Fumarate reductase and other mitochondrial activities in Trypanosoma cruzi

Molecular and Biochemical Parasitology, 19 (1986) 163-169 Elsevier

163

MBP 00662

Fumarate reductase and other mitochondrial activities in cruzi Alberto

Trypanosoma

B o v e r i s , Cecilia M. H e r t i g a n d Julio F. T u r r e n s

Instituto de Quimica y Fisicoqmmica Biol6gicas, Facultad de Farmacia y Bioquimica, Junin 956, 1113 Buenos Aires, Argentina

(Received 2 August 1985; accepted 18 December 1985)

Subcellular fractions obtained from Trypanosoma cruzi epimastigotes broken by freezing and thawing were assayed for fumarate reductase activity with reduced methyl viologen as electron donor and fumarate as electron acceptor under anaerobic conditions. Two distinct activities were detected: one in the mitochondrial membranes, 115 mU(mg protein)-1, accounting for 96% of the total and the other in the cytosol, 3 mU(mg protein)-1, accountingfor 3% of the total. The activity of membrane-bound fumarate reductase correlated statistically with either the activity or the amount of mitochondrial markers such as succinate and NADH dehydrogenases, cytochromes b + c558, cytochrome a611 and 5,7-diene sterols in the obtained subcellular fractions (580 × g, 12000 x g, and 105000 x g sediments and supernatant). Mitochondrial fumarate reductase was inhibited by succinate, malonate, cyanide, and 2-thenoyltrifluoroacetone (TITA); whereas the soluble enzyme was inhibited by succinate and not by TITA. The 12000 x g sediment (mitochondrial membranes) showed after dithionite addition, absorption maxima at 611,560 and 530 nm accounting for the presence of cytochromesb560, c558and a611. A CO-binding cytochromeo was also detected. A scheme of the T. cruzi mitochondrial respiratory chain is presented. Key words: Fumarate reductase; Mitochondria; Succinate dehydrogenase; Trypanosoma cruzi

Introduction When cells of the parasitic hemoflagellate Tryp a n o s o m a c r u z i are cultured in a glucose-containing medium they release a considerable amount of succinate into the medium, which accounts for 25-30% of glucose utilization [1]. Succinate is apparently generated from glycolytic phosphoenolpyruvate which is carboxylated to oxaloacetate, and by a reverse tricarboxylic acid cycle transformed into fumarate, which in turn is reduced to succinate [1,2]. However, no report is available either on the capacity of T. c r u z i mitochondrial succinate dehydrogenase to act as fumarate reductase or on the existence of a specific anaerobic fumarate reductase. On [he other hand, T. cruzi shows a functional tricarboxylic acid cycle and a respiratory chain, with cytochromes of the b, c and a types, which is able to catalyze oxidaAbbreviations: SHAM, salicylhydroxamic acid; TrFA, 2thenoyltrifluoroacetone.

tive phosphorylation [3,4]. In the present study we have assayed T. cruzi subcellular fractions for fumarate reductase activity and mitochondrial markers such as N A D H and succinate dehydrogenases and cytochromes.

Material and Methods T. cruzi cultures. The Tulahuen strain of T. cruzi was grown in a liquid medium consisting of 37 g 1-1 of brain-heart infusion (Difco Labs., Detroit, MI); 20 mg 1-1 hemin chlorhydrate, and 100 ml 1-1 bovine serum [5]. Cell fractionation. Subcellular fractions from T. cruzi epimastigotes were obtained after disruption of the cells suspended at 10 mg protein m1-1 in 230 mM mannitol, 70 mM sucrose, 1 mM E D T A and 10 mM Tris-HC1 (pH 7.2) by freezing (at - 1 6 ° C ) and thawing three times. After each thawing the suspension was homogenized by t h r e e or four passages through a 24-gauge hypo-

0166-6851/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

164 dermic needle attached to a syringe [6,7]. The homogenate was fractionated in a Sorvall RC-2B centrifuge at 0-2°C. A heavy fraction (containing nuclei, flagella and large cell fragments) was collected by a 10 min centrifugation at 480 x g. A fraction rich in mitochondrial membranes was isolated from the 480 x g supernatant by centrifugation at 12000 × g for 10 min. A particulate fraction was isolated from the 12 000 x g supernatant by centrifugation at 105 000 x g for 50 min. The 105 000 ×g supernatant was used as cytosolic fraction.

