R. Seaman of Texas
It has generally been stated that the metabolism of trypanosomes does not involve the reactions of the tricarboxylic acid cycle (von Brand, 1951). Marshall (1948) and Harvey (1949) were unable to demonstrate the presence of succinic or malic dehydrogenase in ruptured cell preparations of Trypanosoma evansi or of Trypanosoma hippicum, respectively. In like manner, Moulder (1948) found that neither pyruvate, succinate, fumarate, nor cr-ketoglutrate were oxidized by Trypanosoma Eewisi.The inability of living T. lewisi to metabolize compounds of the oxidative cycle has recently been confirmed by Ryley (1951); it was found however, that homogenates of the organisms are readily capable of oxidizing these metabolites. The inability of earlier investigators to demonstrate oxidative enzymes in T. lewisi thus appears merely to be a function of the impermeability of the cell membrane. A similar situation has been found to obtain for other protozoans, particularly the free-living ciliate Tetrahymena (Seaman and Houlihan, 1950), and the acetate flagellates (Hutner and Provasoli, 1951). Such an explanation for failure to obtain oxidation of metabolites cannot, however, be applied to the data referred to above obtained with T. evansi and T. hippicum. Investigations on the metabolism of Trypanosoma cruzi have been fragmentary as to the role and/or occurrence of oxidative enzymes. This organism, however, is sensitive to fluoroacetate inhibition (von Brand, Tobie, and Mehlman, 1950), and in consideration of the apparent mode of action of this metabolic poison (Bartlett and Barron, 1947), it would be anticipated that the usual array of oxidative enzymes are present. In addition, Chang (1948) found that whereas cultures of the “oak-leaf like” crithidia forms of T. cruzi accumulated succinic, pyruvic, lactic, and formic acids, the “slender” crithidia and trypanosome forms of the organisms are apparently capable of utilizing these acids and in cultures containing these forms t,here occurs no accumulattion of acids. Raernstein 1 This invedigxtion was :ddctl t1.v :t grmt, from t)hc N:tl ima IIealth, Fctlerd Hccurit,y Agency, Bet hrstln, Maryk~ntl. 236
Inst.it utcs of
and Tobie (1951) however have indicated that the succinic and lactic dehydrogenases are absent in this organism. On the other hand, a stable aerobic malic dehydrogenase has recently been isolated from this species (Baernstein, 1952).
FIG. 1. Oxidation of succinate by extracts of Trypanosoma cruzi as measured with methylene blue as hydrogen acceptor. Incubation mixture: 3.7 X 10e4 M succinate, 4.2 X 10m6 fiil AIC13, 3 X 1OP M C&I,, 5 X 1OF M phosphate buffer, pH 7.4, 1 X IO-* M KC%, methylene blue 1:4.5 X 106. Carbon dioxide absorbed by KCN-KOH mixture (Krebs, 1935)2. Temperature, 25.O”C, pH 7.4. Curve 1, incubation mixture as above. Curve 2, blank, incubation mixture as above but without succinate.
In view of the apparent
data concerning the presence
2 Although the Ca(CN)2&a(OH)2 mixtures described by Robbie (1948) are more accurate in maintaining the cyanide concentration and should be used in studies involving cyanide inhibition, the more convenient KCN-KOH mixtures were adequate for the present investigation, in which it was necessary only t,o assure that the cytochrome oxidase system was completely inhibited.
