Chapter 26 Neurotoxins from Sea Snake and Other Vertebrate Venoms
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Anthony T. Tu Department of Biochemistry, Colorado State University, Fort Collins,CO80523 Neurotoxins present in sea snake venoms are summarized. All sea snake venoms are extremely toxic, with low LD values. Most sea snake neurotoxins consist of only 60-62 amino acid residues with 4 disulfide bonds, while some consist of 70 amino acids with 5 disulfide bonds. The origin of toxicity is due to the attachment of 2 neurotoxin molecules to 2 α subunits of an acetylcholine receptor that is com posed of α ßγδ subunits. The complete structure of several of the sea snake neurotoxins have been worked out. Through chemical modifica tion studies the invariant tryptophan and tyrosine residues of post -synaptic neurotoxins were shown to be of a critical nature to the toxi city function of the molecule. Lysine and arginine are also believed to be important. Other marine vertebrate venoms are not well known. All evidence indicates that the fish venoms are composed of proteins. 50
2
There are many venomous marine vertebrates in the seas, notably sea snakes and fishes. Venoms of sea snakes have been studied much more thoroughly than fish venoms. In this chapter, sea snake venom is described in greater detail than fish venoms simply because there is much more scientific information available. The sea snake is a marine-adapted serpent belonging to the family of Hydrophiidae. There are many varieties of sea snakes with different colors, shapes, and sizes. They are well adapted for the marine environment and have a flat tail and a salt gland. Sea snakes are widely distributed in tropical and subtropical waters along the coasts of the Indian and Pacific Oceans. They are not found in the Atlantic Ocean. All sea snakes are poisonous and their venoms are extremely toxic. The LD for crude sea snake venom can be as low as 0.10 ug/g mouse body weight (2). For purified toxin the LD is even lower, suggesting the high toxicity of sea snake tox ins and venoms. This toxicity is derived from the presence of potent neurotoxins. Compared to snake venoms of terrestrial origin, sea snake venoms have been studied less. Different enzymes reported to be present or absent are summarized in Table I. 50
50
Primary Structure of Sea Snake Neurotoxins Before discussing the structure of the neurotoxins, it is necessary to define the types of neurotoxins. Three types of neurotoxins have been found so far in snake venoms. The first one is a postsynaptic neurotoxin, the second is a presynaptic neurotoxin, and the last is a cholinesterase inhibiting neurotoxin. Most sea snake venoms seem to contain only the postsynaptic neurotoxin. Only in Enhydrina
0097-6156/90/0418-0336S06.00/0 o 1990 American Chemical Society
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
26.
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Neurotoxins from Sea Snake and Other Vertebrate Venoms
Table I.
Presence a n d Absence o f Enzymes i n Sea Snake V e n o m s
Enzyme Acetylcholinesterase Hyaluronidase L e u c i n e aminopeptidase 5'-Nucleotidase
Phosphodiesterase Phosphomonoesterase
Phospholipase A
L - a m i n o acid oxidase
Venom
A r g i n i n e esterase
Comment
6, 33 34 6 6
Activity Activity Activity Activity
E. schistosa H. cyanocinctus Laticauda semifasciata E. schistosa H. cyanocinctus E. schistosa H. cyanocinctus L. semifasciata
Activity detected Activity detected Activity detected
33 34 35
Activity Activity Activity Activity Activity Isolated Activity Isolated Activity Isolated Activity Activity detected Activity detected Activity detected Activity detected Activity detected
detected not
6,35 34 34 34 35 36 6,37,38 39,40 34,41 42-46 41 33
not
34
not
33
not
34
not
47
E. schistosa H. cyanocinctus L. semifasciata Pelamis platurus E. schistosa
E. schistosa H. cyanocinctus L. semifasciata
detected detected detected detected
Reference
Enhydrina schistosa Hydrophis cyanocinctus E. schistosa E. schistosa
H. cyanocinctus
Protease
337
detected detected detected detected detected detected detected