Fumarate reductase. Fumarate reductase activity was measured spectrophotometrically at either 550 or 500 nm as the fumarate-dependent univalent oxidation (bleaching) of the (colored) reduced methyl viologen (E550=6 mM-1 cm-1 and E500=2.2 mM-lcm -1) [8-10]. The reaction medium consisted of 1 mM methyl viologen, 15 mM fumarate, 90 mM phosphate buffer (pH 7.0). It was determined that methyl viologen concentrations larger than 0.3 mM were required for a maximal rate of reaction. The subcellular fractions (0.05-0.3 mg protein m1-1) and methyl viologen were mixed with the reaction medium in a completely filled and Parafilm- and cap-closed cuvette to avoid a large air chamber. Methyl viologen was reduced up to 90% (checked in relation to the maximal absorption change) by addition of a freshly made dithionite solution delivered with a microsyringe through the Parafilm. The anaerobic condition was expressed by the lack of methyl viologen oxidation (stability of the absorption). The reaction was started by addition of fumarate through the Parafilm and mixing by gentle inversion of the cuvette. The assay avoided the use of an oxygen-free atmosphere [10]. Surface oxidation of methyl viologen did not affect the determination over the ca. 5 min in which the reaction was followed. Activities are expressed as nmol of methyl viologen oxidized min-l(mg protein) -1. The reaction was carried out at 30°C. Enzymatic activities. NADH oxidase, NADH-fumarate reductase and NADH-cytochrome c reductase were measured spectrophotometrically at 340 nm(E = 6.2 mM-lcm -1) in a reaction medium consisting of 100 I~M NADH and 90 mM

phosphate buffer (pH 7.0). For the two latter activities, either 15 mM fumarate or 30 p,M horse heart cytochrome c were added, respectively. NADH-ferricyanide reductase was measured in the same reaction medium in the presence of 0.1-1 mM potassium ferricyanide and the reaction followed at 420 nm (E = 1 mM-lcm-1). These enzymatic activities are expressed as nmol of NADH oxidized min-l(mg protein) -1. Succinate oxidase was measured polarographically with a Clark-type oxygen electrode in a reaction medium consisting of 10 mM succinate and 90 mM phosphate buffer (pH 7.0). Succinate dehydrogenase was measured spectrophotometrically at 600 nm (E = 20.5 mM-acm -1) in the presence of 10 mM succinate, 50 ~M 2,6-dichlorophenolindophenol and 0.5 mM phenazine methosulfate in 90 mM phosphate buffer (pH 7.0). The activities were expressed as nmol succinate oxidized min-l(mg protein) -1. Cytochrome oxidase was measured at 550 nm in the presence of 50 IxM horse heart ferrocytochrome c (obtained by ascorbate reduction of the oxidized form) and 90 mM phosphate buffer (pH 7.0). All the determinations of enzyme activity were performed at 30°C.

Mitochondrial cytochromes and markers. The spectral analysis of the subcellular fractions was performed with a split-beam spectrophotometer. Cytochrome b + c558 were determined at 560-575 nm (E = 20 mM-lcm -1) and cytochrome a611 at 611-630 nm (E = 16 mM-lcm -1) after reduction by dithionite. 5,7-Diene sterols were extracted with petroleum ether and their absorption spectra recorded in the 250-320 nm range (E281_193=2.5 mM-lcm -1) [11]. Protein concentration was determined by the Folin reaction with bovine serum albumin as standard [12]. Chemicals. Cytochrome c (horse heart, type VI), 2,6-dichlorophenolindophenol, methyl viologen, phenazine methosulfate, potassium ferricyanide, sodium fumarate and sodium succinate were purchased from Sigma Chemical Co. (Saint Louis, MO). Salicylhydroxamic acid was from Aldrich Chemical Co. (Milwaukee, WI) and 2-thenoyltrifluoroacetone (TFFA) was from K & K Laboratories Inc. (Plainview, NY).