of the tricarboxylic acid cycle in Trypanosoma cruzi, attempts were made to obtain evidence for the presence in this form of the succinic dehydrogenase system, which occupies such a central position in the oxidative metabolism of most tissues. METHODS Trypanosoma crz& was cultured in 2-liter Erlenmeyer flasks containing 800 ml of peptone-blood coagulum medium (Little and SubbaRow, 1945) ; approximately 2.5 X lo4 organisms were used as inoculum, and the cultures were maintained at 25°C until turbidity of the peptone solution was evident, at which time the population was approximately 5 X lo5 organisms/ml. The coagulum was removed by centrifugation at 70 X g for 5 min. at room temperature. Trypanosomes were concentrated from the supernatant by centrifugation at 1,609 X g for 10 min., and were washed twice with 0.9% NaCl. After the final wash the organisms were concentrated t.o as smaI1 a volume as possible and were removed to a chiIled mortar and ground with powdered quartz to a pasty consistency. The paste was immediately transferred to a centrifuge tube (a small volume of 0.1 M sodium succinate, pH 7.4 was used as washing fluid to facilitate quantitative transfer), and centrifuged at 1,600 X g for 5 min. The clear, cell free supernatant was used for enzymatic examination. All manipulations subsequent to the removal of the blood coagulum were carried out at a temperature of 4°C. Succinic dehydrogenase act,ivity was assayed both manometrically with the use of methylene blue as an hydrogen acceptor, and spectrophotometrically by following the rate of reduction of cytochrome c (Cooperstein, Lazarow, and Kurfess, 1950). Manometric measurements were carried out in a modified Gregg respirometer (Seaman, 1952). The concentrations of reactants used in the assays were based on those found to be optimal for the enzyme from the ciliated freeliving protozoan, Tetrahymena (Seaman, 1951), rather than those reported optimal for mammalian tissue. The protein content of the enzyme preparations was determined by the method outlined by Kalckar (1947) and the solution diluted to contain 10 pg protein/ml. RESULTS
The reduction of cytochrome c in the course of succinate oxidation involves one additional factor @later factor (Slater, 1949)) than does the reduction of methylene blue. This is shown in the scheme below which illustrates electron transport in the system. Succinate
Slater factor 1( Cytochrome c -+ Cytochrome
blue -+ 01
a Cultures of T. cruzi were kindly furnished by Drs. W. L. McRary and A. C. Cuckler. Organisms from both sources yielded preparations of identical activity. Data obtained from the two strains are therefore combined.
The cell free preparation of 7’. cruzi reduces cytochrome c in the presence of suecinate as a hydrogen donor (Fig. 1); the preparation utilizes oxygen when methylene blue serves as a hydrogen acceptor for the succinate oxidation (Fig. 2). The extract thus contains Slater factor as well as the dehydrogenase. Baernstein and Tobie (1951) have presented evidence which indicates
FIG. 2. Succinate dehydrogenase activity of extracts of T. cruzi as measured by reduction of cytochrome c. Incubation mixture: 3.7 X lo-* M succinate, 4.2 X 10e6 M AIC13, 3 X 10m4M C:tCl?, 1 X 1OP 111KCN, 1 X 10m6M cytochrome c., 5 X 1O-2 M phosphate buBer, pH 7.4. Total enzyme content was I rg protein. Temperat,ure, 25”C, pH 7.4. Curve 1, incubation mixture as above. Curve 2, blank, incub:ttion mixture as above I)ut) without sucrimttc. I), is the density of the solution upon complete reduciion with sodium hydrosulfite and 1)l is the reading at the indicatrtl time during i.hr course of thr rearlion. Ortlinnlc is plolletl on a logarithmic scale.
