSWRCTGNVPTCNSI~G-CKSFVCACDAAAAKCFAKAPYKKENYNIDTKKRCKCYEQAGKK-GCFPKLTLYSWRCTG~ NSRPG-C~ "~- ", CACDAAAAICCFAKAPYKKENYNIDTRKRCKC GK~GC1>PKLT/YSüCTGiPTCN~-CI~FVCACDAAAARCFAKAP I CKC CACDAAAAKCF IDT CRC RLT/YSÜ"CTG~PTCti1l~-CP~FVCACDAAAAKCFAKA~ P~IID~CKCYDEAGRR-GCFPKMSAYDYYCGENGPYCRNIKKKCLRFVCDCDVEAAFCFAKAPYNNANWNIDTRRRCQCYGEAEKQ-GCYPKMLMYDYYCGSNGPYCRNVKKKCNRKVCDCDVAAAECFARNAYNNANYNIDTRRRCKCYGQAERR-GCFPFLTLYNFICFPGGPTCDRGTT-CQRFVCDCDIQAAFCFARSPYNNKNYNINISKRCKCYGEAEKM-GCYPKLTMYNYYCGTQSPTCDDKTG-CQRYVCACDLEAAKCFARSPYNNKNYNIDTSKRCKCYGQAEKM-GCYPKLTMYNYYCGTQSPTCDNKTG-CQRYVCACDLEIIAKCFARSPYNNKNYNIDTSRRCKCYGEAEKM-GCYPKWTLYTYDCSTEEPNCSTKTG-000FVCACDLEAARCFARSPYNNKNYNIDTSRRCKCYEKAGRM-GCWPYLTLYKYKCSQGKLTCSGGNSKCGAAVCNCDLVAANCFAGARYIDANYNINFKKRCQCYGEAEKL-GCWPYLTLYKYECSQGKLTCSGGNNKCEAAVCNCDLVAANCFAGAPYIDANYNVNLKERCQCYNEAEKISGCWPYFRTYSYECSQGTLTCKGGNNCAAAAVCDCDRLAAICFAGAPYNDNDYNINLRSRCQE CYSDAEKISGCRPYFKTYSYDCTKGRLTCKEGNNECAAFVCKCDRLAAICFAGAHYNDNNNYIDLARHCQ-
0
00
0
00000000
0
0
0
LD 50 (uq/g mice) 0 .13 0 .13 0 .21 0 .21 0 .09 0 .09 4 .7 0 .18 0 .24 0 .23 0 .22 6 .8 3 .4 3 .4 0 .017 0 .11 0 .045 >6 .0 >6 .0 9 .0 0 .63 2 .6 8 .6 4 .9
The amino acid sequences were taken from : ash), j), k), m) and n) (the present paper), i) and 1) NISHIDA et al., 1985b, o) HALPERT and ERKER, 1975, p) LIND and ERKER, 1981, q) TAKASAKI et al., 1988x, r) and s) NISHIDA et al ., 1982, t) TAKASAKI et al., 1988b, u) ERKER, 1978, v) JoueexT, 1977, w) TSAI et al., 1981, x) JOUBERT, 1975. The gaps (-) are introduced in order to get maximal alignments of half~ystine and maximal degree of homology . The numbering is based on Psettdechis australis Pa-3a. Note that gaps introduced in Pa-3a sequence do not affect the numbering and are indicated with an asterisk ("). Phospholipases AZ Pa-9C, LcPLA-II, LsPLA III and LsPLA IV which are indicated with a sharp (~) show biphasic kinetics due to activation by the reaction product (fatty acid). Triangles (~) above amino acid residues indicate the common residues in the group I enzymes (HEINRIKSON et al., 1977) except ß-bungarotoxins . The shadowed residues indicate the amino acid residues which differ from those of Pa-1 l . Open circles (O) below amino acid residues indicate the neurotoxic residues pointed out by DUFfON and HIRER (1983) .
Amino Acid Sequences of Snake Venom Phospholipases A,
335 co
zo
NLIßFGNM1QCANKGSRPSLDYADYGCYCGWGGSGTPVDE L1
IF
L 2
Y1 C3
~C~~i
C5
60
BC
LDRCCQTHDNCYEQAGKKGCFPKLTLYSWKCTGNAPTCNS L3
V
-
2
t
L4
V 3
p
L4
:C6r
C 7 116
100
KPGCKRFVCACDAAAAKCFAKAPYKKENYNIDTKKRCK
L1C-+LiI-~ L 7 i-LB--F-L9 ~ f- L6 -1 r
I~CB
Y4
~F-C9~-C10~-C11
~
rt~
FIG. ~. THE AMINO ACID SEQUENCE OF PHOSPHOLIPASEA= Pa-12C. See legend of Fig. 1 for further details .
m
co
NILQFRKMIßCANKGSRAAWHYLDYGCYCGPGGRGTPVDE
~ L 1 ---~~ L 2 -~F rC1~~C2
L3
~f-C3 -
C4
60
60
LDRCCKIHDDCYfEAGKDGCYPKLTWYSWQCTGDAPTCNP IH- L c ~~ L s -u Ls -
L
C5
KSKCKDFVCACDAAAAKCFAKApYNKANWNIDTKTRCK
~ L7 p L6 :
9
rt -~~ L 10 --~ L l'. -J~- L 1 2 ~H L 13 ~ 1=L 11 -1 -~ F-C6~~--C7 ~ CB I- C6~~
FIG . 8 . THE AMINO ACID SEQUENCE OF PHOSPHOLIPASE Az Pa-I S . See legend of Fig. I for further details .
Sequence studies on Pa-10A, Pa-12A, Pa-12C and Pa-15 are shown in Figs 5-8, respectively . Pa-15 was a mixture of Pa-15a ([Ala-103]Pa-15) and Pa-15b ([Pro-103]Pa-15) which could not be separated with the reversed phase chromatography at pH 2.0, 5.3 or 6.3.
DISCUSSION
The amino acid sequences of P. australis PLAZ enzymes are compared with each other and with some of the known PLAN sequences in Table 1 . Pa-1G, Pa-3 and Pa-15 showed micro-heterogenity at 103rd position with Thr/Ala, Thr/Pro and Ala/Pro, respectively . Pa5 was separated into two components with almost the same activities, Pa-Sa ([Pro-18 and