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY
schistosa v e n o m , w h i c h also possesses a postsynaptic toxin, was a presynaptic type f o u n d a n d identified as p h o s p h o l i p a s e A . T o x i n s from t h e venoms o f t h e subfamily H y d r o p h i i n a e are extremely similar i n a m i n o acid sequence (Table II). Similarly, t h e sequences i n toxins from t h e subfamily o f L a t i c a u d i n a e are also very similar a m o n g themselves (Table II). A l t h o u g h c o m p a r i s o n o f t h e sequences o f toxins from t w o subfamilies shows there are c o n s i d erable similarities, differences also become noticeable. T h e r e are four disulfide bonds i n short-chain (Type I) neurotoxins. T h i s means that there are eight half-cystines. H o w e v e r , a l l H y d r o p h i i n a e toxins have n i n e halfcystines w i t h o n e cysteine residue. A n extra cysteine residue c a n b e readily detected from t h e R a m a n spectrum as t h e sulfhydryl g r o u p shows a distinct S - H stretching v i b r a t i o n at 2578 cm* . S o m e L a t i c a u d i n a e toxins d o n o t have a free cysteine resid u e as i n t h e cases o f L. laticaudata a n d L. semifasciata toxins. I n l o n g toxins (Type II) there are five disulfide bonds (Table III). A n o t h e r type o f n e u r o t o x i n f o u n d i n sea snake venoms is a hybrid type structurally situated between t h e short-chain a n d l o n g - c h a i n types. A s can be seen i n T a b l e I V , t w o toxins s h o w n here have a l o n g stretch o f segment 4, yet there is n o disulfide b o n d i n this p o r t i o n . 1
Secondary Structure I n order to understand t h e exact mechanism o f t h e n e u r o t o x i c a c t i o n , it is i m p o r tant t o k n o w t h e secondary structure o f t h e neurotoxins as w e l l . It is n o w k n o w n that postsynaptic neurotoxins attach to t h e a-subunits o f acetylcholine receptor (AChR). It is a s u p p o s i t i o n that the £ - s h e e t structure o f n e u r o t o x i n is a n essential structural element for b i n d i n g to t h e receptor. T h e presence o f £ - s h e e t structure was f o u n d by R a m a n spectroscopic analysis o f a sea snake n e u r o t o x i n (2). T h e amide I b a n d a n d III b a n d for Enhydrina schistosa toxin were at 1672 c m a n d 1242 c m " , respectively. These wave numbers are characteristic for anti-parallel ^-sheet structure. T h e presence o f £ - s h e e t structure f o u n d by R a m a n spectroscopic study was later c o n f i r m e d by X - r a y diffraction study o n Laticauda semifasciata t o x i n b. Sea snake short-chain toxins have a m o l e c u l a r weight o f o n l y 6,800. T h e small size w i t h four disulfide bonds makes these toxins very c o m p a c t a n d stable molecules. Therefore, w h e n t h e Pelamis platurus t o x i n is subjected t o heat treatment at 1 0 0 C and subsequent c o o l i n g , it does n o t change its c o n f o r m a t i o n substantially. A m i d e I and III bands a n d S-S stretching v i b r a t i o n d i d n o t change by heat treatment. F o u r disulfide bonds are clustered i n o n e area a n d there is a p r o t r u d i n g l o o p . It is suspected that this l o o p is t h e o n e that plays an i m p o r t a n t r o l e i n b i n d i n g to the A C h R . T h e four disulfide bonds are believed t o be i m p o r t a n t for m a i n t a i n i n g t h e specific c o n f o r m a t i o n a n d have been studied extensively. T h e c o n f o r m a t i o n o f t h e disulfide b o n d i n C - C - S - S - C - C n e t w o r k is gauche-gauche-gauche c o n f o r m a t i o n at t h e S-S stretching v i b r a t i o n appearing at 510-512 c m " . 1
0
1
Structure-Function Relationship T h e a m i n o acid residues i n neurotoxins w h i c h are i m p o r t a n t for n e u r o t o x i c a c t i o n are still n o t entirely clarified. S o m e neurotoxins c o n t a i n o n e free S H group, w h i l e others d o n o t . F r o m this fact, it w o u l d b e logical t o assume t h e sulfhydryl group is n o t essential. T h i s was actually p r o v e n t o be t h e case. W h e n J V , / ^ - l , 4 - p h e n y l e n e d i m a l e i m i d e was used for modifying the sulfhydryl g r o u p i n Pelamis t o x i n , 2 m o l o f toxins c o m b i n e d w i t h 1 m o l o f t h e reagent. W i t h
