Vol. 179, No. 2, 1991 September 16, 1991





M. Jenkins,

*+’ Thomas E. Gorrell *’ Miklds P.D.J. Weitzman +4

*The Rockefeller +Department


of Biochemistry,



New York, NY 10021

University of Bath, Bath BA2 7AY, U.K.

Received July 29, 1991

Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria possess distinct ‘small’ (dimeric) thiokinase enzymes which are specific for adenine (ADP) or guanine (GDP) nucleotides, whereas Gram-ne ative bacteria contain a single ‘large’ (tetrameric) enzyme which utilizes bo t% nucleotides. Succinate thiokinase activities, both ADP- and GDP-dependent, were shown to be hydrogenosomal in Tritrichatonas f&us and Trichomonusnug&zalis. Surprisingly, the ‘small’ enzyme was found in T. fietus whereas T. vaginatis contained a ‘large’ enzyme. 0 1991Academic Press. Inc. SUMMARY:

Trichomonads are anaerobic eukaryotes that depend upon the substratelevel phosphorylation reaction of succinate thiokinase to harness the energy liberated from the oxidative decarboxylation of pyruvate. These organisms lack mitochondria (STK), also known as but contain hydrogenosomes (1). Succinate thiokinase succinyl-CoA synthetase (EC and, is one of the few enzymes that carry out substrate-level phosphorylation. The enzyme catalyses the following reversible reaction:

succinyl-CoA where NDP respectively.




+ NDP + Pi x2 represent


succinate + COA + NTP diphosphate

. ‘Present address: Genetics and Biochemistry Branch, of Health, Bethesda, MD 208920. 2 Present address: EPCOInc., !Voodbury, CT 06798.



NIDDK, National


‘Present address: The Rockefeller University, New York, NY 10021. 4To whomcorrespondence should be addressed at present address: Faculty of Life Sciences, Cardiff Institute of Higher Education, Cardiff CFS ZSG, UK. Abbreviation:

STK, succinate thiokinase. OCO6-291x/91 $1.50 892

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.




2, 1991






Weitzman & Kinghorn (2) have noted a taxonomic pattern in the molecular sizes of STKs. Gram-negative bacteria possess a ‘large’ (tetrameric) enzyme of M, -150,000, whereas Gram-positive bacteria and eukaryotes (in their mitochondria) contain a ‘small’ (dimeric) enzyme of M, -75,000. Diversity in nucleotide specificity has also been reported (3-5). The utilization of adenine and guanine nucleotides occurs on a single ‘large’ STK in Gram-negative bacteria but, in both eukaryotes and Gram-positive bacteria, two ‘small’ STKs occur which appear to be specific for their particular nucleotides. Lindmark (6) detected acetate and succinate thiokinase activities in Tritrichomonas foetus, an anaerobic eukaryotic microorganism which lacks mitochondria but contains hydrogenosomes. In this organism, STK was proposed as a key step in the conservation of the energy liberated by the oxidative decarboxylation of pyruvate in the hydrogenosomes (1,6). In this paper we confirm the presence of STK in T. foetus, show that it is also present in T. vaginalis and present cell fractionation results which strongly suggest that the enzyme is an integral component of hydrogenosomes. Surprisingly, the molecular size of the enzyme was found to differ in the two trichomonad species. Preliminary results of this investigation have been reported (7). METHODS Organisms and cultivation. Tritrichomonas foetus (KV strain, e uivalent to ATCC 30924) and Trichomonas vaginafis (NIH Cl strain, A k CC 3000 I > were grown in tr ptone-yeast extract-maltose medium, harvested and washed as described earlier 037. Preparation of hvdroeenosomal extracts. Homogenization of cells sus ended in 250 n&-sucrose and the fractionation of the homogenate into nuclear, Parge particle, small particle and final supernatant fractions, as well as the sub-fractionation of the large particle fraction by isopycnic centrifu ation in a sucrose density gradient, were erformed by established procedures (8 ). For characterization of STK, the Rydrogenosome-enriched lar e particle fraction was suspr ip O.y-T+/HCl buffer, pH 7.5, containing 17g mM-KCl, 1 mM-EDTA, 1 0 p ml- leu eptme, and sonicated with a Branson sonifier equi ped with a microtip 0 W for x 15 s, with coolin in an ice-bath). The sonicate cf material was centrifuged at 100,OOOg for 60 min; ta e clear supernatant contained >90% of the T. foefus or >75% of the T. vaginalis STK activities. Characterisation of STK. The molecular size of STK was determined by gel filtration. Solubilized extract was applied to a Superose-6 column connected to a Pharmacia FPLC system. The column was calibrated with a mixture of roteins extending in M from 12,500 to 450,000. The mobile hase contained 0.1 dTris/HCl buffer, p$I 7.8, 1 mM-EDTA, 20% tv/v) glycerol, !k mM-ascorbate, applied at 0.3 ml min- . Fractions were collected and assayed for enzyme activity. Enzyme and urotein assavs. STK was assayed by an electrochemical method based on the polarographic uantitation of the free sulfhydryl grou s liberated on conversion of succinyl -z oA to CoA (9). Enzyme activities use B as markers of various subcellular components (malate dehydro enase (decarboxylating) for hydrogenosomes, acid hos hatase and PN-acetyl-g Pucosaminidase for hydrolasecontaining particles an cr NA g H oxidase for the non-sedimentable corn nent of the cytoplasm) and protein were determined by methods previously descri ic d (8,10-12). 893

