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BIOCHEMICAL EFFECTS OF TETRAMISOLE
8
B I O C H E M I C A L E F F E C T S OF T E T R A M I S O L E H. Van den Bossche Department of Comparative Biochemistry Janssen Pharmaceutica - Research Laboratories Beerse - Belgium.
Tetramisole* is an antinematodal drug effective b o t h in animals and ( 1 ) m a n . The laevo-isomer** has been shown in animals and man t o be approximately twice as active as the racemic m i x t u r e , and depending on ( 2 ) the parasite, several times more active than the d e x t r o - i s o m e r . In 1967 and 1969, we forwarded the hypothesis t h a t the a n t h e l m i n t i c effect, of tetramisole may be due t o the i n h i b i t i o n of fumarate reductase ( 2 3) in sensitive helminths . T h i s hypothesis was based on the f o l l o w i n g observations. Incubation of Ascaridia galli f o r 15 hours at 37° in an atmosphere of 95 % N2 and 5 % CO2 resulted in a decreased succinate p r o d u c t i o n when tetramisole was added t o the i n c u b a t i o n m i x t u r e . 63.4 % i n h i b i t i o n was obtained at a tetramisole concentration of 2.5 M g / m l . This decreased succinate p r o d u c t i o n is associated w i t h a decrease (4) in the A T P c o n t e n t of the in v i t r o incubated p a r a s i t e s . Evidence has been presented t h a t succinate is one of the major metabolic end-products in several parasites. Succinate is f o r m e d f r o m fumarate in ( 5) the presence of N A D H and of the e n z y m e , fumarate r e d u c t a s e . Since fumarate reductase w o u l d appear t o be a key enzyme in the energy yielding pathway, we measured the effect of tetramisole on this enzyme. The results presented in Fig. 1 indicate t h a t b o t h optical isomers, i.e. laevo- and dextro-tetramisole, i n h i b i t the N A D H - c o u p l e d fumaratesuccinate system of Ascaris muscle. The laevo-isomer proved t o be a more potent i n h i b i t o r of the enzyme system, a fact compatible w i t h its more potent a n t h e l m i n t i c action. T o investigate f u r t h e r the mechanism o f the tetramisole-induced i n h i b i t i o n of the fumarate reductase mechanism, * Generic name for the h y d r o c h l o r i d e of 2, 3, 5, 6-tetrahydro-6-phenyl-imidazo [ 2 , 1 b ] thiazole. * * levamisole
117
H. V A N den BOSSCHE
we isolated particles (R2) containing succinoxidase activity f r o m muscle ( 6) strips of Ascaris suum, as described by Kmetec and B u e d i n g . Fumarate reductase activity was measured by f o l l o w i n g the anaerobic o x i d a t i o n of N A D H in the presence of fumarate. The Lineweaver-Burk plots shown in Fig. 2 indicate t h a t tetramisole is a non-competitive i n h i b i t o r ( K s = 4 _ 4 1.4 χ 1 0 " M ; Kj = 4.4 χ 1 0 M ) . Incubation of Ascaris m i t o c h o n d r i a in the presence of A D P , malate and inorganic phosphate results in the ( 7) f o r m a t i o n of A T P . Since A T P p r o d u c t i o n is associated w i t h the fumarate reduction, any i n h i b i t i o n o f fumarate reductase w o u l d conseq u e n t l y result in a decreased incorporation of inorganic phosphate into A T P . The results shown in Fig. 3 indicate t h a t laevo- and dextro-tetramisole i n h i b i t the malate-induced 32p incorporation i n t o organic phosphate in m i t o c h o n d r i a isolated f r o m Ascaris muscle by the m e t h o d ( 8) of Saz and Lescure . The concentration of laevo- and d e x t r o - t e t r a m i sole, needed to obtain 50 % i n h i b i t i o n were 0.9 and 4 . 3 m M respectively. These experiments c o n f i r m e d t h a t tetramisole is an i n h i b i t o r of the energy yielding pathway in Ascaris. Reinecke has shown that tetramisole at a level of 15 mg/kg of b o d y weight was 89 t o 1 0 0 % effective against all stages o f development of (9) Haemonchus contortus . Laevo-tetramisole inhibits the fumarate re( 1 )0 , and as ductase mechanism in third-stage larvae of this nematode shown in Table 1, also affects the anaerobic o x i d a t i o n of N A D H in the presence of fumarate in a particulate fraction of fourth-stage larvae. A l l the results presented so far seem t o suggest t h a t the antinematodal activity o f tetramisole may be due t o i n h i b i t i o n o f the fumarate reductase mechanism in