165 Results

A

B

reset '

Furnarate reductase activity. Addition of fumarate to reduced methyl viologen and mitochondrial fractions from T. cruzi produced a rapid oxida-

tion of the dye (Fig. 1A). Appropriate controls lacking either enzyme or fumarate showed that the absorption decrease was both enzyme- and fumarate-dependent. The rate of viologen oxidation at 550 nm was proportional to protein concentration and was used for the determination of fumarate reductase activity. This wavelength, although it does not provide maximal sensitivity (see methyl viologen spectrum, Fig. 1B), was preferred. It should be noted that two molecules of methyl viologen are required to reduce one molecule of fumarate. The distribution of fumarate reductase in the subcellular fractions of T. cruzi showed a maximal activity in the mitochondrion-rich fraction (Table I). Mitochondrial markers, such as succinate and N A D H dehydrogenases, cytochromes b + c558, cytochrome a611 and 5,7-diene sterols had a similar distribution (Table I). Statistical correlation between fumarate reductase and either succinate dehydrogenase (r = 0.98), or N A D H dehydrogenase (r = 0.94), or cytochrome b + c558 (r = 0.87), or cytochrome a611 ( r = 0.92), or 5,7diene sterols (r = 0.96) were found to be significant (P < 0.05). The m e m b r a n e - b o u n d fumarate reductase activity found in the 480 × g and 105 000 x g fractions were considered to be contamination of these fractions by mitochondrial membranes. However, the soluble fumarate reductase activity of the 105 000 × g supernatant could not be sim-

~

8

± 4

Z~A: 0.1 T

i

i

00

600 nrn

1 mM mehyl dithionite viologen~ ~

2min,,

Fig. 1. (A) Fumarate reductase activity of the mitochondrion-rich fraction of T. cruzi. Determination at 550 nm with 75 p,g protein m1-1. Other experimental conditions are described in Materials and Methods. The number near the trace indicates activity as nmol of methyl viologen oxidized min-l(mg protein)-1. (B) Spectrum of reduced methyl viologen. ilarly explained. The effect of succinate and T I ' F A distinguished between the two enzymatic forms. The soluble enzyme was more sensitive to succinate; a ca. 4.3 times lower succinate concentration was required to bring its activity to one half as compared with a similar effect on the membrane-bound enzyme (Fig. 2). The soluble fumarate reductase was insensitive to T I T A which inhibited 75% of the activity of the membranebound enzyme (Fig. 2). The activity of fumarate reductase of the soluble fraction was about 2-3% of that in the mitochondrion-rich fraction. Since the fractions had about the same protein content (Table I), the contribution of the soluble fumarate reductase to

TABLE I Fumarate reductase and other mitochondrial activities and markers in subcellular fractions from T. cruzia Fraction

Total protein (%)

Fumarate reductaseb

Succinate NADH Cytochromes Cytochrome dehydrogenaseb dehydrogenaseb b + c558c a611c

480 × g 22 14 4.0 52 12000 x g 37 110 15.2 165 105000× g 6 24 3.4 64 Supernatant 35 3 0.1 7 a Valuesare the average from three different experiments. b Valuesgiven are nmol substrate oxidized min-1 (mg protein)-1. c Valuesgiven are nmol (mg protein)-1.

