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Enzyme variation in man and trypanosomes
TRYPANOSOMIASISSEMINAR
119
minutes. Fraction V bovine serum albumin was found to be an effective substitute for cow serum on a weight basis. This non-homogeneous fraction is under investigation. P r e s e n c e and properties of dihydrofolate reductases in T r y p a n o s o m a t i d flagellate
W. E. G U T T E R I D G E , J. J. JAFFE AND J. J. McCORMACK, JR. National Institute for Medical Research, Mill Hill, London; Nuffield Institute of Comparative Medicine, London, and Department of Pharmacology, University of Vermont, U.S.A. After the demonstrations of dihydrofolate reductases in Trypanosoma equiperdum (JAFFE and MCCORMACK, 1967) and T. cruzi (GUTTERIDGE and SENIOR, 1968), we have now surveyed 7 species, representing all four major sub-groups of the genus Trypanosoma, for the presence of the enzyme. The species studied were rat-adapted bloodstream forms of T. brucei, T. rhodesiense, T. equiperdum, T. congolense, T. vivax, T. lewisi and T. cruzi and the culture forms of T. rhodesiense and T. cruzi. Dihydrofolate reductases were detected in all 7 species. The general properties of these enzymes were similar to those isolated from other sources. Trypanosomal reductases, however, showed a pattern of sensitivity to the inhibitory action of a number of 2, 4-diaminopyrimidines and related heterocycles, which was quite distinct from the patterns shown by the enzymes from bacterial and mammalian cells (see BURCHALLand HITCHINGS, 1965). In addition, we observed that reductases from trypanosomes of African origin had very similar drug sensitivity profiles, clearly distinguishable in certain respects from those of the reductases from two species of non-African origin (T. lewisi and T. cruzi). No significant differences have yet been found in the properties of dihydrofolate reductases from bloodstream and culture forms of T. rhodesiense and T. cruzi. The properties of the dihydrofolate reductases from Crithidia fasciculata and C. oncopelti have also been investigated. In their general properties they are similar to reductases from other flagellates; their drug sensitivity profiles to diaminoheterocycles are similar to those of T. lewisi and T. cruzi. Most of the reductase activity in the two organisms occurred in high speed, centrifugal supernatant fractions of organisms disrupted in the Mickle disintegrator. Small amounts of activity have, however, been detected in particulate fractions. The properties, location and function of this additional reductase activity in these flagellates will be discussed.
REFERENCES BURCHALL,J. J. & HITCHINGS, G. H. (1965). Mol. Pharmaeol., 1, 126. GUTTERIDGE,W. E. & SENIOR, D. S. (1968). Trans. R. Soc. trop. Med. Hyg., B2, 135. JAFFE, J. J. & McCORMACK, JR., J. J. (1967). Mol. Pharmacol., 3, 359. E n z y m e variation in m a n and t r y p a n o s o m e s
C. W. PARR AND S. G. WELCH The London Hospital Medical College, London, E. 1 Recent developments in techniques by which small differences between protein and enzyme molecules may be detected (such as single amino-acid changes in the primary structures) have led to the realization that each enzyme within a species, hitherto thought to have been a single entity, may well occur in a number of variant or isoenzymic forms. Thus, the autosomally linked enzyme phosphogluconate dehydrogenase (PGD) in man, first shown in this laboratory to exist in more than one form (FILDES and PARR, 1963) has now been found in no less than 12 different phenotypical modifications, with
120
TRYPANOSOMIASISSEMINAR
varying degrees of activity and stability (PARRand FITCH, 1967; DAVlDSON, 1967; CARTER, FILDES, FITCH and PARR, 1968; KIRK, personal communication; FITCH and PARR, unpublished). Some interesting examples of stability differences concern the 3 human PGD phenotypes that arise from the 2 most common alleles (PGD Aand PGDC): the AA and CC homozygous types and the heterozygous CA variety. (These types occur to the extent of about 96, 0.07 and 4% respectively in Britain; there is a higher incidence of the CA and CC types in certain other parts of the world, including Africa.) Although differences in enzymic activity between these phenotypes are slight, they show considerable differences in stability when mildly stressed (exposure to heat, urea, iodoacetate and ionic detergents). The table summarizes the consequences of exposure under standardized conditions to low concentrations of a secondary aklyl sulphate (a component of the Shell Chemicals U.K. Ltd. product T e e p o l ) : J Inactivation of human PGD by Teepol (HANSHAWand PARR,unpublished)
Phenotype
Percentage inactivation :k standard de~ciation
Range