33 6
C. TAKASAKI et at.
Tyr-61]Pa-5) and Pa-Sb ([Ser-18 and Phe-61]Pa-5) . All of these amino acid substitutions can be explained by single base substitutions in DNA. Pa-1Ga and Pa-1Gb molecules lack the carboxyl-terminal Lys/Gln residue and consist of 117 amino acid residues while the other P. australis PLAZ molecules consist of a single chain of 118 amino acid residues including 14 half-cystine residues and all the common residues among PLAZ enzymes from other sources . Five residues, i.e. Trp-69, T'hr-72, Lys85, Ala-93 and Lys-101 are common in P. australis PLAZ enzymes while they are not in the sequences of other PLA Z enzymes. In the amino acid sequences of the three less active PLAZ enzymes, Pa-9C, Pa-13 and Pa-15, hydrophilic residues appeared at 6th, 7th and 10th positions which are members of the amino-terminal a-helix consisting of the micellar substrate recognition site. The decrease of the hydrophobicity of this region probably diminishes the enzymatic activity . The structure-toxicity relationship in PLAZ enzymes have been discussed. Kn~n and IWANAGA (1986) thought that a hydrophobic region around residues 73-100 was important for the presynaptic neurotoxicity from the comparison studies of hydropathy profiles between presynaptically acting PLAZ enzymes and non-neurotoxic PLAZ enzymes. But in P. australis PLAZ enzymes which show presynaptic neurotoxicity, there is no marked difference of hydrophobicity in this region while presynaptic toxicity differs by 70 times (Tnxasnxt et al., 1990). Tsnt et al. (1987) thought that the charge scores of residues 57, 58, 63, 70 and 86 of the elapid PLAZ correlated with the toxicity, i .e. presynaptically toxic PLAZ enzymes have higher scores of charge (+4 or +3) than the non-toxic PLAZ enzymes (0± 1). In P. australis PLAZ enzymes the scores of highly toxic PLAZ enzymes, Pa-1G and Pa-5 are + 1 while those of less toxic PLAZ enzymes, Pa-1 l, Pa-12A and Pa-12C are +4, +4 and +5, respectively . Koxno et al. (1989) pointed out the triad of basic residues 57, 63 and 86 (the residue numbers are fitted to those of Pa-3a) for high toxicity (ß, to ßa-bungarotoxin A chains, notexin and notechis II-5) and the triad of basic residues 63, 86 and 58/81 for lower toxicity (ßs-bungarotoxin A chain, Enhydrina schistosa myotoxin, LsPLA III and LsPLA IV). However, Lys-63 and Lys-81 appear in all the P. australis PLAZ sequences, while the basic residue at 86 appears in only Pa-12A . Furthermore, LsPLA III and LsPLA IV, which were classified into weak neurotoxins, are non-toxic (TAKASAKI Ct al., 19ß8a) and a highly toxic neurotoxin, LcPLA-II (LD P _ 0.045 hg/g mice) had a phenylalanyl residue at the 63rd position instead of the basic amino acid (TAKASAKI Ct al., 19ß8a) . In contrast to other elapid PLAZ enzymes, ß-bungarotoxins consisted of two dissimilar polypeptides, A (120 amino acid residues) and B (60 residues) chains, crosslinked by an interchain disulfide bond (Koxno et al., 19ß2a, b), therefore, the three-dimensional structures of ß-bungarotoxins cannot be deduced. DuFrcix and HroEtt (1983) pointed out several residues required for toxicity from sequence comparisons of toxic PLAZ enzymes (notexin and Notechis scutatus IIS from an Australian elapid and Enhydrina schistosa myotoxin from a true sea snake) and non-toxic PLAZ enzymes (Laticauda semifasciata PLA I, III and IV from a sea kraft and N. scutatus I 11) (Table 1) . N. scutatus II1 is a PLAZ homologue protein without activity because of the substitution of an invariant residue, Gly-30 to Ser-30 (Lixn and EnKER, 1980), and the other non-toxic PLAZ enzymes are from a sea kraft, so the differences of the sub-family of the snakes probably reflect on the sequence differences. Recently a toxic PLAZ, LcPLA-II has been isolated from the venom of a sea kraft, Laticauda colubrina and its amino acid sequence was determined (Tnxnsnta et al., 19ß8a) . In this paper, we reported the amino acid sequences of several PLAZ enzymes from an Australian elapid, P. australis. In Table 1, we compare the amino acid sequences of single-chain PLAZ enzymes from the venom of
Amino Acid Sequences of Snake Venom Phospholipases A,
FIG .
9.
ZiIE BACKBONE CONFORMAT10N3 OF PHOSPHOLIPASE NEUROTOXICITY .
AZ
337
WI7N SOME IINPORTANT RE4IDUE.4 FOR
Ribbon indicates the backbone conformation of bovine pancreatic phospholipase A, based on data by DUKSTRA et al. (1981). Circles and squares indicate basic (Arg and Lys) and acidic (Asp and Glu) residues, respectively . A triangle indicates Asn-74. Numbering is based on Pseudechis austratis phospholipases A,. Note that 43rd, 46th, 50th, 54th, 58th, 90th and 94th residues are facing up from the paper while the 74th residue is facing the other side.
various elapid snakes including Australian elapids [Pseudechis and Notechis, a)-o)], a true sea snake [Enhydrina, p)], sea kraits [Laticauda, cil-t)] and cobras [Naja and Hemachatus, u)-z)], and found that Asp-50, Lys-58 and Asn-90 seem to be important for the toxicity and another three residues, i.e. Lys-46, Asn-74 and Glu-94 are also predominant in the toxic PLAZ enzymes. There is no X-ray crystallographic data for any elapid PLAZ, but there is data for pancreatic (DuxsTRA et al., 1981) and crotalid PLAZ enzymes (ICEtTIt et al., 1981). Rt?tvErssDF.x et al. (1985) showed the strong similarity in the backbone conformations of these two PLAZ enzymes. According to the classification by HBINRIKSON et al. (1977), the pancreatic and elapid PLAZ enzymes belong to the group I while the crotalid PLAZ enzymes belong to the group II, therefore, the single-chain elapid enzymes also seem to have very similar backbone conformations to those of bovine PLAZ. The six residues pointed out in the above discussion, were drawn on the ribbon model of the bovine PLAZ backbone (Fig . 9). They (except Asn-74) are located close to each other and seem to form a strong hydrophilic domain with two other residues, i.e. one invariant residue, Arg-43, and a variable 54th residue . We propose that the domain for the toxicity of presynaptically toxic PLAZ enzymes of elapid snakes consists of seven hydrophilic residues i.e. Arg-43, Lys-46, Asp-50, Glu-54, Lys-58, Asp-90 and Glu-94, and PLAZ enzymes which possess the complete set of the seven residues are strong neurotoxins while PLAZ enzymes which lack a few of them, especially Asp-50, Lys-58 or Asp-90, are weak neurotoxins . Four PLAZ enzymes Pa-9C, LcPLA-II, LsPLA III and LsPLA IV showed biphasic kinetics due to activation by the reaction products (TAxnsnKr et al., 1990; TAKASAKi et al., 1988a; YosFUnA et al., 1979). The common residue among these four PLAZ enzymes and not in the other PLAZ enzymes is only Asp-79, which is considered to be located close to
338
C. TAKASAKI et xl.
the entrance of the active site pocket of PLA Z 79th residue has not been clarified.
(DIJKSTRA
et al., 1981) and the role of the
Acknowledgements-We are grateful to Professor K. Oautt~ of Tohoku University, Sendai, Japan for the use of the Pharmacia fast protein liquid chromatography system . We thank Mr H. AeE for the amino acid analysis.