0.03 0,17 0.08 -

0.02 0.16 0.05 -

5,7-diene sterolsc 37 97 30 6

166 [T TFA] (mM)( A,,& ) 0

0.5

.0

$

_

80~ ~

-~

~

,2.0

~o

u J ~ 60 t~ =

1.5 T .S am

t~

1.0

<~

.~

40

,0.5 E

-~ E. 20

0

0

10 20 [$UCCINATE ] (raM) ( o , e )

0 30

Fig. 2. Effect of succinate and T I ~ A on the fumarate reductase activity of the mitochondrion-rich fraction (o, A) and of the soluble fraction (o, A) isolated from T. cruzi.

the total fumarate reductase capacity of the cell was considered close to negligible and its study was not pursued any further. The effect of other inhibitors on the mitochondrial fumarate reductase activity was analyzed. Malonate had a marked inhibitory effect, being about 1.7 times more effective than succinate (Table II and Fig.2). Cyanide inhibited partially, about 50%, and salicylhydroxamic acid (SHAM) did not have any effect on the mitochondrial fumarate reductase activity (Table II). Other mitochondrial enzyme activities. The mitochondrion-rich fraction of T. cruzi showed succinate dehydrogenase and N A D H dehydrogen-

ase activities (Tables I and III) similar to those previously reported [13,14]. Activities involving a series of mitochondrial components were also determined. NADH-oxidase, NADH-fumarate reductase and NADH-cytochrome c reductase activities accounted for about 10% of the rate of NADH-ferricyanide reductase activity, implying a rate-limiting step beyond the N A D H dehydrogenase flavoprotein. On the other hand, succinate oxidase and succinate dehydrogenase activities were quantitatively similar. Cytochrome oxidase activity, assayed with horse heart ferrocytochrome c could not be detected (Table III). Mitochondrial cytochromes. The spectrum of the mitochondrion-rich fraction after addition of dithionite (Fig. 3A) showed: (a) a rather broad absorption band centered at 611 nm, (b) a slightly asymmetrical absorption band at 560 nm, and (c) a small absorption band at 530 nm. The 611 nm band could correspond to a terminal oxidase of the a type; since it appears far from the regular absorption range of these pigments, we have distinguished and termed it cytochrome a611. With regard to the position of this band, this report is at variance with Docampo et al. [4] who reported a maximal absorption of the terminal oxidase at 603 nm, but it agrees with Agosin et al. [15] who reported a 610 nm-absorbing pigment in T. cruzi membrane fractions isolated at 105 000 x g. The dithionite-reduced mitochondrion-rich fraction of T. cruzi also showed a CO-binding pigment with absorption maxima at 572, 542 and TABLE III Enzymatic activities of the mitochondrion-rich fraction of T. cruzi

TABLE II Effect of inhibitors on the mitochondrial fumarate reductase activity of T. cruzi

Enzyme activity

Inhibitor

NADH oxidase 16 -+ 2 NADH-ferricyanide reductase 180 - 8 NADH-fumarate reductase 18 -~ 3 NADH-cytochrome c reduc18 ± 2 tase Succinate oxidase 12 -+ 1 Succinate dehydrogenase 17 - 2 Fumarate reductase 94 +- 6 Cytochrome oxidase 0 -+ 0

None Malonate KCN SHAM

mM

5 10 0.3 1 2 5

Fumarate reductase [nmol methyl viologen oxidized min-l(mg protein) -1] 105 60 34 66 54 105 102

Specific activity [nmol substrate oxidized rain -1 (mg protein) -1]

Results express the mean value -+ S.E.M. of 3-5 experiments.

167

Discussion

424

560

\

611

1 54,2 572

1

l 400

I 450

I 500

I 550

,,A=O.OO,, T

l 600

I 650

nm

Fig. 3. Cytochromes of the mitochondrion-rich fraction of T. cruzi. (A) Dithionite-reduced minus oxidized difference spectrum; (B) CO + reduced (dithionite) minus reduced (dithionite) difference spectrum. 2 mm light-path cuvene, 4 mg protein m1-1 for both A and B.