helminths. This was c o n f i r m e d by Prichard in his publication on the mechanism of action of thiabendazole However, most of these biochemical effects were observed at concentrations higher or after incubation periods longer than those needed t o induce contraction of the w o r m s (Van Nueten, this b o o k ) . This may indicate t h a t the effect of tetramisole on muscle tension is the p r i m a r y site o f chemotherapeutic activity in vivo. The tetramisole-stimulated tonus increase in Ascaris is f o l l o w e d by a decrease in tension and irreversible paralysis. These t w o phases may be independent, since the contraction but not the paralysis was inhibited by h e x a m e t h o n i u m (Van Nueten, this book). Based on these observations and on the theories on muscle contraction in higher animals, the f o l l o w i n g hypothesis f o r the mechanism of action o f tetramisole can be suggested (Fig. 4 ) . The activation o f muscle by tetramisole breaks d o w n the myosin-bound A T P t o A D P . The next step is n o r m a l l y the ^ p h o s p h o r y l a t i o n of the resultant b o u n t A D P t o b o u n d
118
C O M P A R A T I V E B I O C H E M I S T R Y OF P A R A S I T E S
ATP. However, our biochemical experiments indicate that in the presence of tetramisole A T P p r o d u c t i o n is i n h i b i t e d and the rephosphorylation o f A D P slowed down or diminished as A T P is exhausted, effects w h i c h may result in the f o r m a t i o n of actomyosin w i t h consequent muscle stiffness. A l t h o u g h we have at present no direct experimental evidence f o r this hypothesis, there are a number of observations w h i c h seem t o lend support t o it. Incubation of Ascaris in the presence of 10 μ$ laevo-tetramisole per ml of incubation m i x t u r e resulted in complete i m m o b i l i z a t i o n of the parasite after o n l y 2 hours, whereas, as shown in the previous c o m m u n i c a t i o n , i t takes o n l y 10 minutes t o obtain paralysis w h e n the w o r m s are connected t o an isotonic lever w i t h a preload of 1 g. The reaction o f the w o r m s t o this artificial position may result in an utilization of A T P . Since we have suggested t h a t paralysis is due t o an effect on the energy yielding pathway, i t is possible t h a t the more rapid response obtained in the latter experiments was the result of a lower energy reserve in the Ascaris muscle. It has also been shown t h a t tetramisole has reversible spasmogenic efffects on guinea pig ileum and rabbit d u o d e n u m . F u r t h e r m o r e , alt h o u g h some side-effects were observed in mammals after administration of tetramisole, these always disappear in a f e w hours. Thus, these i n v i t r o and in vivo reversible effects are in direct contrast t o the irreversible paralysis observed in Ascaris. This difference may be. a t t r i b u t e d t o the fact that tetramisole affected the energy p r o d u c t i o n in Ascaris muscle ( 4 ) but not in mammalian tissues . ( 1 )2 The experiments of Denham also support our hypothesis. Denham compared the in v i t r o effects of m e t h y r i d i n e and tetramisole on the m o t i l i t y and development of Ostertagia circumcincta. Both methyridine and tetramisole had a paralysing effect on the exsheated third-stage larvae of this nematode. When the larvae treated w i t h m e t h y r i d i n e were washed and incubated w i t h o u t the drug for 18 hours at 3 7 ° , all were as active as those in the c o n t r o l tubes. A similar experiment w i t h tetramisole however, showed that after incubation, the washed larvae were moving in a rather sluggish manner as compared w i t h the controls. This difference in recovery may be explained by d i f f e r e n t mechanisms o f a c t i o n ; ( 1 3 ) methyridine is said t o act as a neuromuscular b l o c k e r , whereas tetramisole inhibits the fumarate reductase mechanism and also affects the neuromuscular system of the parasites. The same author has shown t h a t when larvae exposed t o m e t h y r i d i n e were washed and cultured in v i t r o , they grew as well as the control larvae. Exposure of larvae t o tetramisole on the other hand, decreased the growth-rate when compared w i t h controls, and no fifth-stage larvae developed during the test period.