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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Neurotoxins from Sea Snake and Other Vertebrate Venoms
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1
t h e sulfhydryl g r o u p modified, t h e S - H stretching v i b r a t i o n a l b a n d at 2578 c m " disappeared. T h e m o d i f i c a t i o n o f t h e single sulfhydryl g r o u p d i d n o t alter t h e b i n d i n g ability t o A C h R o r toxicity (3). D i s u l f i d e bonds, however, are i m p o r t a n t i n m a i n t a i n i n g t h e particular toxin structure a n d have been s h o w n t o be essential for toxicity. W h e n a l l four disulfide bonds are reduced a n d alkylated, t h e n e u r o t o x i n loses its toxicity (4). T h e o n e residue most extensively studied is t r y p t o p h a n . It is very easily m o d i fied, i n d i c a t i n g that t r y p t o p h a n residue is exposed (5-8). R a m a n spectroscopic analysis o f a sea snake n e u r o t o x i n indicated that a single t r y p t o p h a n residue is i n d e e d exposed (2). T h e t r y p t o p h a n residue lies i n the i m p o r t a n t l o o p consisting o f segment 4. M o d i f i c a t i o n o f t h e t r y p t o p h a n residue induces t h e loss o f A C h R b i n d i n g ability as w e l l as t h e loss o f toxicity (5-8). T h e r e is o n l y o n e tyrosine residue i n s o m e sea snake neurotoxins. T h i s residue is usually q u i t e difficult t o modify, but o n c e it is modified, t h e toxicity is lost (9). H i s t i d i n e seems n o t t o b e essential as t h e c h e m i c a l m o d i f i c a t i o n o f this residue does n o t affect t h e toxicity (70). A r g i n e a n d lysine are believed to be i m p o r t a n t , but results are n o t clear because sea snake neurotoxins c o n t a i n several residues o f these a m i n o acids (7). A c l o n e d c o m p l e m e n t a r y D N A to a n e u r o t o x i n precursor R N A extracted from t h e v e n o m glands o f Laticauda semifasciata was isolated a n d its n u c l e o t i d e sequence was identified (77). T h e c l o n i n g o f n e u r o t o x i n s h o u l d a i d t h e understanding o f structure—function relationship eventually.
Comparison to Other Snake Toxins T h e similarity o f t h e primary structure o f different sea snake venoms has already been discussed. Postsynaptic neurotoxins from E l a p i d a e v e n o m have been extensively studied. E l a p i d a e i n c l u d e w e l l - k n o w n snakes such as cobra, krait, mambas, c o r a l snakes, a n d a l l A u s t r a l i a n snakes. L i k e sea snake toxins, E l a p i d a e toxins can also be g r o u p e d i n t o short-chain (Type I) a n d l o n g - c h a i n (Type II) toxins. M o r e over, t w o types o f neurotoxins are also similar to cardiotoxins, especially i n the p o s i t i o n s o f disulfide bonds. H o w e v e r , a m i n o acid sequences between cardiotoxins and sea snake a n d E l a p i d a e neurotoxins are quite different. I n c o m p a r i n g the sequence o f sea snake a n d E l a p i d a e neurotoxins, there is a considerable conservation i n a m i n o acid sequence, but the difference is greater t h a n a m o n g t h e various sea snake toxins. S i m i l a r i t y o f v e n o m s a m o n g different sea snakes a n d E l a p i d a e c a n also be detected i m m u n o l o g i c a l l y . F o r instance, t h e antibody for Enhydrina schistosa showed cross reactivity w i t h t h e venoms o f Hydrophis cyanocinctus, Lapemis hardwkkii, a n d Pelamis platurus (12). T h e sea snake antivenin n o t o n l y neutralizes t h e toxicity o f various sea snake venoms, but also Naja naja atra (Taiwan cobra) v e n o m (13-16). T h e reverse is also true; namely, some E l a p i d a e antivenins are also effective for n e u t r a l i z i n g sea snake v e n o m lethality (17-19).