418 nm and a trough at 430 nm in the differential spectrum (Fig. 3B); also in agreement with a previous report [15]. These peak positions identify a cytochrome o, present in the amount of 0.1 nmol (mg protein) -1 (E418_429 = 160 mM-lcm -1) [16,17].

Cytochromes of the endoplasmic reticulum. The fraction isolated at 105 000 x g and usually called microsomal fraction [13,15] showed absorption bands at 611 nm, 558 nm and a shoulder at 530 nm (Fig. 4). From the relative absorption at 611 and 560 nm and by comparison with the mitochondrion-rich fraction (Fig. 3A) it can be estimated that about 50% of the membranes isolated in this fraction are of mitochondrial origin. Other fractionation procedures, such as the one utilized by Frasch et al. [14], are likely to give more contamination of the 105 000 x g pellet by mitochondrial fragments. The remnant of the 560 nm absorption seems to correspond to the endoplasmic reticulum pigments: cytochromes b 5 and P-450 [13,15]. Considering the amount of protein isolated in the 105000 × g fraction, the content of endoplasmic reticulum cytochromes could be estimated in about 3% relative to 97% of the mitochondrial cytochromes for the whole cell.

The membrane-bound fumarate reductase activity of T. cruzi appears to be associated with the succinate dehydrogenase activity [18,19], implying that both are probably activities of the same enzyme. Both activities and some mitochondrial markers distribute similarly in the subcellular fractions. The ratio succinate dehydrogenase/fumarate reductase, which is about 0.28 (2 x 15.2/110; Table I), seems to indicate an important physiological role for the fumarate reductase activity in T. cruzi. The mitochondrial enzyme from aerobic sources such as mammals and plants has a high ratio of succinate dehydrogenase/fumarate reductase; for beef heart the value is about 60 [18]. In contrast, the enzyme from obligate anaerobes shows low ratios; the one from Micrococcus lactideus has a value of 0.03 [20] and the one from anaerobically grown Escherichia coli shows a value of 0.06 [21]. Facultative parasitic aerobes such as the metazoa Fasciola hepatica and Ascaris lumbricoides have values of 2.3 and 2.0 [21,22 I. In addition to its participation in energy and carbohydrate metabolism, mitochondrial fumarate reductase may be important in uracil biosynthesis for the oxidation of dihydroorotate by fumarate [23]. The soluble fumarate reductase activity (Table I and Fig. 2) could be due to the NADPH-dependent flavoprotein that gives H202 upon autoxidation [7]. A soluble fumarate reductase in anaerobically grown yeast has been described [24,25]. 421

S60 10

& A -T- 0"004

~

611

I

I

I

I

I

I

4.00

450

500

550

600

650

nm

Fig. 4. Cytochromes of the microsomal fraction of T. cruzi. Dithionite-reduced minus oxidized difference spectrum. 2 mm light-path cuvette, 8 mg protein ml -l.

168 It is worth noting that T. cruzi cells, in spite of their active oxygen uptake [10 nmol min-l(mg protein)-1; at 30°C] which is comparable to the ones of rat liver and T. brucei cells [20 and 35 nmol min-l(mg protein) -1, respectively at the same temperature, ref. 27] show features of facultative aerobes such as the low succinate dehydrogenase/fumarate reductase ratio and the presence of superoxide dismutase with absence of catalase [28]. It is apparent that a major pathway of carbohydrate metabolism in T. cruzi involves COz fixation into phosphoenolpyruvate to yield oxaloacetate [3] in the glycosomes [29,30]; this latter converted to succinate, by a partial mitochondrial tricarboxylic acid cycle functioning in reverse [30] and to fumarate by mitochondrial fumarate reductase. Oxaloacetate may inhibit succinate dehydrogenase and hence the classical operation of the tricarboxylic acid cycle. If fumarate reductase is less inhibited by oxaloacetate [24], the reverse pathway producing fumarate from phosphoenolpyruvate will be unaffected. There is evidence that when oxygen is largely available and CO z is kept low, a considerable portion of carbohydrate metabolism functions via the tricarboxylic acid cycle which feeds N A D H to a respiratory chain with functional oxidative phosphorylation [3,4,26]. However, in conditions of low oxygen and high CO 2, close to the situation in the host tissues, regulation of T. cruzi succinate dehydrogenase by oxaloacetate may be crucial to change the metabolic pathways. The mitochondrial respiratory chain of T. cruzi has been far less studied than those of T. mega and T. brucei [27], The African trypanosomes show a marked dimorphism of their respiratory mechanisms during their life cycle. The bloodstream forms degrade glucose to pyruvate, which they cannot further metabolize, and oxidize N A D H via a cyanide-insensitive a-glycerophosphate oxidase. The vector and culture forms have a cyanide- and antimycin-sensitive electron transfer system with two terminal oxidases, cytochromes aa 3 and o [27]. T. cruzi does not change its respiratory mechanisms during the life cycle; cyanide sensitivity is similar in the three main stages of differentiation: amastigotes, epimastigotes and trypomastigotes [31]. The sensitivity to