Vol. 179, No. 2, 1991






Homogenates of both trichomonad species liberated free CoA from succinylCoA under standard assay conditions. Added ADP or GDP produced a significant enhancement of the rate of this process, indicating the presence of STK activity. As the enzymes were found to be localized in the hydrogenosomes (see below), their properties were studied with the use of either hydrogenosome-enriched large particle fractions or a solubilized fraction prepared from them. STK of either species showed an absolute requirement for Pi, as well as a strong preference for ADP. Km values for ADP of 48 PM and 27 PM were obtained with T. foetus and T. vaginalis respectively. Both species could also utilize GDP as a phosphoryl acceptor but with a much lower affinity (Km > 500 l&I). If the assays were performed at saturating NDP concentrations, the specific activities differed only slightly. Fast protein liquid chromatography on a calibrated Superose-6 gel filtration column gave a single peak of enzyme activity for each species (Fig. 1). The results show that the migration of the activity differs for the two organisms. The ADPdependent STK of T. f&us has an apparent M, of -75,000, whereas that of T. vaginalis is -150,000. Although the activity of the enzymes is much lower when measured with GDP, it was sufficient to establish that the GDP-dependent activity co-migrates with the ADP-dependent activity. Figure 2 shows the distribution of STK activity and marker enzymes after differential centrifugation of homogenates of T. vaginalis and after isopycnic centrifugation of the large particle fraction in a sucrose gradient. STK had a distribution pattern similar to malate dehydrogenase (decarboxylating) and different from those of the hydrolases studied and of NADH oxidase. Similar distribution patterns were obtained with T. fcetus. These results indicate a hydrogenosomal localization of the STK activity. These studies demonstrate the presence of an active STK in two trichomonad species and strongly suggest their subcellular localization in hydrogenosomes, characteristic organelles of trichomonads and several other anaerobic protists (6). The earlier demonstration that the size of STK is associated with the taxonomic position of the organism (2) predicted that determination of this property would yield molecular size values similar to the ‘small’ eukary@tic enzyme. To our surprise, the two species possessed STKs of different size. The typical ‘small eukaryotic enzyme was found in ‘I. f&us, whereas T.zxzgidis contained the ‘large’ enzyme previously found only in Gram-negative bacteria. It is pertinent to recall that, in aerobic eukaryotes, STKs are mitochondrial, whereas in trichomonads, which are anaerobic, they are hydrogenosomal. However, in view of the strict molecular size pattern exhibited by STKs throughout Nature and the similarity between the properties of the hydrogenosomes of the two species, this result is both remarkable and intriguing. However, our findings may reflect the 894



179, No. 2, 1991



4 6.0 -

3 2 I A


5.5 -

7.3 C ? \ I


4.0 I















5b Percent

of protein





1'3 density

Fi . 1. Gel filtration on Superose6 column of sonicatedextracts of T. zxzginalisand T. $iiTlarge y anule fractions. Standard roteins and M, values: A: ferritin, 450,000; B: aldolase, 58,000;C: albumin, 45,000;8: chyrnotrypsinogen, 25,000;E: cytochrome C, 12,500.

metabolic differences that are known to occur with respect to glucose fermentation (13). Subtle differences may also occur between the two species in their intrahydrogenosomal organization and metabolism which may require such a difference in molecular size. The physiological role of STK in both species is thought to be that of energy conservation. STK activity in conjunction with the enzyme succinate:acetate CoAtransferase, detected in the large granule fraction (6), can account for the liberation of acetate, a known end-product

of trichomonad metabolism (l), from acetyl-CoA

formed in the hydrogenosomal oxidative decarboxylation of pyruvate.