REFERENCES Dlncszlu, B. W., KALK, K. H., Hot, W. G. J. and DRENTH, l. (1981) Structure of bovine pancreatic phospholipase A, at I .7 ~ resolution. J. molec. Biol. 147, 97-123 . Dut;roN, M. J. and HIUEIt, R. C. (1983) Classification of phospholipase Az according to sequence . Evolutionary and pharmacological implications . Ew. J. Biochem. 137, 545-551. EAKER, D. (1978) Studies of presynaptically neurotoxic and myotoxic phospholipaseA. In: Versatility of Proteins, pp. 41331 (LI, C . H ., Ed .) New York: Academic Press. HALPERT, J. and EAxEx, D. (1975) Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutxtus acetates (Australian tiger snake) . J. biol. Chem . 2511, 6990997. HnxxIKSOx, R. L., KREUGER, E. T. and KEIM, P. S. (1977) Amino acid sequence of phospholipase A,-a from the venom of Crotxlus adxmanteus. A new classification of phospholipases A, based upon structural determinants. J. biol. Chem. 252, 4913951. HEwtCK, R. M., HtnvtcArILLEß, M. W., Hoon, L. E. and DIeEVEIe, W. J. (1981) A gas-liquid solid phase peptide and protein sequenator . J. biol. Chem . 256, 7990-7996. JOUBERT, F. J. (1975) Hemachatus haemachatus (ringhals) venom. Purification, some properties and the aminoacid sequence of phospholipase A (fraction DE-I). Ew. J. Biochem. 52, 539-554. Jo~Exz, F, J. (1977) Naja mossambica mossambica venom. Purification, some properties and the amino-acid sequence of three phospholipasea A (CM-I, CM-II and CM-III). Biochim . biophys. Acts 493, 216-227. KEIrtI, C., FEIDbrAN, D. S., DEGAxELCO, S., GLICK, J., WAItn, K. B., Joxrs, E. O. and SIGI-Ex, P. B. (1981) The 2.5 A crystal structure of a dimeric phospholipase A2 from the venom of Crotales atrox. J. biol. Chem . 256, 8602-8607 . Klt~n, R. M. and IWANAGA, S. (1986) Structure-function relationships of phospholipases I: prediction of presynaptic neurotoxicity . Toxicon 24, 527-541. Koxno, K., TODA, H., NAIeIrA, K. and LEE, C.-Y . (1982x) Amino acid sequence of ßibungarotoxin from Bungarus multicinctus venom. The amino acid substitutions in the B chains . J. Biol., Tokyo 91, 1519-L530. Koxno, K., TODA, H., NAxrrA, K. and LEE, C:Y. (19826) Amino acid sequence of three ß-bungarotoxins (ß,-, ß,-, and ß; bungarotoxins) from Bungarus multicinctus venom. Amino acid substitutions in the A chains . J. Biol ., Tokyo 91, 1531-1548 . Koxno, K., ZHANO, J.-K., Xu, K. and KACAIrDYAMA, H . (1989) Amino acid sequence of a presynaptic neurotoxin, agkiatrodotoxin from the venom of Agkistrodon hxlys papas. J. Biochem., Tokyo 105, 196-203 . Llrtn, P. and FAKER, D. (1980) Complete amino acid sequence of non-neurotoxic, non-enzymatic phospholipase A, homolog from the venom of the Australian tiger snake (Notechis acetates acetates). Ew . J. Biochem. 111, 403(19. Llrm, P. and FAKER, D. (1981) Amino acid sequence of a lethal myotoxic phospholipase A, from the venom of the common sea snake (Enhydrina schistosa) . Toxicon 19, 11-24. NlstnnA, S,, Knt, H. S. and TAt~mrA, N. (1982) Amino acid sequences of three phospholipasesA I, III and IV from the venom of sea snake, Laticauda semijxsciata. Biochem. J. 207, 589-594. NISIünA, S., T~RASFm~tA, M., SrnstAZU, T., TAKASAKI, C. and TAMIYA, N. (1985x) Isolation and properties of two phospholipases A, from the venom of an Australian elapid snake (Pseudechis australis). Toxicon 23, 87-104. NISIflnA, S., T~RAS}nI`IA, M. AND TAMIYA, N. (19856) Amino acid sequences of phospholipases A2 from the venom of an Australian elapid snake (king brown snake, Pseudechis austrxlis) . Toxicon 23, 87-104. REwErsEnEtz, R., BRUx~, S., DuKSZRA, B. W., DRENTH, J. and $IDLER, P. B. (1985) A comparison of the crystal structures of phospholipase A, from bovine pancreas and Crotales atrox venom. J. biol. Chem. 260, 11627-11634. TAKASAKI, C. and TAMIYA, N. (1982) Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis. Biochem. J. 203, 269-276. TAKASAKI, C., KIMtIRA, S., KoKUSUIV, Y. AND TAMIYA, N. (1988x) Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticaeda colubrina, from the Solomon Islands. Biochem. J. 253, 869-875. TAKASAKI, C., KURAA(OCHI, H., SH~NAZU, T. and TAMIIA, N. (19886) Correction of amino acid sequence of phospholipase A, I from the venom of Laticaudx semifasciata (erabu sea snake) . Toxicon 26, 747-749.
Amino Acid Sequences of Snake Venom Phospholipases A,
33 9
T~xaseKt, C., Suzuxt, J. and T~tmva, N. (1990) Purification and properties of several phospholipases A, from the venom of Australian king brown snake (Pseudechis australis). Toxicon 28, 319-327. TsN, L-H., Wu, S.-H. and Lo, T.-B. (1981) Complete amino acid sequence of a phospholipaseA, from the venom of Naja naja atra (Taiwan cobra). Toxicon 19, 141-152. Tsn~, L-H., L~rt, H.-C . and CHANG, T. (1987) Toxicity domain in presynaptically toxic phospholipaseA, of snake venom. Biochim. bioPhys . Acta 916, 94-99. Yost~nn~, H., Kuno, T., SfnNtut, W. and T~tve, N. (1979) Phospholipase A of sea snake Laticauda semifasciata venom. Isolation and properties of novel forms lacking tryptophan . J. Biochem ., Tokyo 85, 379-388.
0041-0101/90 53.00 + .00 ® 1990 Pcgenwn Pros pk
Printed in Oreat Britain.
AMINO ACID SEQUENCES OF EIGHT PHOSPHOLIPASES A2 FROM THE VENOM OF AUSTRALIAN KING BROWN SNAKE, PSEUDECHIS A USTRALIS GüIKAHISA TAKASAKI, FUJIKO YUTANI
and
TSUYOSHI KAJIYASHIKI
Dt:partment of Cht:alistry, Faculty of Science, Tohoku University, Aobayama, Sendai 980, Japan (Acceptedfor publication 15 .4agust 1989)
C. TAKASAKI, F. YUTANI and T. KAJIYASHIKI . Amino acid sequences of eight phospholipases A2 from the venom of Australian king brown snake, Pseudechis australis. Toxicon 28, 329-339, 1990.-The amino acid sequences of eight phospholipases A2 (Pa-1G, Pa-3, Pa-5, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-15) which had been isolated from the venom of Australian king brown snake (Pseudechis australis) were elucidated . Pa-1G, Pa-3 and Pa-15 showed micro-heterogeneity at the 103rd position and Pa-5 was separated into two components, Pa-Sa ([Pro-18 and Tyr-61]Pa-S) and Pa-Sb ([Ser-18 and Phe61]Pa-5). All the phospholipase A2 molecules except Pa-1Ga and Pa-1Gb which lack the 118th residue, consisted of a single chain of 118 amino acid residues including 14 half-cystine residues and all the common residues among phospholipases A2 from other sources. From comparison studies, Asp-50, Lys58 and Asp-90 seem to be important for the toxicity, and we propose that the domain for the presynaptic toxicity consists of seven hydrophilic residues, i.e. Arg-43, Lys-46, Asp-50, Glu-54, Lys-58, Asp-90 and Glu-94 .