Pharmacological and Biological Activities W h e n a nerve-muscle p r e p a r a t i o n is stimulated i n t h e presence o f a sea snake neur o t o x i n , there is n o twitch. H o w e v e r , w h e n t h e muscle itself is s t i m u l a t e d directly i n t h e presence o f a n e u r o t o x i n , t h e muscle contracts. T h i s means that n e u r o t o x i n does n o t i n h i b i t t h e muscle itself. M o r e o v e r , postsynaptic n e u r o t o x i n does n o t i n h i bit t h e release o f acetylcholine from the nerve ending. Therefore, the site o f snake t o x i n i n h i b i t i o n must be i n t h e postsynaptic site (20). L a t e r it was s h o w n that a n e u r o t o x i n strongly binds to t h e acetylcholine receptor ( A C h R ) .
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
Toxin
Aipysurus laevis toxin a toxin b toxin c L. laticaudata laticotoxin a L. semifasciata erabutoxin a erabutoxin b erabutoxin c toxin b
Laticaudinae
L L L R R R R R
T T T R I I I I
c
C c c c c c
C
Segment 2
c c c F F F F F
N N N N N N N N Q Q q H q q q q
q q q p H H H H
s s s s s s s s
s s s s s s s s q q q q q q q q
p p p p p p p p K q q q q q
K
K
T T T T T T T T
T T T N T T T T
T T T K K K K K
C N Q Q S S Q P K T T T
Segment 1
Acatyptophis peronii major minor Astroitia stokesii toxin a Enhydrina schistosa toxin 4 toxin 5 Hydrophis hydrophitoxin cyanocinctus a hydrophitoxin M T C b Lapemis hardwickii lapemis toxin Pelamis platwus pelamitoxin a pelamis toxin b
Hydrophiinae
Snake
D D D S T T T T
Table II. Amino Acid Sequence of Sea Snake Neurotoxins (Type I or Short Chain)
C c c c c c c
C
p p p p p
E E E
S S S S S S
A D N s A D N s A D N s p G E N s s G q S s s G q S s s G q s s s G q s s
A A A
S
c c c c c c c c
C
G N S G N S G N S E SS E SS E S S C
A E
A A A A A A
Segment 3
H H H H H H
RGTI IERG RGTI IERG R G T I I E R G R G T R I E R G R G T R I E R G R G T R I E R G
Y Y Y Y Y Y Y Y
K K N N H H H
K
K M K K K K K K
T T T q q q q q
wq wR wK wR wS wS wS wS
K K T W S K K T W S
D D D D D D D D
H H H H F F F F
D H D H K K T W SD H
G G G G G G G G
T T T T T T T T
R R R I I I I I
I I I T I I I I
T R I T R I T R I
E E E E E E E E
E E E
R R R R R R R R
R R R
G G G G G G G G
G G G
Continued on next page
R R R R R R R R
R G R G R G
Y K K T W S D R G T R I E R G
K K T W SD K K T W SD K K T W SD K K T W S D K K T W SD D YKKTW
Segment 4
o
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
Toxin
Laticaudinae Aipysurus laevis toxin a toxin b toxin c L. laticaudata laticotoxin a L. semifasciata erabutoxin a erabutoxin b erabutoxin c toxin b