This process

is expected to be connected to the substrate-level phosphorylation reaction of STK, the occurrence of which has been confirmed in anaerobic (8) and respiring (14) intact hydrogenosomes. The finding of ADP- and GDP-dependent STK activities in both 7’. foefus and

T. vaginalis may be significant.

Previous studies on the ‘small’ animal STKs have indicated that not only are they absolutely specific for their respective nucleotide 895


179, No. 2, 1991




(ADP or GDP), but the different enzymes are also associated with separate metabolic roles, i.e. the citric acid cycle, ketone body activation and the biosynthesis of porphyrins (15-17). It is possible that the ‘large’ STK present in T. vaginalis can utilize both adenine and guanine nucleotides, whereas T. feetus may contain two distinct ‘small’ STK enzymes. Further work is required to test this proposal and to investigate the reason for the occurrence of an anomalous ‘large’ STK in T. vaginalis. Preliminary experiments establishin Acknowledzments: the presence and some properties of succinate thiokinase in T. vaginalis were %one on material kindl rovided by Dr. W.E. Gutterid e (Universit of Kent, U.K.). A visit b M.M. to Bat K t; niversity m 1983 and of T.M. 7 . to The Rot 1:efeller Universi in 198fJ enabled us to corn lete this collaborative effort. The assistance of Dr. J. ‘A:ckers and Miss Jackie Sou t!t erland (London School of Hygiene and Tropical Medicine, U.K.) in maintaining protozoan cultures over an extended riod was of great he1 . Conscientious assistance was provided in New York r y Mr. J. Liebelson, Ms. E. DiDomenico, Ms. D. Mulread and Mr. A. Pelaschier. Sup rt by U.S. Public Health Service ants AI 11942 and LR 07065 and by National r cience Foundation grants PCM 80 $8713 and PCM 8110606 is gratefully acknowledged. REFERENCES

:: 2 2: 7. ki lb. 11. E 14: E17:

Miiller, M. (1988) Ann. Rev. Microbial. 42,465-488. Weitzman, P.D.J., and Kinghorn, H.A. (1980) FEBS Lett. 114,225-227. Weitzman, P.D.J., and Jaskowska-Hodges, H. (1982) FEBS Lett. 143,237-240. Weitzman, P.D.J., Jenkins, T.M., Else, A.J., and Holt, R.A. (1986) FEBS Lett. 199,57-60. Jenkins, T.M., and Weitzman, P.D.J. (1987) Biochem. Sot. Trans. 15,839. Lindmark, D.G. (1976) in Biochemistry of Parasites and Host-Parasite Relationships (H. Van den Bossche, Ed.), pp. 16-21. North-Holland, Amsterdam. Miiller, M., Weitzman, P.D.J., and Gorrell, T.E. (1984) Abstracts 84th Meeting Amer. Sot. Microbial. . 153. Steinbiichel, A., and J uller, M. (1986) Mol. Biochem. Parasitol. 20,57-65. Weitzman, P.D. J., and Kinghorn, H.A. (1978) FEBS Lett. 88,255-258. Mtiller, M. (1973) J. Cell Biol. 57,453-474. Lindmark, D.G., and Muller, M. (1973) J. Biol. Chem. 248,7724-7728. Lindmark, D.G., Miiller, M., and Shio, H. (1975) J. Parasitol. 61,552-554. einbiichel, A. and Mtiller, M. (1986) Mol. B&hem. Parasitol. 20,45-55. f! erkasov, J., ?erkasovov& A., Kulda, J., and Vilhelmovl, D. (1978) J. Biol. Chem. 253,1207-1214. Jenkins, T.M., and Weitzman, P.D.J. (1986) FEBS Lett. 205,215-218. Jenkins, T.M., and Weitzman, P.D.J. (1988) FEBS I.&. 230,6-8. Jenkins, T.M., Eisenthal, R., and Weitzman, P.D. J. (1988) B&hem. Biophys. Res. Commun. 151,257-261.


Hydrogenosomal succinate thiokinase in Tritrichomonas foetus and Trichomonas vaginalis.

Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria posses...
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