INTRODUCTION
A NUMBER of phospholipases A2 (PLA2, EC 3.1 .1.4) have been purified, and their amino acid sequences determined, from the venoms of various snakes, including those belonging to the three families Elapidae, Crotalidae and Viperidae. All the sequences show a high degree of homology: Some of the basic PLA2s show presynaptic neurotoxicity while other basic PLA2s or acidic PLA2s do not show this type of toxicity (for a review, see EAKER, 1978). The structure-function relationship in PLA2 enzymes have been discussed (for example; DuIrroN and HinsR, 1983; KINI and IWANAGA, 1986; T3AI et al., 1987 ; KoNno et al., 1989). Two PLA2 enzymes, Pa-11 and Pa-13 have been purified from the venom of Australian king brown snake (Pseudechis australis, NI3HIDA et al., 1985a) and sequenced (NISHIDA et al., 19856) . In addition, 13 isoenzymes of PLA2 were isolated from the venom o~ _P. australisal including Pa-1G, an acidic PLA2 with neurotoxicity (TAKASAKI et ., 1990). The present paper describes the complete amino acid sequences of eight PLA2 enzymes (Pa-1G, Pa-3, Pa-5, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-15) from the venom of P. australis. 329
330 Phospholipases A,
C. TAKASAKI et al. MATERIALS AND METHODS
Phospholipases A Pa-1G, Pa-3, Pa-S, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-IS were purified from the venom of king brown snake (Pseudechis oonfmlin), as already described (TAKASAKI et al ., 1990).
Proteinases
Lysyl~ndopeptidase from Achrornobacter lyticus, Staphylococcus aweus V8 proteinase, chymotrypsin and carboxypeptidase Y from yeast were purchased from Wako Pure Chemical industries (Osaka, Japan), Miles Laboratories Inc. (Elkhart, IN, U.S .A.), Worthington Biochemical Corp . (Freehold, NJ, U.S.A .) and Oriental Yeast Co . (Osaka, Japan), respectively.
Enzyme digestions
Lysyl~ndopeptidase digestions of reduced and S-carboxymethylated (RCM) proteins were carried out in 50 mM Tris-HCl buffer, pH 9.1 at an enzyme/substrate ratio of 1 :200 (w/w) and 37°C for 4 hr . V8 proteinase digestions were carried out in 50 mM NH,HCO, at an enzyme/substrate ratio of 1 :100 (w/w) and 37°C for I S hr. Chymotryptic digestions were carried out in 50 mM NH,HCO, at an enzyme/substrate ratio of 1 :250 (wJw) and '37°C for 2 hr . The digestions of RCM-proteins or peptides (20 nmole) with carboxypeptidase Y (0.5 hg) were carried ôut in 50 mM citrate buffer, pH 6.0 for 30 min to 6 hr, and aliquots of the digests were taken out at suitable intervals, added with an equal volume of 2 M acetic acid and directly applied to the amino acid analyzer.
Pur~cation of peptides
Peptides were separated with a Pharmacia fast protein liquid chromatography system (FPLC) equipped with a column of Pcp RPC HR 5/5 (Pharmacia, Uppsala, Sweden). Elution was performed with a linear gradient of acetonitrile in 0.1 % trifluoroacetic acid from 0 to 30% in 30 min, then 30 to 40% in 5 min at a flow rate of 1.2 ml/min . The fractionated peptides were manually collected by monitoring the absorbance at 214 nm. If necessary, rechromatography was carried out on the same column with a linear gradient of acetonitrile in 10 mM ammonium formate, pH 6.3 . In some cases, gel filtration with a Sephadex G-50 or a Bio Gel P-2 column or ion exchange chromatography with a DEAE~ellulose DE-52 column were carried out.
Amino acid analysis and Edman degradation
Amino acid analysis and the manual Edman degradation were tamed out as described previously (TAKASAKI and T~uv~, 1982). Automated Edman degradation with a gas-phase sequencer (model 470A ; Applied Hiosystems, Foster, CA, U.S .A .) on RCM-Pa-S, peptides C6 from Pa-3, V3 from Pa-5, C3 and C4 from Pa-10A, C6 from Pa-12A and C4 and CS from Pa-15 was carried out essentially as described by HEWICIC et al. (1981) .
RESULTS
Amino acid sequence of phospholipaseA~ Pa-IG RCM-Pa-1G (about 100 nmoles each) was digested with lysyl-endopeptidase or V8 proteinase, and the digests were separated using the FPLC reversed phase system . RCMPa-1G (130 nmole) was also degraded with 6 mg BrCN in 1 .0 ml of 70% formic acid (v/v) at 37 °C for 24 hr. The reaction mixture was applied to a column (1 .2 ctn x 130 cm) of Sephadex G-25 equilibrated with 5 mM HCI. Peptides B 1, B2 and B3 were eluted at distribution coefficient (Kp) values of 0.80, 0.35 and 0.05 respectively . Peptide B3 (95 nmole) was digested with chymotrypsin . Sequence studies on Pa-IG are summarized in Fig. 1 . Both threonine and alanine residues were detected at the 103rd position, therefore, Pa-1G was a mixture of Pa-1Ga ([Tltr-103]Pa-1G) and Pa-1Gb ([Ala-103]Pa-1G) . They did not separate from each other by reversed phase chromatography at pH 2.0, 5.3 or 6.3.
Amino Acid Sequences of Snake Venom Phospholipases A2
33l
zo 40 NLIOFGNMIQCANKGSRPTRHYMDYGCYCGWGGSGTPVDE ' rB3C1-a ~ B3Ci ~---81-~ ~ B 2V
1
60
BO
LDRCCQTHDDCYGEAEKKGCYPKLTLYSWDCTGNVPICSP ~ L3
L4
--17
-
.~
V2 Y 3-4 _ -~ ________ VT3 _--____----_____ 100
B3 C3
Y4
117
KAECKDFVCACDAEAAKCFAKAÂYNDAMIVNIDTK~RÇ ~1-LS~ ~
L6
T L7--I ~
L6 ~ ~L9~
' :-B3 C4 ~-B3 CS---1~- B 3 C 6 ----1
FIG. I . THE AMINO ACID sDQUENCE OF PHOSPHOLIPASE A= Pa-IG. B, L, V, C and T refer to the peptides derived by cyanogen bromide degradation, lysylendopeptidase, Staphylococcal proteinase, chymotryptic and Cryptic digestions, respectively . Underlines and arrows indicate amino acid residues detected by Edman degradation and carbozypeptidase Y digestion, respectively.
Amino acid sequence
of phospholipase A1
Pa-3
RCM-Pa-3 (about 100 nmoles each) was digested with lysyl-endopeptidase, V8 proteinase or chymotrypsin and the digests were applied to the FPLC reversed phase system . Sequence studies on Pa-3 are summarized in Fig . 2. Pa-3 was a mixture of Pa-3a ([Thr103]Pa-3) and Pa-3b ([Pro-103]Pa-3) and they were partially separated from each other by reversed phase chromatography at pH 6.3. There were no marked düïerences in the enzymatic or lethal activities between Pa-3a and Pa-3b.
20
40
60
BO
NLIQFGNMIOCANKGSRPTRHYMDYGCYCGWGGSGTPVDE
~--
L
C ~ -~~- C 1--~_ C 3
LDRCCKVHDDCYGEAEKKGCYPKLTLYSWDCTGNVPICSP L 3 --~:-L 4 -1 ,
V 2
6
V
3 A_
C6 -
KAECKDFVCACDAEAAKCFAKA~YNDANWNIDTKTRC I K L6 -~; ~ :-
-I :
___
L 7
-_
~ r L B 1F- L 9
___-_ _ ____V 5 1~
t~
-, H L 10 ---I
C 7 -~~ C 8 -I ~- C 9
FIG . 2. THE AMINO ACn) SEQUENCE OF PHOSPHOLIPASE
See legend of Fig. 1 for further details.
A2 Pa-3 .
332
C . TAKASAKI et al.