Lapemis hardwickii lapemis toxin Pelamis platuruspelamitoxin a pelamis toxin b
Acatyptophis peronii major minor Astroitia stokesiitoxin a Enhydrina schistosa toxin 4 toxin 5 Hydrophis hydrophitoxin cyanocinctus a hydrophitoxin
Hydrophiinae
Snake
Table II. Continued
c c c c c c c c G G G G G G G G
c G c G c G
C p q V K S G I K L E c
C C C c c c c c p p p p p p p p q q q T T T T T
V V V V V V V V
K K K K K K K K
P P P P P P P P
G G G G G G G G I I I I I I I I
K K K K K K N K
L L L L L L L L
E E E T S S S S
T T T T T
NE N E NE NE NE
c K T NE
c H T NE c H T NE c H T NE
c H T NE
c H c H c H c H c H
c c c c c c
c c c c c c
K q E E E E
T NE S E D S E V sE V s E V s E R
c c KT NE
c
C p q V K P G I K L E c C p q V K S G I K L E c C p q V K S G I K L E c
c c
c
C
C
c G
V K S G I K L E q V K S G I K L E q V K P G I K L E q V K S G I K L E q V K K G I K L E
C p C p C p C p C p C p
C C C C c c c c
N N N N N N N N
N N N N N N N N
C N N C N N C N N
C N N
C N N C N N C N N
C N N
C N N C N N
56 56 56 57 50 50 50 58
53 54 55
52
48 49 50 51 51 52
Segment 7 Segment 8 Ref.
QV K S G I K L E C c H T N E
Segment 6
C G c G c G c G c G c G
Segment 5
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
toxin b toxin c
Toxin
toxin b toxin c
Toxin
Laticauda semifasciata L S I I I
Laticaudinae
Astroitia stokesii
Hydrophiinae
Snake
Table III. Continued
Laticauda semifasciata L S I I I
Laticaudinae
Astroitia stokesii
Hydrophiinae
Snake
C A A T
C A A T C A A T
Segment 5
R E C
L S C L S C
Segment 1
Segment 3
C P S V N T G T E I K
Segment 7
C C S A D K C
NTYP
Segment 4c
57
59 59
Ref
C S S R G K V L E F G
C B T R G E R I I M G CSTRGERIVGM
Segment 8
CNAW
CDGF C D A F
Segment 4b
N I Y A K W G Ser-NHj N I Y T K W G S G R-NH^
CYVKSW
CFVKTW CFVKTW
Segment 4a
C C S T D N C C C S T D N C
C P S G Q E I
CPPGENV C P P G E N V
C P T A K S G V H I A C P T A K S G V H I A
Segment 6
YLNPHDTQT
YLGYKHSQT YLGYKHSQT
Segment 2
Table III. A m i n o A c i d Sequences o f Sea Snake N e u r o t o x i n (Type II, L o n g C h a i n )
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
Laticauda colubrina
Snake
Table IV. Continued
Laticauda colubrina
Snake
Toxin
Toxin
C C
C C
A A T A A T
Segment 5
R I R I
Segment 1
C C
I I
C C
P T V K PG I D I K P T V K R GI H I K
Segment 6
Y L A P R D T Q Y L A P R D T Q
Segment 2
Table IV. Amino Acid Sequences of Sea Snake Neurotoxins (Hybrid Type) Segment 3
C C
C C
C C
S T D K S T D K
Segment 7
A P G Q E I A P G Q E I
Segment 4
C C
N P H P K L A N P H P K L A
Segment 8
60 60
Ref.