Number of observations
AA
7±4%
2-9 %
12
CA
52 J: 8%
40-58%
12
CC
83 + 5%
78-86%
12
These results constitute an interesting model of a possible mechanism for the hypersensitivity to certain drugs, sometimes observed in some individuals but not in others (such for example as the report of APTED (1960) that nitrofurazone was sometimes effective, but in other cases toxic, when used in the treatment of trypanos0miasis). Another of the variant phenotypes for human PGD discovered in this laboratory, the "Whitechapel" variant, is characterized by an enzyme so unstable that only about 5% of the usual level of enzymic activity can be detected in unstressed red cells. Primaquine has been shown to cause erythrocytes with this deficiency to haemolyse prematurely in vivo (CARSON, 1968). Thus it would appear that PGD deficiency (as well as glucose-6phosphate dehydrogenase deficiency) causes potentially haemolytic drugs (of which nitrofurazone is one) to exert their haemolytic action. An electrophoretic investigation of the isoenzymes of PGD, phosphoglucose isomerase and phosphoglucomutase in extracts of Trypanosoma rhodesiense, T. brucei and Crithidia oncopelti showed identity of the various isoenzyme components for all three enzymes in the cases of the two trypanosome extracts, and quite different patterns in the case of the crithidia extract. This suggests that, genetically, T. rhodesiense and T. brucei are very closely related. It illustrates the sort of taxonomic problems in which the study of isoenzymes may be of value. REFERENCES
APTED, F. I. C. (1960). Trans. R. Soc. trop. Meg. Hyg., 54, 225. CARSON,P. E. (!968). In Hereditary Disorders of Erythrocyte Metabolism. Ed. by Beutler, E. New York: Grune & Stratton. p. 204. CARTER,N. D., FrLgES, R. A., FITCH, L. I. & PARR,'C. W. (I968). Acta genet. Basel, 18, 109.
TRYPANOSOMIASIS SEMINAR
121
DAVlDSON, R. G. (1967). Ann. hum. Genet., 30, 355. FILDES, R. A. & PARR, C. W. (1963). Nature, Lond., 200, 890. PARR, C. w . & FITCH, L. I. (1967). Ann. hum. Genet., 80, 339. Further studies on the mode of action of arsenicals on trypanosome pyruvate kinase
I. W. FLYNN AND I. B. R. BOWMAN Department of Biochemistry, University of Edinburgh Work reported at a previous seminar (FLYNN and BOWMAN,1968) suggested that arsenical drugs act in vitro upon the terminal glycolytic enzyme, pyruvate kinase (PK). This enzyme has a negative temperature coefficient of activity--a property of hydrophobic enzyme proteins--in contrast to the mammalian and yeast PKs which remain stable at -t-2°C. (HAECKELet al., 1968). In order to allow the purification of this trypanosome enzyme, the following substances were tested for stabilization of the enzyme: ADP and PEP as cosubstrates, Mg 2+and K + as activating ions, TEA, (NH4)2SO~, D T T , glutathione and glycerol. Maximal enzymic activity is retained only in the presence of 25% glycerol. Methods of purification by (NH~)2SO4 fractionation, DEAE-cellulose, Sephadex G.200 and Agarose gel chromatography will be described. Cysteine, glutathione or D T T stimulate the activity of the crude trypanosomal enzyme although they do not prevent the inactivation of the enzyme on storage. These data are consistent with the involvement of - - S H groups at the active centre of the enzyme. Furthermore, the inhibitions by trivalent arsenicals and Cd ~+ ions suggest that a disulphydryl grouping participates in the formation of the enzyme-substrate complex. The effects of these reagents on the mammalian enzyme are markedly different and will be discussed. It has been shown that melarsen oxide inhibition decreases the number of free, fit_ratable - - S H groups in the crude enz~/me preparation. This also may reflect the participation of enzyme - - S H groups in the inhibition by arsenical trypanocidal drugs. Attempts are now being made to confirm these observations on a purified enzyme preparation. TEA ~ Triethanolamine D T T = Dithiothreitol REFERENCES
FLYNN,I. W. & BOWMAN,I. B. R. (1968). Trans.R. Soc. trop. Med Hyg, 62, 132. HAECKEL,R. et al. (1968). Hoppe-SeyIer'sZ. physiol. Chem., 349, 699. PATHOGENICITY:
PATHOLOGY
E v i d e n c e o f c o n t i n u e d p r e s e n c e o f t r y p a n o s o m e s i n b l o o d after c h a l l e n g e w i t h
Trypanosoma cruz/ S. SEAH AND P. D. MARSDEN London School of Hygiene and Tropical Medicine
In this experiment mice were given intraperitoneally 1 x 105 T. cruzi, Peru strain, isolated from a mouse after 97 passages in mice. Tail blood was examined at regular intervals, and the blood of mice in which no parasites were found was subinoculated daily into clean mice. By these means trypanosomes were demonstrated continuously in the circulation from a few hours after challenge until death.