NLIQFSNMIQCANKGSR~SLDYADYGCYCGWGGSGTPVDE
LDRCCKVHDDCYAEAGKKGCFPKLTLYSWDCTGNVPICNP
100
118
KTECKDFTCACDAEAAKCFAKAPYKKENWNIDTKTRCK V
V 4
6
V 6 -
i~C5~~C6~ ~- C7--
rr
FIG . 3 . TIIE AMINO ACID ~QUFNCE OF PH08PHOLIPASE Az PS-S .
See legend of Fig . 1 for further details .
Amino acid sequence ofphospholipase AZ Pa-S RCM-Pa-S (about 700 nmoles each) was digested with V8 proteinase or chymotrypsin, and the digests were applied to a column (1.S cm x 18 cm) of DEAE-cellulose DE-S2 equilibrated with 0.01 MNH,HCO,. A linear concentration gradient elution with NH,HC03 was carried out from 0.01 M to 0.6 M over 1 liter. An expected chymotryptic peptide (C1, positions 1-5) was not obtained, probably because of its low solubility . Sequence studies on Pa-5 are summarized in Fig. 3. Pa-5 was a mixture of Pa-Sa (35%) and Pa-Sb (6S%) which were eluted at 27% and 29% acetonitrile, respectively, by reversed phase chromatography at pH 5 .3. Pa-Sa and Pa-Sb were separately reduced, S-carboxymethylated and digested with V8 proteinase, and the digests were separated with reversed phase chromatography at pH 5.3. The amino acid compositions suggested that Pa-Sa and Pa-Sb are [Pro-18 and Tyr-61]Pa-5 and [Ser-18 and Phe-61]Pa-5, respectively . There were no marked differences in the activities of Pa-Sa and Pa-Sb. 20
40
NLIQFKSIIECANRGSRRWLDYADYGCYCGWGGSGTPVDE
~~
i-L I L 2 Li F-L2T1 -~ h Ti~ ~ ~C 1W
C 2
T3 -
~~ C 3 ---; - C4-~~-
C 5
60
80
LDRCCKVHDECYGEAVKQGCFPKLTVYSWKCTENVPICDS .-i ~- L 3 -~F-- L 4 V 3
0
~~ L 5
-~I---- L 6
V 4
,-
100
IIB
RSKCKDFVCACDAAAAKCFAKAPYNKDNYNIDTKTRCQ
--iiL7a7
L B --~H L 9 ~H L10-IF-- L 11
-,H L12--î
V 5 I~C7 ---IF-CB~~ C9 FIG . 4 . THE AMINO ACID SEQUENCE OF PHOÔPHOLIPASE A z
See legend of Fig . 1 for further details .
rr
Pa-9C.
333
Amino Acid Sequences of Snake Venom Phospholipases A, 20
40
60
80
NLI(~FSNMIDCANKGSRPSLHYADYGCYCGWGGSGTPVDE
LORCCKVHDDCYDQAGKKGCFPKLTLYSWDCTGNVPICNP }-- L 7
irL4
/
L5
1~
I IB
KSKCKDFVCACDAAAAKCFAKAPYNKANWNIDTKTRCK ~-L s~k ~a---- L e "r- L s -F-- L I o -+--- L I I ~-L I z -I ' F-cs ~- cs~--cz -~ FIG . 5 . THE Aanxo ACW 3EQUENCE OF PHOSPHOLIPASE AZ Pa-10A. See legend of Fig. 1 for further details.
Amino acid sequence ofphospholipase A? Pa-9C
RCM-Pa-9C (about 100 nmoles each) was digested with lysyl-endopeptidase, V8 proteinase or chymotrypsin and the digests were separated with the FPLC reversed phase system . Peptide L2 (40 nmole) was digested with trypsin in 0.2 M NH4 HC0 3 at an enzyme/substrate ratio of 1 :100 and 37°C for 16 hr, and the digests were applied to a column (1 .2 cm x 136 cm) of Sephadex G-50 equilibrated with 0 .02 M NH4HC0, and peptides L2T1, L2T2 and L2T3 were eluted at KD values of 0.85, 1 .08 and 0.60, respectively . Sequence studies on Pa-9C are summarized in Fig. 4. Amino acid sequence ofphospholipase A? Pa-10A, Pa-12A, Pa-12C and Pa-15
RCM-Pa-lOA, PCM-Pa-12A, RCM-Pa-12C or RCM-Pa-15 (about 100 nmoles each) was separately digested with lysyl-endopeptidase, V8 proteinase or chymotrypsin . 20
40
NLI~FGNMIQCANKGSRPSLNYADYGCYCGWGGSGTPVDE L 1
--1f------- L 2 V 1
~ C 1 -~ C 2
v-
C 3 -ij-C 4~- C 5 60
80
LDRCC(~VHDNCYE(~AGKKGCFPKLTLYSWKCTGNVPTCNS J-_- L__ 3 ~F--- L 4 -V ~ t 7 __L 4 --~F _ C6 I(D
11B
KTGCKSFVCACDAAAAKCFAKAPYKKENYNIDTKKRCK -~L6 --~
L9 --'~ f-LB-IFL9~r
L10--~L11-~
'~- Y 4 C 7 -H C B
.r- C 9 -
FIG. 6. THE AMINO ACID SEQUENCE OF PHOSPHOLIPASEA= Pa-12A. See legend of Fig. 1 for further details.
33 4
C. TAKASAKI et al .
TABLE
a) b) c) d) e) f) h) i) i) k) 1) m) n) o) P) 4) s) tl u) v) w) x)
1.