119
H. V A N den BOSSCHE
These observations are also considered t o lend some support t o our hypothesis as it is k n o w n that i n h i b i t i o n of energy p r o d u c t i o n resulted in a decreased protein synthesis. A l t h o u g h our hypothesis lacks direct c o n f i r m a t i o n , we believe t h a t this proposed mechanism of action explains the different tetramisole-induced ( 34 ) effects better than the hypothesis we presented previously ' .
Acknowledgements The author whishes t o express his thanks t o Dr. Paul A . J . Janssen f o r his constant interest; t o Mrs. Horemans, Mr. Goossens and Mr. Vermeiren f o r their technical assistance, and t o Mr. & Mrs. Scott f o r their help in preparing the manuscript.
References. 1. T H I E N P O N T , D., V A N P A R I J S , O.F.J., R A E Y M A E K E R S , A . H . M . , V A N D E N B E R K , J., D E M O E N , P.J.A., A L L E W I J N , F.T.N., M A R S B O O M , R.P.H., N I E M E GEERS, C.J.E., S C H E L L E K E N S , K.H.L. and JANSSEN, P.A.J., Nature 209, 1084 (1966). 2. B U L L O C K , M.W., H A N D , J.J. and W A L E T Z K Y , E., J. Med. Chem. 1 1 , 169 (1968). 3. V A N DEN BOSSCHE, H. and J A N S S E N , P.A.J., Life Sei. 6, 1781 (1967). 4. V A N DEN BOSSCHE, H. and J A N S S E N , P.A.J., Biochem. Pharmacol. 18, 35 (1969). 5. S A Z , H.J. and B U E D I N G , E., Pharmacol. Rev. 18, 871 (1966). 6. K M E T E C , E. and B U E D I N G , E., J. biol. Chem. 2 3 6 , 584 (1961). 7. S A Z , H J . , Comp. Biochem. Physiol. 39B, 627 (1971). 8. S A Z , H.J. and LESCURE, O.L., Comp. Biochem. Physiol. 30, 4 9 (1969). 9. R E I N E C K E , R.K., J.S. A f r . vet. med. Ass. 37, 27 (1966). 10. V A N DEN BOSSCHE, H., V A N P A R I J S , O.F.J, and T H I E N P O N T , D., Life Sei. 8, 1047 (1969). 11. P R I C H A R D , R.K., Nature 228, 684 (1970). 12. D E N H A M , D.A., E x p t l . Parasitol. 28, 493 (1970). 13. B R O O M E , A.W.J., Brit. J. Pharmacol. 17, 327 (1961).
120
C O M P A R A T I V E B I O C H E M I S T R Y OF P A R A S I T E S
Table 1. Effect o f L-tetramisole upon reduced-NAD: fumarate reductase a c t i v i t y in a particulate f r a c t i o n o f fourth-stage H. contortus larvae
L-tetramisole ^moles/ml)
3
NADH oxidized Absolute ( n m o l e s / m i n / m g protein)
0
8.71
0.580
3.42 (2.45 -
(7.02 - 10.65)
number of experiments: 3.