119
minutes. Fraction V bovine serum albumin was found to be an effective substitute for cow serum on a weight basis. This non-homogeneous fraction is under investigation. P r e s e n c e and properties of dihydrofolate reductases in T r y p a n o s o m a t i d flagellate
W. E. G U T T E R I D G E , J. J. JAFFE AND J. J. McCORMACK, JR. National Institute for Medical Research, Mill Hill, London; Nuffield Institute of Comparative Medicine, London, and Department of Pharmacology, University of Vermont, U.S.A. After the demonstrations of dihydrofolate reductases in Trypanosoma equiperdum (JAFFE and MCCORMACK, 1967) and T. cruzi (GUTTERIDGE and SENIOR, 1968), we have now surveyed 7 species, representing all four major sub-groups of the genus Trypanosoma, for the presence of the enzyme. The species studied were rat-adapted bloodstream forms of T. brucei, T. rhodesiense, T. equiperdum, T. congolense, T. vivax, T. lewisi and T. cruzi and the culture forms of T. rhodesiense and T. cruzi. Dihydrofolate reductases were detected in all 7 species. The general properties of these enzymes were similar to those isolated from other sources. Trypanosomal reductases, however, showed a pattern of sensitivity to the inhibitory action of a number of 2, 4-diaminopyrimidines and related heterocycles, which was quite distinct from the patterns shown by the enzymes from bacterial and mammalian cells (see BURCHALLand HITCHINGS, 1965). In addition, we observed that reductases from trypanosomes of African origin had very similar drug sensitivity profiles, clearly distinguishable in certain respects from those of the reductases from two species of non-African origin (T. lewisi and T. cruzi). No significant differences have yet been found in the properties of dihydrofolate reductases from bloodstream and culture forms of T. rhodesiense and T. cruzi. The properties of the dihydrofolate reductases from Crithidia fasciculata and C. oncopelti have also been investigated. In their general properties they are similar to reductases from other flagellates; their drug sensitivity profiles to diaminoheterocycles are similar to those of T. lewisi and T. cruzi. Most of the reductase activity in the two organisms occurred in high speed, centrifugal supernatant fractions of organisms disrupted in the Mickle disintegrator. Small amounts of activity have, however, been detected in particulate fractions. The properties, location and function of this additional reductase activity in these flagellates will be discussed.
REFERENCES BURCHALL,J. J. & HITCHINGS, G. H. (1965). Mol. Pharmaeol., 1, 126. GUTTERIDGE,W. E. & SENIOR, D. S. (1968). Trans. R. Soc. trop. Med. Hyg., B2, 135. JAFFE, J. J. & McCORMACK, JR., J. J. (1967). Mol. Pharmacol., 3, 359. E n z y m e variation in m a n and t r y p a n o s o m e s
C. W. PARR AND S. G. WELCH The London Hospital Medical College, London, E. 1 Recent developments in techniques by which small differences between protein and enzyme molecules may be detected (such as single amino-acid changes in the primary structures) have led to the realization that each enzyme within a species, hitherto thought to have been a single entity, may well occur in a number of variant or isoenzymic forms. Thus, the autosomally linked enzyme phosphogluconate dehydrogenase (PGD) in man, first shown in this laboratory to exist in more than one form (FILDES and PARR, 1963) has now been found in no less than 12 different phenotypical modifications, with
120
TRYPANOSOMIASISSEMINAR
varying degrees of activity and stability (PARRand FITCH, 1967; DAVlDSON, 1967; CARTER, FILDES, FITCH and PARR, 1968; KIRK, personal communication; FITCH and PARR, unpublished). Some interesting examples of stability differences concern the 3 human PGD phenotypes that arise from the 2 most common alleles (PGD Aand PGDC): the AA and CC homozygous types and the heterozygous CA variety. (These types occur to the extent of about 96, 0.07 and 4% respectively in Britain; there is a higher incidence of the CA and CC types in certain other parts of the world, including Africa.) Although differences in enzymic activity between these phenotypes are slight, they show considerable differences in stability when mildly stressed (exposure to heat, urea, iodoacetate and ionic detergents). The table summarizes the consequences of exposure under standardized conditions to low concentrations of a secondary aklyl sulphate (a component of the Shell Chemicals U.K. Ltd. product T e e p o l ) : J Inactivation of human PGD by Teepol (HANSHAWand PARR,unpublished)
Phenotype
Percentage inactivation :k standard de~ciation
Range
Number of observations
AA
7±4%
2-9 %
12
CA
52 J: 8%
40-58%