COMPARISON OF THE AMINO ACID SEQUENCE OF VARIOUS PHOBPHOLIPASES SNAKES
Paeudechis auatralia
Pa-1Ga Pa-1Gb Pa-3a Pa-3b Pa-Sa Pa-Sb Pa-9C~ Pa-10A Pa-11 Pa-12A Pa-12C Pa-13 Pa-15a Pa-15b Notechis s . acutatus Notexin Enhvdrinâ échiatosa Myotoxin Laticauda c0lubrina LcPLA-II~~ Laticauda semifaaciata LsPLA III LePLA I IPV LsPLA I Na a niaricollie basic PLA Na a m . moesambica CM-I Na a na a atra acidic PLA Hemachatua haemachatua DE-I
h) i) k) 1) m) n) o) P) 41 r) s) t) u) v) w) x)
FROM THE VENOM OF ELAPIll
NLIyhGNMÎQCANKGSRP~Y~Y~YCGW~S~~~VDEÎ~D~tCC~ 0 NLIQFGNMIQCANKGSRp~y~YGCYCGWGGSGTPVDELDRCC NLIQFGNMIQCANKGSR YGCYCGWGGSGTPVDELDRC~~ NLIQFGNMIQCANRGSRP~Y YGCYCGWGGSGTPVDELDRCC/vH NLIQ MIQCANRGSRPSLDYADYGCYCGWGGSGTPVDELDRCCQVHDN NLIQ MIQCANRGS SLDYADYGCYCGWGGSGTPVDELDRCCQVHDN NLIQ~IICAI~GS~DYADYGCYCGWGGSGTPVDELDRC~~ NLIQ MIQCANKGSRPSI~SIADYGCYCGWGGSGTPVDELDRC NLIQFGNMIQCANKGSRPSLDYADYGCYCGWGGSGTPVDELDRCCQVHDN NLIQFGNMIQCANRGSRPSL/YADYGCYCGWGGSGTPVDELDRCCQVHDN NLIQFGNMIQCANKGSRPSLDYADYGCYCGWGGSGTPVDELDRCCQ7HDN ~IQCANKGSR DYGCY~TPVDELDRCC NLVQFSYLIQCANHGKRPTWHYMDYGCYCGAGGSGTPVDELDRCCKIHDD NLVQFSYVITCANHNRRSSLDYADYGCYCGAGGSGTPVDELDRCCKIHDD NLIQFSELIQCANRGKRATYYYMDYGCYCGKGGSGTPVDDLDRCCKTHDD NLVQFTYLIQCANSGKRASYHYADYGCYCGAGGSGTPVDELDRCCKIHDN NLVQFSYLIQCANTGKRASYHYADYGCYCGAGGSGTPVDELORCCKIHDN NLVQFSNLIQCNVKGSRASYHYADYGCYCGAGGSGTpVDELDRCCKIHDN NLYQFHNMIHCTVP-SRPWWHFADYGCYCGRGGRGTPVDDLDRCCQVHDN NLYQFKNMIHCTVP-SRPWWHFADYGCYCGRGGKGTAVDDLDRCCQVHDN NLYQFRNMIQCTVP-SRSWWDFADYGCYCGRGGSGTpVDDLDRCCQVHDN NLYQFKNMIKCTVP-SRSWWHFANYGCYCGRGGSGTPVDDLDRCCQTHDN
1 0
9 a) b) c) d) e) f)
AZ
1 1
1 1 _9*
0 _ ~1l~RK-G~KLTLYSFI" C7GN -~K~V~A~L~A~ÂÂK~~A CY(~i/!tK-G KLTLYS =CI~VCACD CFA K-G KLTLYS C~yCACD CFAK I CRK-G KLTLYS CACD CFARAP I CKKCFAKAPYKRE CKGRK-G KLTLYS CD IDT GKK-GCFPRLTLYSf~CTGNV CD KCFAKAPYKRE ID CKGCFPKLI~1[SWKCT~IV FVCACDAAAAKCFAKAPI~/NYNI C,ÂGKK-GCFPKLTLYSW/CTGN FVCACDAAAAKCFAKAP ~IDT CKCYEQAGRK-GCFPKLTLYSWRCTGNVPTCNSRPG-CRSFVCACDAAAAKCFARAPYRKENYNIDTKKRCKCYEQAGRK-GCFPKLTLYSWRCTGNVPTCNSI~G-CKSFVCACDAAAAKCFAKAPYKKENYNIDTKKRCKCYEQAGKK-GCFPKLTLYSWRCTG~ NSRPG-C~ "~- ", CACDAAAAICCFAKAPYKKENYNIDTRKRCKC GK~GC1>PKLT/YSüCTGiPTCN~-CI~FVCACDAAAARCFAKAP I CKC CACDAAAAKCF IDT CRC RLT/YSÜ"CTG~PTCti1l~-CP~FVCACDAAAAKCFAKA~ P~IID~CKCYDEAGRR-GCFPKMSAYDYYCGENGPYCRNIKKKCLRFVCDCDVEAAFCFAKAPYNNANWNIDTRRRCQCYGEAEKQ-GCYPKMLMYDYYCGSNGPYCRNVKKKCNRKVCDCDVAAAECFARNAYNNANYNIDTRRRCKCYGQAERR-GCFPFLTLYNFICFPGGPTCDRGTT-CQRFVCDCDIQAAFCFARSPYNNKNYNINISKRCKCYGEAEKM-GCYPKLTMYNYYCGTQSPTCDDKTG-CQRYVCACDLEAAKCFARSPYNNKNYNIDTSKRCKCYGQAEKM-GCYPKLTMYNYYCGTQSPTCDNKTG-CQRYVCACDLEIIAKCFARSPYNNKNYNIDTSRRCKCYGEAEKM-GCYPKWTLYTYDCSTEEPNCSTKTG-000FVCACDLEAARCFARSPYNNKNYNIDTSRRCKCYEKAGRM-GCWPYLTLYKYKCSQGKLTCSGGNSKCGAAVCNCDLVAANCFAGARYIDANYNINFKKRCQCYGEAEKL-GCWPYLTLYKYECSQGKLTCSGGNNKCEAAVCNCDLVAANCFAGAPYIDANYNVNLKERCQCYNEAEKISGCWPYFRTYSYECSQGTLTCKGGNNCAAAAVCDCDRLAAICFAGAPYNDNDYNINLRSRCQE CYSDAEKISGCRPYFKTYSYDCTKGRLTCKEGNNECAAFVCKCDRLAAICFAGAHYNDNNNYIDLARHCQ-
0
00
0
00000000
0
0
0
LD 50 (uq/g mice) 0 .13 0 .13 0 .21 0 .21 0 .09 0 .09 4 .7 0 .18 0 .24 0 .23 0 .22 6 .8 3 .4 3 .4 0 .017 0 .11 0 .045 >6 .0 >6 .0 9 .0 0 .63 2 .6 8 .6 4 .9
The amino acid sequences were taken from : ash), j), k), m) and n) (the present paper), i) and 1) NISHIDA et al., 1985b, o) HALPERT and ERKER, 1975, p) LIND and ERKER, 1981, q) TAKASAKI et al., 1988x, r) and s) NISHIDA et al ., 1982, t) TAKASAKI et al., 1988b, u) ERKER, 1978, v) JoueexT, 1977, w) TSAI et al., 1981, x) JOUBERT, 1975. The gaps (-) are introduced in order to get maximal alignments of half~ystine and maximal degree of homology . The numbering is based on Psettdechis australis Pa-3a. Note that gaps introduced in Pa-3a sequence do not affect the numbering and are indicated with an asterisk ("). Phospholipases AZ Pa-9C, LcPLA-II, LsPLA III and LsPLA IV which are indicated with a sharp (~) show biphasic kinetics due to activation by the reaction product (fatty acid). Triangles (~) above amino acid residues indicate the common residues in the group I enzymes (HEINRIKSON et al., 1977) except ß-bungarotoxins . The shadowed residues indicate the amino acid residues which differ from those of Pa-1 l . Open circles (O) below amino acid residues indicate the neurotoxic residues pointed out by DUFfON and HIRER (1983) .
Amino Acid Sequences of Snake Venom Phospholipases A,
335 co
zo
NLIßFGNM1QCANKGSRPSLDYADYGCYCGWGGSGTPVDE L1
IF
L 2
Y1 C3
~C~~i
C5
60
BC
LDRCCQTHDNCYEQAGKKGCFPKLTLYSWKCTGNAPTCNS L3
V
-
2
t
L4
V 3
p
L4
:C6r
C 7 116
100
KPGCKRFVCACDAAAAKCFAKAPYKKENYNIDTKKRCK
L1C-+LiI-~ L 7 i-LB--F-L9 ~ f- L6 -1 r
I~CB
Y4
~F-C9~-C10~-C11
~
rt~
FIG. ~. THE AMINO ACID SEQUENCE OF PHOSPHOLIPASEA= Pa-12C. See legend of Fig. 1 for further details .
m
co
NILQFRKMIßCANKGSRAAWHYLDYGCYCGPGGRGTPVDE
~ L 1 ---~~ L 2 -~F rC1~~C2
L3
~f-C3 -
C4
60
60
LDRCCKIHDDCYfEAGKDGCYPKLTWYSWQCTGDAPTCNP IH- L c ~~ L s -u Ls -
L
C5
KSKCKDFVCACDAAAAKCFAKApYNKANWNIDTKTRCK
~ L7 p L6 :
9
rt -~~ L 10 --~ L l'. -J~- L 1 2 ~H L 13 ~ 1=L 11 -1 -~ F-C6~~--C7 ~ CB I- C6~~
FIG . 8 . THE AMINO ACID SEQUENCE OF PHOSPHOLIPASE Az Pa-I S . See legend of Fig. I for further details .