121
4.61)
%
100 39.2
H. V A N den BOSSCHE
1.5-,
T i m e in m i n u t e s
Fig. 1. Effect of tetramisole on anaerobic o x i d a t i o n of N A D H in the presence of fumarate and a particulate fraction of Ascaris muscle. The fraction used ( 8) treated w i t h Tris-buffer (pH 7.4; 0.1M). was a mitochondrial f r a c t i o n The incubation m i x t u r e consisted o f : 2.8 ml Tris-buffer (pH 8.5; 0 . 0 4 M ) ; 0.1 ml of the particulate fraction (0.84 mg p r o t e i n ) ; 0.1 ml Tris-buffer (pH 7.4; 0.1M) or 0.1 ml o f L- or D-tetramisole (0.44 Mmoles/ml of incubation m i x t u r e ) . A f t e r 30 min of incubation at 3 7 ° , 0.1 ml of fumarate (1.2 Mmoles) and 0.02 ml N A D H (1.068 Mmoles) were added. The reaction was f o l l o w e d at 340 ητιμ f o r 10 min at 37° in an atmosphere of nitrogen. 1 = c o n t r o l ; 2 = D-tetramisole; 3 = L-tetramisole.
122
C O M P A R A T I V E B I O C H E M I S T R Y OF P A R A S I T E S
Fig. 2. Effect of L-tetramisole on N A D H o x i d a t i o n in the presence of fumarate and a r e f r a c t i o n of Ascaris muscle. The incubation m i x t u r e was the same as that described in Fig. 1. The effect of tetramisole was measured w i t h o u t pre-incubation. 1 = c o n t r o l ; 2 = 0.272 μ moles L-tetramisole; 3 = 0.544 μηηοΙβ5 L-tetramisole/ml of incubation m i x t u r e .
123
H. V A N den BOSSCHE
100-1
80
60
19 o Ü0
Drug c o n c e n t r a t i o n
Fig. 3. Effect of Ι_-(·) and D-(o) tetramisole on malate-induced 32p incorporat i o n i n t o organic phosphate by m i t o c h o n d r i a isolated f r o m Ascaris 3 2 ( 7 ) muscle. P incorporation was measured by the method of Saz .
124
C O M P A R A T I V E B I O C H E M I S T R Y OF P A R A S I T E S
Tetramisole
induced contraction
A c t i n • A T P - Myosin
Actin«
ADP
ADP-Myosin
ATP
4 I n h i b i t e d by T e t r a m i s o l e Fumarate •
NADH2
Succinate • NAD
Fig. 4. Hypothesis f o r the mechanism of action of tetramisole.
125
B I O C H E M I C A L E F F E C T S OF T E T R A M I S O L E H. Van den Bossche Department of Comparative Biochemistry Janssen Pharmaceutica - Research Laboratories Beerse - Belgium.
Tetramisole* is an antinematodal drug effective b o t h in animals and ( 1 ) m a n . The laevo-isomer** has been shown in animals and man t o be approximately twice as active as the racemic m i x t u r e , and depending on ( 2 ) the parasite, several times more active than the d e x t r o - i s o m e r . In 1967 and 1969, we forwarded the hypothesis t h a t the a n t h e l m i n t i c effect, of tetramisole may be due t o the i n h i b i t i o n of fumarate reductase ( 2 3) in sensitive helminths . T h i s hypothesis was based on the f o l l o w i n g observations. Incubation of Ascaridia galli f o r 15 hours at 37° in an atmosphere of 95 % N2 and 5 % CO2 resulted in a decreased succinate p r o d u c t i o n when tetramisole was added t o the i n c u b a t i o n m i x t u r e . 63.4 % i n h i b i t i o n was obtained at a tetramisole concentration of 2.5 M g / m l . This decreased succinate p r o d u c t i o n is associated w i t h a decrease (4) in the A T P c o n t e n t of the in v i t r o incubated p a r a s i t e s . Evidence has been presented t h a t succinate is one of the major metabolic end-products in several parasites. Succinate is f o r m e d f r o m fumarate in ( 5) the presence of N A D H and of the e n z y m e , fumarate r e d u c t a s e . Since fumarate reductase w o u l d appear t o be a key enzyme in the energy yielding pathway, we measured the effect of tetramisole on this enzyme. The results presented in Fig. 1 indicate t h a t b o t h optical isomers, i.e. laevo- and dextro-tetramisole, i n h i b i t the N A D H - c o u p l e d fumaratesuccinate system of Ascaris muscle. The laevo-isomer proved t o be a more potent i n h i b i t o r of the enzyme system, a fact compatible w i t h its more potent a n t h e l m i n t i c action. T o investigate f u r t h e r the mechanism o f the tetramisole-induced i n h i b i t i o n of the fumarate reductase mechanism, * Generic name for the h y d r o c h l o r i d e of 2, 3, 5, 6-tetrahydro-6-phenyl-imidazo [ 2 , 1 b ] thiazole. * * levamisole