that T. cruzi contains neither cytochrome c nor cytochrome ox&se. They did observe a pigment, the properties of which indicate it to be identical with cytochrome b. These investigators also observed that added cytochrome c was readily reduced by their extract, but was not oxidized by the oxidase in the preparation. In consideration of these” data, it appears that electron transport in T. cruzi normally proceeds over cytochrome b to a carrier, other than cytochrome c, possibly a flavoprotein. Since Slater factor is present in this organism, the factor probably mediates the transfer from reduced cytochrome b to the acceptor, whatever it may be. As has been observed with preparations of Trypanosoma hippicum (Harvey, 1949), extracts of T. cruzi held in the absence of substrate for any length of time failed to show dehydrogenase activity. A similar but less pronounced necessity of early addition of succinate to the enzyme has been observed with mammalian tissue (Cooperstein, Lazarow, and Kurfess, 1950). Extracts of T. cruzi deprived of succinate for as short a time as 4 min. after preparation of the cell paste failed to show significan dehydrogenase activity, as measured either by oxygen uptake or by the reduction of cytochrome c. Thus in all cases (except in the preparation of blanks) succinate was added to the cell paste immediately after the grinding procedure. The preparations serving as blanks were made up in the same manner as the experimentals except that 0.9 % NaCl was used in transferring the paste from the mortar. SUMMARY
Cell free extracts of Tryparwsoma cruzi were prepared which show succinic dehydrogenase activity as measured by oxygen uptake in the presence of methylene blue and by the reduction of cytochrome c. REFEREXCES BARTLETT, G. R., AND BARRON, E. S. G. 1947. The effect of fluoroscetate symes and on tissue metabolism. Its use for the study of the oxidation way of pyruvate metabolism. J. Biol. Chem. 170, 67-82. BAERNSTEIN, H. D. 1952. Malate system of Trypanosoma cruzi. Federation
11, 183. BAERNSTEIN, H. D., AND TOBIE, E. J. 1951. Cytochrome system of Trypanosoma cruzi. Federation Proc. 10, 159. VON BRAND, T. 1951. Metabolism of Trypnnosomidne and Bodonidae. In Biochemistry and Physiology of Protozoa. Ed. by A. LWOFF, Academic Press Inc., New York, pp. 177-234. VON BRAND, T., TOBIE, E. J., AND MEHLMAN, R. 1950. The influence of some
mme on the oxygen consumption of some Comp. Physiol. 36, 273-300. trypanosomes. J. Cellular CHANO, S. L. 1948. Studies on hemoflagellates. IV. Observations concerning some biochemical activities in culture and respiration of three species of Leishmanias and Trl/panoscma cruzi. .I. Znfectioz~s Diseases 82, 109-118. COOPERSTEIN,~. J., L.~z~Ro~,A.,ANI~ KURFESS, N.J.l950..\microspectrophotometric method for the drtermination of succinic drhydrogennsc. J. Biol. Chem. 188, 129-139. hippiocm. 1. HARVEY, S. C. 1949. The cnrb0hydrat.e m&t.bolism of Tr~ypanosoma Biol. Chem. 179, 435-453. HUTNER, S. II., ASD PROVASOLI, I,. 1951. In Biochemistry and Physiology of Protozoa. Ed. by A. LWOFF, Academic Press Inc., New York, pp. 27-128. KALCKAR, H. M. 1947. Differential spectrophotometry of purine compounds by means of specific enzymes III. Studies on the enzymes of purine metabolism. J. Biol. Chem. 187, 461-475. KREBS, H. A. 1935. Metabolism of Amino-a,cids III. Deamination of aminoacids. Biochem.
J. 29, 1620-1644.
LITTLE, P. A., AND SUBBAROW, T. 1945. A practical liquid medium for cultivation of Trypanosoma cruzi in large volumes. J. Back 60,57-60. MARSHAI~L, P. B. 1948. The glucose metabolism of T. rounsi and the action of 3, S-14. trypanocides. Brit. J. Phurmucol. MOULDER, J. W. 1948. The oxidative metabolism of l’rypanosoma Eewisi in a phosphate-saline medium. J. Znfectiotts Diseuses, 83, 33-41. ROBBIE, W. A. 1948. Use of cyanide in tissue respiration studies. Methods in Med. Research 1, 307-316. RYLEY, J. F. 1951. Studies on the metabolism of the protozoa I. Metabolism of the parasitic flagellate, Trypanosoma lewisi. Biochem. J. 49, 577-585. SEAMAN, G. R. 1951. Enzyme systems in Tetrahymena geleii S I. Anerobic dehydro34, 775-783. genases concerned with carbohydrate oxidation. J. Gen. Physiol. SEAMAN, G. R. 1952. A modified Gregg type respirometer. Texas Repls. Biol. Med. 10, 92-95. SEA~IAW, G. R ., AND HOULIFIAN, R. I<. 1950. Trans.l,2-cyclopentanedicarboxylic acid, a succinic acid analog ‘affecting the permeability of the cell membrane. Arch.
SLATER, E. C. 1949. ,4 respirat,ory catalyst required for t,he reduction J. 46. 14-30. chrome c by cytochrome h. B&hem.