Sequence studies on Pa-10A, Pa-12A, Pa-12C and Pa-15 are shown in Figs 5-8, respectively . Pa-15 was a mixture of Pa-15a ([Ala-103]Pa-15) and Pa-15b ([Pro-103]Pa-15) which could not be separated with the reversed phase chromatography at pH 2.0, 5.3 or 6.3.
DISCUSSION
The amino acid sequences of P. australis PLAZ enzymes are compared with each other and with some of the known PLAN sequences in Table 1 . Pa-1G, Pa-3 and Pa-15 showed micro-heterogenity at 103rd position with Thr/Ala, Thr/Pro and Ala/Pro, respectively . Pa5 was separated into two components with almost the same activities, Pa-Sa ([Pro-18 and
33 6
C. TAKASAKI et at.
Tyr-61]Pa-5) and Pa-Sb ([Ser-18 and Phe-61]Pa-5) . All of these amino acid substitutions can be explained by single base substitutions in DNA. Pa-1Ga and Pa-1Gb molecules lack the carboxyl-terminal Lys/Gln residue and consist of 117 amino acid residues while the other P. australis PLAZ molecules consist of a single chain of 118 amino acid residues including 14 half-cystine residues and all the common residues among PLAZ enzymes from other sources . Five residues, i.e. Trp-69, T'hr-72, Lys85, Ala-93 and Lys-101 are common in P. australis PLAZ enzymes while they are not in the sequences of other PLA Z enzymes. In the amino acid sequences of the three less active PLAZ enzymes, Pa-9C, Pa-13 and Pa-15, hydrophilic residues appeared at 6th, 7th and 10th positions which are members of the amino-terminal a-helix consisting of the micellar substrate recognition site. The decrease of the hydrophobicity of this region probably diminishes the enzymatic activity . The structure-toxicity relationship in PLAZ enzymes have been discussed. Kn~n and IWANAGA (1986) thought that a hydrophobic region around residues 73-100 was important for the presynaptic neurotoxicity from the comparison studies of hydropathy profiles between presynaptically acting PLAZ enzymes and non-neurotoxic PLAZ enzymes. But in P. australis PLAZ enzymes which show presynaptic neurotoxicity, there is no marked difference of hydrophobicity in this region while presynaptic toxicity differs by 70 times (Tnxasnxt et al., 1990). Tsnt et al. (1987) thought that the charge scores of residues 57, 58, 63, 70 and 86 of the elapid PLAZ correlated with the toxicity, i .e. presynaptically toxic PLAZ enzymes have higher scores of charge (+4 or +3) than the non-toxic PLAZ enzymes (0± 1). In P. australis PLAZ enzymes the scores of highly toxic PLAZ enzymes, Pa-1G and Pa-5 are + 1 while those of less toxic PLAZ enzymes, Pa-1 l, Pa-12A and Pa-12C are +4, +4 and +5, respectively . Koxno et al. (1989) pointed out the triad of basic residues 57, 63 and 86 (the residue numbers are fitted to those of Pa-3a) for high toxicity (ß, to ßa-bungarotoxin A chains, notexin and notechis II-5) and the triad of basic residues 63, 86 and 58/81 for lower toxicity (ßs-bungarotoxin A chain, Enhydrina schistosa myotoxin, LsPLA III and LsPLA IV). However, Lys-63 and Lys-81 appear in all the P. australis PLAZ sequences, while the basic residue at 86 appears in only Pa-12A . Furthermore, LsPLA III and LsPLA IV, which were classified into weak neurotoxins, are non-toxic (TAKASAKI Ct al., 19ß8a) and a highly toxic neurotoxin, LcPLA-II (LD P _ 0.045 hg/g mice) had a phenylalanyl residue at the 63rd position instead of the basic amino acid (TAKASAKI Ct al., 19ß8a) . In contrast to other elapid PLAZ enzymes, ß-bungarotoxins consisted of two dissimilar polypeptides, A (120 amino acid residues) and B (60 residues) chains, crosslinked by an interchain disulfide bond (Koxno et al., 19ß2a, b), therefore, the three-dimensional structures of ß-bungarotoxins cannot be deduced. DuFrcix and HroEtt (1983) pointed out several residues required for toxicity from sequence comparisons of toxic PLAZ enzymes (notexin and Notechis scutatus IIS from an Australian elapid and Enhydrina schistosa myotoxin from a true sea snake) and non-toxic PLAZ enzymes (Laticauda semifasciata PLA I, III and IV from a sea kraft and N. scutatus I 11) (Table 1) . N. scutatus II1 is a PLAZ homologue protein without activity because of the substitution of an invariant residue, Gly-30 to Ser-30 (Lixn and EnKER, 1980), and the other non-toxic PLAZ enzymes are from a sea kraft, so the differences of the sub-family of the snakes probably reflect on the sequence differences. Recently a toxic PLAZ, LcPLA-II has been isolated from the venom of a sea kraft, Laticauda colubrina and its amino acid sequence was determined (Tnxnsnta et al., 19ß8a) . In this paper, we reported the amino acid sequences of several PLAZ enzymes from an Australian elapid, P. australis. In Table 1, we compare the amino acid sequences of single-chain PLAZ enzymes from the venom of
Amino Acid Sequences of Snake Venom Phospholipases A,
FIG .
9.
ZiIE BACKBONE CONFORMAT10N3 OF PHOSPHOLIPASE NEUROTOXICITY .
AZ
337
WI7N SOME IINPORTANT RE4IDUE.4 FOR
Ribbon indicates the backbone conformation of bovine pancreatic phospholipase A, based on data by DUKSTRA et al. (1981). Circles and squares indicate basic (Arg and Lys) and acidic (Asp and Glu) residues, respectively . A triangle indicates Asn-74. Numbering is based on Pseudechis austratis phospholipases A,. Note that 43rd, 46th, 50th, 54th, 58th, 90th and 94th residues are facing up from the paper while the 74th residue is facing the other side.
various elapid snakes including Australian elapids [Pseudechis and Notechis, a)-o)], a true sea snake [Enhydrina, p)], sea kraits [Laticauda, cil-t)] and cobras [Naja and Hemachatus, u)-z)], and found that Asp-50, Lys-58 and Asn-90 seem to be important for the toxicity and another three residues, i.e. Lys-46, Asn-74 and Glu-94 are also predominant in the toxic PLAZ enzymes. There is no X-ray crystallographic data for any elapid PLAZ, but there is data for pancreatic (DuxsTRA et al., 1981) and crotalid PLAZ enzymes (ICEtTIt et al., 1981). Rt?tvErssDF.x et al. (1985) showed the strong similarity in the backbone conformations of these two PLAZ enzymes. According to the classification by HBINRIKSON et al. (1977), the pancreatic and elapid PLAZ enzymes belong to the group I while the crotalid PLAZ enzymes belong to the group II, therefore, the single-chain elapid enzymes also seem to have very similar backbone conformations to those of bovine PLAZ. The six residues pointed out in the above discussion, were drawn on the ribbon model of the bovine PLAZ backbone (Fig . 9). They (except Asn-74) are located close to each other and seem to form a strong hydrophilic domain with two other residues, i.e. one invariant residue, Arg-43, and a variable 54th residue . We propose that the domain for the toxicity of presynaptically toxic PLAZ enzymes of elapid snakes consists of seven hydrophilic residues i.e. Arg-43, Lys-46, Asp-50, Glu-54, Lys-58, Asp-90 and Glu-94, and PLAZ enzymes which possess the complete set of the seven residues are strong neurotoxins while PLAZ enzymes which lack a few of them, especially Asp-50, Lys-58 or Asp-90, are weak neurotoxins . Four PLAZ enzymes Pa-9C, LcPLA-II, LsPLA III and LsPLA IV showed biphasic kinetics due to activation by the reaction products (TAxnsnKr et al., 1990; TAKASAKi et al., 1988a; YosFUnA et al., 1979). The common residue among these four PLAZ enzymes and not in the other PLAZ enzymes is only Asp-79, which is considered to be located close to
338
C. TAKASAKI et xl.
the entrance of the active site pocket of PLA Z 79th residue has not been clarified.