117
H. V A N den BOSSCHE
we isolated particles (R2) containing succinoxidase activity f r o m muscle ( 6) strips of Ascaris suum, as described by Kmetec and B u e d i n g . Fumarate reductase activity was measured by f o l l o w i n g the anaerobic o x i d a t i o n of N A D H in the presence of fumarate. The Lineweaver-Burk plots shown in Fig. 2 indicate t h a t tetramisole is a non-competitive i n h i b i t o r ( K s = 4 _ 4 1.4 χ 1 0 " M ; Kj = 4.4 χ 1 0 M ) . Incubation of Ascaris m i t o c h o n d r i a in the presence of A D P , malate and inorganic phosphate results in the ( 7) f o r m a t i o n of A T P . Since A T P p r o d u c t i o n is associated w i t h the fumarate reduction, any i n h i b i t i o n o f fumarate reductase w o u l d conseq u e n t l y result in a decreased incorporation of inorganic phosphate into A T P . The results shown in Fig. 3 indicate t h a t laevo- and dextro-tetramisole i n h i b i t the malate-induced 32p incorporation i n t o organic phosphate in m i t o c h o n d r i a isolated f r o m Ascaris muscle by the m e t h o d ( 8) of Saz and Lescure . The concentration of laevo- and d e x t r o - t e t r a m i sole, needed to obtain 50 % i n h i b i t i o n were 0.9 and 4 . 3 m M respectively. These experiments c o n f i r m e d t h a t tetramisole is an i n h i b i t o r of the energy yielding pathway in Ascaris. Reinecke has shown that tetramisole at a level of 15 mg/kg of b o d y weight was 89 t o 1 0 0 % effective against all stages o f development of (9) Haemonchus contortus . Laevo-tetramisole inhibits the fumarate re( 1 )0 , and as ductase mechanism in third-stage larvae of this nematode shown in Table 1, also affects the anaerobic o x i d a t i o n of N A D H in the presence of fumarate in a particulate fraction of fourth-stage larvae. A l l the results presented so far seem t o suggest t h a t the antinematodal activity o f tetramisole may be due t o i n h i b i t i o n o f the fumarate reductase mechanism in helminths. This was c o n f i r m e d by Prichard in his publication on the mechanism of action of thiabendazole However, most of these biochemical effects were observed at concentrations higher or after incubation periods longer than those needed t o induce contraction of the w o r m s (Van Nueten, this b o o k ) . This may indicate t h a t the effect of tetramisole on muscle tension is the p r i m a r y site o f chemotherapeutic activity in vivo. The tetramisole-stimulated tonus increase in Ascaris is f o l l o w e d by a decrease in tension and irreversible paralysis. These t w o phases may be independent, since the contraction but not the paralysis was inhibited by h e x a m e t h o n i u m (Van Nueten, this book). Based on these observations and on the theories on muscle contraction in higher animals, the f o l l o w i n g hypothesis f o r the mechanism of action o f tetramisole can be suggested (Fig. 4 ) . The activation o f muscle by tetramisole breaks d o w n the myosin-bound A T P t o A D P . The next step is n o r m a l l y the ^ p h o s p h o r y l a t i o n of the resultant b o u n t A D P t o b o u n d
118
C O M P A R A T I V E B I O C H E M I S T R Y OF P A R A S I T E S