12
CC
83 + 5%
78-86%
12
These results constitute an interesting model of a possible mechanism for the hypersensitivity to certain drugs, sometimes observed in some individuals but not in others (such for example as the report of APTED (1960) that nitrofurazone was sometimes effective, but in other cases toxic, when used in the treatment of trypanos0miasis). Another of the variant phenotypes for human PGD discovered in this laboratory, the "Whitechapel" variant, is characterized by an enzyme so unstable that only about 5% of the usual level of enzymic activity can be detected in unstressed red cells. Primaquine has been shown to cause erythrocytes with this deficiency to haemolyse prematurely in vivo (CARSON, 1968). Thus it would appear that PGD deficiency (as well as glucose-6phosphate dehydrogenase deficiency) causes potentially haemolytic drugs (of which nitrofurazone is one) to exert their haemolytic action. An electrophoretic investigation of the isoenzymes of PGD, phosphoglucose isomerase and phosphoglucomutase in extracts of Trypanosoma rhodesiense, T. brucei and Crithidia oncopelti showed identity of the various isoenzyme components for all three enzymes in the cases of the two trypanosome extracts, and quite different patterns in the case of the crithidia extract. This suggests that, genetically, T. rhodesiense and T. brucei are very closely related. It illustrates the sort of taxonomic problems in which the study of isoenzymes may be of value. REFERENCES
APTED, F. I. C. (1960). Trans. R. Soc. trop. Meg. Hyg., 54, 225. CARSON,P. E. (!968). In Hereditary Disorders of Erythrocyte Metabolism. Ed. by Beutler, E. New York: Grune & Stratton. p. 204. CARTER,N. D., FrLgES, R. A., FITCH, L. I. & PARR,'C. W. (I968). Acta genet. Basel, 18, 109.
TRYPANOSOMIASIS SEMINAR
121
DAVlDSON, R. G. (1967). Ann. hum. Genet., 30, 355. FILDES, R. A. & PARR, C. W. (1963). Nature, Lond., 200, 890. PARR, C. w . & FITCH, L. I. (1967). Ann. hum. Genet., 80, 339. Further studies on the mode of action of arsenicals on trypanosome pyruvate kinase
I. W. FLYNN AND I. B. R. BOWMAN Department of Biochemistry, University of Edinburgh Work reported at a previous seminar (FLYNN and BOWMAN,1968) suggested that arsenical drugs act in vitro upon the terminal glycolytic enzyme, pyruvate kinase (PK). This enzyme has a negative temperature coefficient of activity--a property of hydrophobic enzyme proteins--in contrast to the mammalian and yeast PKs which remain stable at -t-2°C. (HAECKELet al., 1968). In order to allow the purification of this trypanosome enzyme, the following substances were tested for stabilization of the enzyme: ADP and PEP as cosubstrates, Mg 2+and K + as activating ions, TEA, (NH4)2SO~, D T T , glutathione and glycerol. Maximal enzymic activity is retained only in the presence of 25% glycerol. Methods of purification by (NH~)2SO4 fractionation, DEAE-cellulose, Sephadex G.200 and Agarose gel chromatography will be described. Cysteine, glutathione or D T T stimulate the activity of the crude trypanosomal enzyme although they do not prevent the inactivation of the enzyme on storage. These data are consistent with the involvement of - - S H groups at the active centre of the enzyme. Furthermore, the inhibitions by trivalent arsenicals and Cd ~+ ions suggest that a disulphydryl grouping participates in the formation of the enzyme-substrate complex. The effects of these reagents on the mammalian enzyme are markedly different and will be discussed. It has been shown that melarsen oxide inhibition decreases the number of free, fit_ratable - - S H groups in the crude enz~/me preparation. This also may reflect the participation of enzyme - - S H groups in the inhibition by arsenical trypanocidal drugs. Attempts are now being made to confirm these observations on a purified enzyme preparation. TEA ~ Triethanolamine D T T = Dithiothreitol REFERENCES
FLYNN,I. W. & BOWMAN,I. B. R. (1968). Trans.R. Soc. trop. Med Hyg, 62, 132. HAECKEL,R. et al. (1968). Hoppe-SeyIer'sZ. physiol. Chem., 349, 699. PATHOGENICITY:
PATHOLOGY
E v i d e n c e o f c o n t i n u e d p r e s e n c e o f t r y p a n o s o m e s i n b l o o d after c h a l l e n g e w i t h
Trypanosoma cruz/ S. SEAH AND P. D. MARSDEN London School of Hygiene and Tropical Medicine
In this experiment mice were given intraperitoneally 1 x 105 T. cruzi, Peru strain, isolated from a mouse after 97 passages in mice. Tail blood was examined at regular intervals, and the blood of mice in which no parasites were found was subinoculated daily into clean mice. By these means trypanosomes were demonstrated continuously in the circulation from a few hours after challenge until death.