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Presence and properties of dihydrofolate reductases in Trypanosomatid flagellate
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
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