ATP. However, our biochemical experiments indicate that in the presence of tetramisole A T P p r o d u c t i o n is i n h i b i t e d and the rephosphorylation o f A D P slowed down or diminished as A T P is exhausted, effects w h i c h may result in the f o r m a t i o n of actomyosin w i t h consequent muscle stiffness. A l t h o u g h we have at present no direct experimental evidence f o r this hypothesis, there are a number of observations w h i c h seem t o lend support t o it. Incubation of Ascaris in the presence of 10 μ$ laevo-tetramisole per ml of incubation m i x t u r e resulted in complete i m m o b i l i z a t i o n of the parasite after o n l y 2 hours, whereas, as shown in the previous c o m m u n i c a t i o n , i t takes o n l y 10 minutes t o obtain paralysis w h e n the w o r m s are connected t o an isotonic lever w i t h a preload of 1 g. The reaction o f the w o r m s t o this artificial position may result in an utilization of A T P . Since we have suggested t h a t paralysis is due t o an effect on the energy yielding pathway, i t is possible t h a t the more rapid response obtained in the latter experiments was the result of a lower energy reserve in the Ascaris muscle. It has also been shown t h a t tetramisole has reversible spasmogenic efffects on guinea pig ileum and rabbit d u o d e n u m . F u r t h e r m o r e , alt h o u g h some side-effects were observed in mammals after administration of tetramisole, these always disappear in a f e w hours. Thus, these i n v i t r o and in vivo reversible effects are in direct contrast t o the irreversible paralysis observed in Ascaris. This difference may be. a t t r i b u t e d t o the fact that tetramisole affected the energy p r o d u c t i o n in Ascaris muscle ( 4 ) but not in mammalian tissues . ( 1 )2 The experiments of Denham also support our hypothesis. Denham compared the in v i t r o effects of m e t h y r i d i n e and tetramisole on the m o t i l i t y and development of Ostertagia circumcincta. Both methyridine and tetramisole had a paralysing effect on the exsheated third-stage larvae of this nematode. When the larvae treated w i t h m e t h y r i d i n e were washed and incubated w i t h o u t the drug for 18 hours at 3 7 ° , all were as active as those in the c o n t r o l tubes. A similar experiment w i t h tetramisole however, showed that after incubation, the washed larvae were moving in a rather sluggish manner as compared w i t h the controls. This difference in recovery may be explained by d i f f e r e n t mechanisms o f a c t i o n ; ( 1 3 ) methyridine is said t o act as a neuromuscular b l o c k e r , whereas tetramisole inhibits the fumarate reductase mechanism and also affects the neuromuscular system of the parasites. The same author has shown t h a t when larvae exposed t o m e t h y r i d i n e were washed and cultured in v i t r o , they grew as well as the control larvae. Exposure of larvae t o tetramisole on the other hand, decreased the growth-rate when compared w i t h controls, and no fifth-stage larvae developed during the test period.
119
H. V A N den BOSSCHE
These observations are also considered t o lend some support t o our hypothesis as it is k n o w n that i n h i b i t i o n of energy p r o d u c t i o n resulted in a decreased protein synthesis. A l t h o u g h our hypothesis lacks direct c o n f i r m a t i o n , we believe t h a t this proposed mechanism of action explains the different tetramisole-induced ( 34 ) effects better than the hypothesis we presented previously ' .
Acknowledgements The author whishes t o express his thanks t o Dr. Paul A . J . Janssen f o r his constant interest; t o Mrs. Horemans, Mr. Goossens and Mr. Vermeiren f o r their technical assistance, and t o Mr. & Mrs. Scott f o r their help in preparing the manuscript.