Y L K S W D D G T G F L K G N R L E F G Y L K S W D D C T G S I R G N R L E F G
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T h e A C h R is c o m p o s e d o f five subunits, a Pl8. A n e u r o t o x i n attaches to t h e a subunit. S i n c e there are 2 m o l o f t h e a subunits, 2 m o l o f neurotoxins attach to 1 mol of AChR. A neurotransmitter, acetylcholine ( A C h ) , also attaches t o t h e a subunit. W h e n t h e A C h attaches to t h e A C h R , t h e A C h R changes c o n f o r m a t i o n , o p e n i n g u p t h e transmembrane p o r e so that cations ( N a , K ) c a n pass t h r o u g h . B y this m e c h a n i s m t h e d e p o l a r i z a t i o n wave from a nerve is n o w conveyed t o a muscle. T h e difference between n e u r o t o x i n a n d A C h is that t h e former's attachment does n o t o p e n t h e transmembrane pore. A s a consequence, t h e nerve i m p u l s e from a nerve cannot b e transmitted t h r o u g h t h e postsynaptic site (21). 2
+
+
A t t h e m o m e n t , it is n o t k n o w n whether each toxin attaches t o t h e same site AChR. Laticauda semifasciata v e n o m added to t h e outside bathing s o l u t i o n o f frog skin causes an increase i n transmural p o t e n t i a l difference a n d short-circuit current, i n d i cating t h e change i n t h e N a transport system. T h e v e n o m - i n d u c e d stimulatory effects c a n b e explained as being either due to an increase i n N a permeability o f the o u t e r m e m b r a n e o r by an increase i n the activity o f t h e N a - p u m p (22). W h i l e most investigations show that sea snake neurotoxins are postsynaptic type, G a w a d e a n d G a i t o n d e (23) stated that Enhydrina schistosa major toxin has d u a l actions o r postsynaptic as well as presynaptic toxicity. E. schistosa v e n o m p h o s p h o lipase A is b o t h n e u r o t o x i c and myotoxic. N e u r o t o x i c a c t i o n o f t h e enzyme is weak so that there is sufficient t i m e for m y o n e c r o t i c a c t i o n to take place (24). S e a snake, L. semifasciata, t o x i n also inhibits transmission i n a u t o n o m i c ganglia, but has n o effect o n transmission i n c h o r o i d neurons. in
+
+
+
Other Vertebrate Venoms S i n c e sea snake venoms are discussed here, it is appropriate to review o t h e r vertebrate v e n o m s also. U n f o r t u n a t e l y , very few investigations have been d o n e o n t h e v e n o m s o f o t h e r m a r i n e vertebrates. It is k n o w n that s o m e fish secrete venoms from t h e i r spines. T h e fishes k n o w n to have venoms are t h e s c o r p i o n fish (family: Scorpaenidae), weever fish (family: Trachinidae), catfish (order: S i l u r i f o r m e s ; there are 31 families), stargazers (family: U r a n o s c o p i d a e ) , toad fish (family: B a t r a c h o i d i dae), a n d stingrays (suborder: M y l i o b a t o i d e a ) . V e n o m is secreted from the dorsal, pelvic a n d anal spines. A review o f o r i g i n a l papers indicates that m a n y papers have failed to specify from w h i c h spine t h e v e n o m was obtained. Therefore, some publications are meaningless scientifically. N o t a single c o m p o n e n t o f fish venoms has been characterized for t h e a m i n o acid sequence yet. E v e n t h e m o l e c u l a r weight o f fish toxins is n o t clear. D e a k i n s a n d Saunders (25) c o n c l u d e d that the m o l e c u l a r weight o f Scorpaena t o x i n was 150,000, w h i l e Schaeffer et a l . (26) c o n c l u d e d that it h a d a m o l e c u l a r weight range o f 50,000 to 800,000. T h e r e is 5-hydroxytryptamine i n weever fish v e n o m besides p r o t e i n . It is believed that l o c a l p a i n is attributed t o the presence o f 5-hydroxytryptamine (27). O t h e r s m a l l c o m p o u n d s such as histamine, adrenaline, a n d n o r a d r e n a l i n e are also present i n t h e weever fish (28). A s f o r t h e stingray v e n o m , n o t m u c h is k n o w n . T h e r e was a report o n t h e presence o f 5'-nucleotidase a n d phosphodiesterase i n stingray, Urolophus halleri, v e n o m (29). S p i n e v e n o m o f catfish, Ictalurus catus, contains toxins w i t h a m o l e c u l a r weight o f 10,000 a n d isoelectric points o f 3.8 a n d 7.8 (30). P e c t o r a l v e n o m o f Arias thailasinus contains a l k a l i n e phosphatase (31). T h e v e n o m is a m i x t u r e o f at least 30 proteins.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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Summary From this brief review of marine vertebrate venoms, it is obvious that very few biochemical investigations have been done. The technology to study marine vertebrate venom components is available. There are simply not enough scientists interested enough to enter the field. The first task is to isolate the toxic principles and identify the amino acid sequences. Pharmacological investigation should be done on the purified toxic principle and not on the crude venom, which is a mixture of many proteins and nonproteins.