(DIJKSTRA
et al., 1981) and the role of the
Acknowledgements-We are grateful to Professor K. Oautt~ of Tohoku University, Sendai, Japan for the use of the Pharmacia fast protein liquid chromatography system . We thank Mr H. AeE for the amino acid analysis.
REFERENCES Dlncszlu, B. W., KALK, K. H., Hot, W. G. J. and DRENTH, l. (1981) Structure of bovine pancreatic phospholipase A, at I .7 ~ resolution. J. molec. Biol. 147, 97-123 . Dut;roN, M. J. and HIUEIt, R. C. (1983) Classification of phospholipase Az according to sequence . Evolutionary and pharmacological implications . Ew. J. Biochem. 137, 545-551. EAKER, D. (1978) Studies of presynaptically neurotoxic and myotoxic phospholipaseA. In: Versatility of Proteins, pp. 41331 (LI, C . H ., Ed .) New York: Academic Press. HALPERT, J. and EAxEx, D. (1975) Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutxtus acetates (Australian tiger snake) . J. biol. Chem . 2511, 6990997. HnxxIKSOx, R. L., KREUGER, E. T. and KEIM, P. S. (1977) Amino acid sequence of phospholipase A,-a from the venom of Crotxlus adxmanteus. A new classification of phospholipases A, based upon structural determinants. J. biol. Chem. 252, 4913951. HEwtCK, R. M., HtnvtcArILLEß, M. W., Hoon, L. E. and DIeEVEIe, W. J. (1981) A gas-liquid solid phase peptide and protein sequenator . J. biol. Chem . 256, 7990-7996. JOUBERT, F. J. (1975) Hemachatus haemachatus (ringhals) venom. Purification, some properties and the aminoacid sequence of phospholipase A (fraction DE-I). Ew. J. Biochem. 52, 539-554. Jo~Exz, F, J. (1977) Naja mossambica mossambica venom. Purification, some properties and the amino-acid sequence of three phospholipasea A (CM-I, CM-II and CM-III). Biochim . biophys. Acts 493, 216-227. KEIrtI, C., FEIDbrAN, D. S., DEGAxELCO, S., GLICK, J., WAItn, K. B., Joxrs, E. O. and SIGI-Ex, P. B. (1981) The 2.5 A crystal structure of a dimeric phospholipase A2 from the venom of Crotales atrox. J. biol. Chem . 256, 8602-8607 . Klt~n, R. M. and IWANAGA, S. (1986) Structure-function relationships of phospholipases I: prediction of presynaptic neurotoxicity . Toxicon 24, 527-541. Koxno, K., TODA, H., NAIeIrA, K. and LEE, C.-Y . (1982x) Amino acid sequence of ßibungarotoxin from Bungarus multicinctus venom. The amino acid substitutions in the B chains . J. Biol., Tokyo 91, 1519-L530. Koxno, K., TODA, H., NAxrrA, K. and LEE, C:Y. (19826) Amino acid sequence of three ß-bungarotoxins (ß,-, ß,-, and ß; bungarotoxins) from Bungarus multicinctus venom. Amino acid substitutions in the A chains . J. Biol ., Tokyo 91, 1531-1548 . Koxno, K., ZHANO, J.-K., Xu, K. and KACAIrDYAMA, H . (1989) Amino acid sequence of a presynaptic neurotoxin, agkiatrodotoxin from the venom of Agkistrodon hxlys papas. J. Biochem., Tokyo 105, 196-203 . Llrtn, P. and FAKER, D. (1980) Complete amino acid sequence of non-neurotoxic, non-enzymatic phospholipase A, homolog from the venom of the Australian tiger snake (Notechis acetates acetates). Ew . J. Biochem. 111, 403(19. Llrm, P. and FAKER, D. (1981) Amino acid sequence of a lethal myotoxic phospholipase A, from the venom of the common sea snake (Enhydrina schistosa) . Toxicon 19, 11-24. NlstnnA, S,, Knt, H. S. and TAt~mrA, N. (1982) Amino acid sequences of three phospholipasesA I, III and IV from the venom of sea snake, Laticauda semijxsciata. Biochem. J. 207, 589-594. NISIünA, S., T~RASFm~tA, M., SrnstAZU, T., TAKASAKI, C. and TAMIYA, N. (1985x) Isolation and properties of two phospholipases A, from the venom of an Australian elapid snake (Pseudechis australis). Toxicon 23, 87-104. NISIflnA, S., T~RAS}nI`IA, M. AND TAMIYA, N. (19856) Amino acid sequences of phospholipases A2 from the venom of an Australian elapid snake (king brown snake, Pseudechis austrxlis) . Toxicon 23, 87-104. REwErsEnEtz, R., BRUx~, S., DuKSZRA, B. W., DRENTH, J. and $IDLER, P. B. (1985) A comparison of the crystal structures of phospholipase A, from bovine pancreas and Crotales atrox venom. J. biol. Chem. 260, 11627-11634. TAKASAKI, C. and TAMIYA, N. (1982) Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis. Biochem. J. 203, 269-276. TAKASAKI, C., KIMtIRA, S., KoKUSUIV, Y. AND TAMIYA, N. (1988x) Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticaeda colubrina, from the Solomon Islands. Biochem. J. 253, 869-875. TAKASAKI, C., KURAA(OCHI, H., SH~NAZU, T. and TAMIIA, N. (19886) Correction of amino acid sequence of phospholipase A, I from the venom of Laticaudx semifasciata (erabu sea snake) . Toxicon 26, 747-749.
Amino Acid Sequences of Snake Venom Phospholipases A,
33 9
T~xaseKt, C., Suzuxt, J. and T~tmva, N. (1990) Purification and properties of several phospholipases A, from the venom of Australian king brown snake (Pseudechis australis). Toxicon 28, 319-327. TsN, L-H., Wu, S.-H. and Lo, T.-B. (1981) Complete amino acid sequence of a phospholipaseA, from the venom of Naja naja atra (Taiwan cobra). Toxicon 19, 141-152. Tsn~, L-H., L~rt, H.-C . and CHANG, T. (1987) Toxicity domain in presynaptically toxic phospholipaseA, of snake venom. Biochim. bioPhys . Acta 916, 94-99. Yost~nn~, H., Kuno, T., SfnNtut, W. and T~tve, N. (1979) Phospholipase A of sea snake Laticauda semifasciata venom. Isolation and properties of novel forms lacking tryptophan . J. Biochem ., Tokyo 85, 379-388.