References. 1. T H I E N P O N T , D., V A N P A R I J S , O.F.J., R A E Y M A E K E R S , A . H . M . , V A N D E N B E R K , J., D E M O E N , P.J.A., A L L E W I J N , F.T.N., M A R S B O O M , R.P.H., N I E M E GEERS, C.J.E., S C H E L L E K E N S , K.H.L. and JANSSEN, P.A.J., Nature 209, 1084 (1966). 2. B U L L O C K , M.W., H A N D , J.J. and W A L E T Z K Y , E., J. Med. Chem. 1 1 , 169 (1968). 3. V A N DEN BOSSCHE, H. and J A N S S E N , P.A.J., Life Sei. 6, 1781 (1967). 4. V A N DEN BOSSCHE, H. and J A N S S E N , P.A.J., Biochem. Pharmacol. 18, 35 (1969). 5. S A Z , H.J. and B U E D I N G , E., Pharmacol. Rev. 18, 871 (1966). 6. K M E T E C , E. and B U E D I N G , E., J. biol. Chem. 2 3 6 , 584 (1961). 7. S A Z , H J . , Comp. Biochem. Physiol. 39B, 627 (1971). 8. S A Z , H.J. and LESCURE, O.L., Comp. Biochem. Physiol. 30, 4 9 (1969). 9. R E I N E C K E , R.K., J.S. A f r . vet. med. Ass. 37, 27 (1966). 10. V A N DEN BOSSCHE, H., V A N P A R I J S , O.F.J, and T H I E N P O N T , D., Life Sei. 8, 1047 (1969). 11. P R I C H A R D , R.K., Nature 228, 684 (1970). 12. D E N H A M , D.A., E x p t l . Parasitol. 28, 493 (1970). 13. B R O O M E , A.W.J., Brit. J. Pharmacol. 17, 327 (1961).
120
C O M P A R A T I V E B I O C H E M I S T R Y OF P A R A S I T E S
Table 1. Effect o f L-tetramisole upon reduced-NAD: fumarate reductase a c t i v i t y in a particulate f r a c t i o n o f fourth-stage H. contortus larvae
L-tetramisole ^moles/ml)
3
NADH oxidized Absolute ( n m o l e s / m i n / m g protein)
0
8.71
0.580
3.42 (2.45 -
(7.02 - 10.65)
number of experiments: 3.
121
4.61)
%
100 39.2
H. V A N den BOSSCHE
1.5-,
T i m e in m i n u t e s
Fig. 1. Effect of tetramisole on anaerobic o x i d a t i o n of N A D H in the presence of fumarate and a particulate fraction of Ascaris muscle. The fraction used ( 8) treated w i t h Tris-buffer (pH 7.4; 0.1M). was a mitochondrial f r a c t i o n The incubation m i x t u r e consisted o f : 2.8 ml Tris-buffer (pH 8.5; 0 . 0 4 M ) ; 0.1 ml of the particulate fraction (0.84 mg p r o t e i n ) ; 0.1 ml Tris-buffer (pH 7.4; 0.1M) or 0.1 ml o f L- or D-tetramisole (0.44 Mmoles/ml of incubation m i x t u r e ) . A f t e r 30 min of incubation at 3 7 ° , 0.1 ml of fumarate (1.2 Mmoles) and 0.02 ml N A D H (1.068 Mmoles) were added. The reaction was f o l l o w e d at 340 ητιμ f o r 10 min at 37° in an atmosphere of nitrogen. 1 = c o n t r o l ; 2 = D-tetramisole; 3 = L-tetramisole.
122
C O M P A R A T I V E B I O C H E M I S T R Y OF P A R A S I T E S
Fig. 2. Effect of L-tetramisole on N A D H o x i d a t i o n in the presence of fumarate and a r e f r a c t i o n of Ascaris muscle. The incubation m i x t u r e was the same as that described in Fig. 1. The effect of tetramisole was measured w i t h o u t pre-incubation. 1 = c o n t r o l ; 2 = 0.272 μ moles L-tetramisole; 3 = 0.544 μηηοΙβ5 L-tetramisole/ml of incubation m i x t u r e .
123
H. V A N den BOSSCHE
100-1
80
60
19 o Ü0
Drug c o n c e n t r a t i o n
Fig. 3. Effect of Ι_-(·) and D-(o) tetramisole on malate-induced 32p incorporat i o n i n t o organic phosphate by m i t o c h o n d r i a isolated f r o m Ascaris 3 2 ( 7 ) muscle. P incorporation was measured by the method of Saz .
124
C O M P A R A T I V E B I O C H E M I S T R Y OF P A R A S I T E S
Tetramisole
induced contraction
A c t i n • A T P - Myosin
Actin«
ADP
ADP-Myosin
ATP
4 I n h i b i t e d by T e t r a m i s o l e Fumarate •
NADH2
Succinate • NAD
Fig. 4. Hypothesis f o r the mechanism of action of tetramisole.
125