Acknowledgment This work was supported by U. S. Army Medical Research and Development tract DAMD17-81-C-6063.
Con-
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Tu, A. T. Handbook of Natural Toxins, Vol. 3, Marine Toxins and Venoms; 1988, 379-444. Yu, N. T.; Lin, T. S.; Tu, A. T. J. Biol. Chem. 1975, 250, 1782. Ishizaki, H.; Allen, M.; Tu, A. T. J. Pharm. Pharmacol. 1984, 36, 36. Tu, A. T.; Lin, T. S.; Bieber, A. L. Biochemistry 1975, 14, 3408. Seto, A.; Sato, S.; Tamiya, N. Biochim. Biophys. Acta 1970, 214, 483. Tu, A. T.; Toom, P. M. J. Biol. Chem. 1971, 246, 1012. Tu, A. T.; Hong, B. S. J. Biol. Chem. 1971, 246, 2772. Tu, A. T.; Hong, B. S.; Solie, T. N. Biochemistry 1971, 10, 1295. Raymond, M. L.; Tu, A. T. Biochim. Biophys. Acta 1972, 285, 498. Sato, S.; Tamiya, N. J. Biochem. 1970, 68, 867. Tamiya, T.; Lamouroux, A.; Julien, J. F.; Grima, B.; Mallet, J.; Fromageot, P.; Menez, A. Biochimie 1985, 67, 185. Tu, A. T.; Ganthavorn, S. Am. J. Trop. Med. Hyg. 1969, 18, 151. Tu, A. T.; Salafranca, E. S. Am. J. Trop. Med. Hyg. 1974, 23, 135. Kaire, G. H. Med. J. Aust. 1964, 2, 729. Gawade, S. P.; Budak, D. P.; Gaitonde, B. B. Ind. J. Med. Res. 1980, 72, 747. Okonogi, T.; Hattori, Z.; Watanabe, M.; Amagai, E. Snake, 1970, 2, 18. Coulter, A. R.; Harris, R. D.; Sutherland, S. K. Proc. Melbourne Herp. Symp. 1981, 39. Baxter, E. H.; Gallichio, H. A. Toxicon 1976, 14, 347. Madsen, T.; Lundstroem, H. Toxicon 1979, 17, 326. Tu, A. T. Venoms: Chemistry and Molecular Biology, John Wiley, New York,
1977 . 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Cash, D. J.; Hess, G. P. Proc. Nat. Acad. Sci., USA 1980, 77, 842. Gerencser, G. A.; Loo, S. Y. Comp. Biochem. Physiol. 1982, 72A, 727. Gawade, S. P.; Gaitonde, B. B. Toxicon 1982, 20, 797. Lind, P.; Eaker, D. Toxicon 1981, 19, 11. Deakins, D. E.; Saunders, P. R. Toxicon 1967, 4, 257. Schaeffer, R. C.; Jr.; Carlson, R. W.; Russell, F. E. Toxicon 1971, 9, 69. Carlisle, D. B. J. Mar. Biol. Assoc., U. K. 1962, 42, 155. Haavaldsen, R.; Fonnum, F. Nature 1963, 199, 286. Russell, F. E.; Panos, T. C., Kang, L. W.; Warner, W. M.; Colket, T. C. Am. J. Med. Sci. 1958, 235, 566. Calton, G. J.; Burnett, J. W. Toxicon 1975, 13, 399.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
346 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.
MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY Thulesius, O.; Al-Hassan, J.; Criddle, R. S.; Thomson, M. Gen. Pharmacol. 1983, 14, 129. Al-Hassan, J. M.; Thomson, M.; Ali, M.; Criddle, R. S. Toxin Rev. 1987, 6, 1. Gawade, S. P.; Bhide, M. B. Bull. Haff. Inst. 1977, 5, 45. Su, B.; Lao, Z.; Sho, Z.; Chang, M.; Zeng, J.; Pan, F.; Wu, S.; Xu, L.; Mo, Y. Redai Haiyang 1984, 3, 41. Setoguchi, Y.; Morisawa, S.; Obo, F. Acta Med. Univ. Kagoshima 1968, 10, 53. Uwatoko-Setoguchi, Y. Acta Med. Univ. Kagoshima 1970, 12, 74. Carey, J. E.; Wright, E. A. Trans. R. Soc. Med. Hyg., 1960, 54, 50. Ibrahim, S. A.; Thompson, R. H. S. Biochim. Biophys. Acta 1965, 99, 331. Fohlman, J.; Eaker, D. Toxicon 1977, 15, 385. Tan, N. H. Biochim. Biophys. Acta 1982, 717, 503. Durkin, J. P.; Pickwell, G. V.; Trotter, J. T.; Shier, W. T. Toxicon 1981, 19, 535. Uwatoko-Setoguchi, Y.; Minamishima, Y.; Obo, F. Acta Med. Univ. Kagoshima 1968, 10, 219. Uwatoko-Setoguchi, Y.; Obo, F. Acta Med. Univ. Kagoshima 1969, 11, 139. Tu, A. T.; Passey, R. B.; Toom, P. M. Arch. Biochem. Biophys. 1970, 140, 96. Yoshida, H.; Kudo, T.; Shinkai, W.; Tamiya, N. J. Biochem. 1979, 85, 379. Nishida, S.; Kim, H. S.; Tamiya, N. Biochem. J. 1982, 207, 589. Uwatoko, Y.; Nomura, Y.; Kojima, K.; Obo, F. Acta Med. Univ. Kagoshima 1966, 8, 141. Mari, N.; Tu, A. T. Arch. Biochem. Biophys. 1988, 260, 10. Mori, N.; Tu, A. T. Biol. Chem. Hoppe-Seyler 1988; 369, 521. Maeda, N.; Tamiya, N. Biochem. J. 1977, 167, 289. Frykland, L.; Eaker, D.; Karlsson, E. Biochemistry 1972, 11, 4633. L i u , C. S.; Blackwell, R. Q. Toxicon 1974, 12, 543. Fox, J. W.; Elzinga, M.; Tu, A. T. FEBS Lett. 1977, 80, 217. Wang, C. L.; Liu, C. S.; Hung, Y. O.; Blackwell, R. Q. Toxicon 1976 14, 459. Mori, N.; Ishizaki, H.; Tu, A. T. unpublished data, 1988. Maeda, N.; Tamiya, N. Biochem. J. 1976, 153, 79. Maeda, N.; Tamiya, N. Biochem. J. 1974, 141, 389. Tsernoglou, D.; Petsko, G. A.; Tu, A. T. Biochim. Biophys. Acta 1977, 491, 605. Maeda, N.; Tamiya, N. Biochem. J. 1978, 175, 507. Kim, H. S.; Tamiya, N. Biochem. J. 1982, 207, 215.
RECEIVED
June 26, 1989
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