- PMS~DCPI

.

NADH~(~

~ 0l

j 0~"

°2

Antimycin

KCN

\HIO

K3(CNI6Fe

Fig. 5. The mitochondrial respiratory chain of T. cruzi. cyanide and antimycin [3,4] points to a respiratory chain with cytochromes of the b, c and a types. The scheme of Fig. 5 accounts for the present knowledge of the mitochondrial respiratory chain of T. cruzi. N A D H dehydrogenase has been documented by its NADH-ferricyanide reductase activity (Table III; ref. 14). Succinate dehydrogenase was determined with dichlorophenolindophenol and phenazine methosulfate as electron acceptors (Table I; refs. 13,14). There is no evidence of ubiquinone in T. cruzi, but the presence of ubiquinone45 in the related parasitic trypanosomatid Crithidia fasciculata [32] makes the possibility likely. Atempts to measure ubiquinone by extraction and ultraviolet adsorption have failed due to the high amounts of 5,7-diene sterols [11]. The existence of cytochrome b and c558 that overlap in the absorption band at 560 nm is well documented (Fig. 4A; refs. 4,15). Moreover, their function seems established since their redox levels are modified by respiratory inhibitors and uncouplers [4]. The terminal oxidase, with maximal absorption at 611 nm, does not oxidize horse heart ferrocytochrome c (Table III); it seems that the a-type terminal oxidases from trypanosomes are specific for ferrocytochrome c558 [27]. Cytochrome o has been identified by its absorption bands (Fig. 3B; ref. 15) but its function as terminal oxidase does not seem important since antimycin inhibits T. cruzi respiration by 97% [4].

Acknowledgments This research was supported by grants from Consejo Nacional de Investigaciones Cientificas y T6cnicas, Argentina. A.B. and J.F.T. are Career Investigators from the same Institution.

169

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18 Kimura, T., Hauber, J. and Singer, T.P. (1967) Studies on succinate dehydrogenase. 13. Reversible activation of the mammalian enzyme. J. Biol. Chem. 242, 4987--4993. 19 Singer, T.P., Kearney, E.B. and Gutman, M. (1972) Regulation of succinate dehydrogenase in mitochondria. In: Biochemical Regulatory Mechanisms in Eukariotic Cells (Kun, E. and Grisolia, S., eds.), pp. 271-301, Wiley, New York. 20 Warringa, M.G.P.J., Smith, O.H., Guiditta, A. and Singer, T.P. (1958) Studies on succinic dehydrogenase. 8. Isolation of a succine dehydrogenase fumaric reductase from an obligate anaerobe. J. Biol. Chem. 230, 97-109. 21 Lara, F.J.S. (1959) The succinic dehydrogenase. Propionibacterium pentosaceum. Biochim. Biophys. Acta 33, 565-567. 22 Barret, J. (1976) Bioenergetics in helminths. In: Biochemistry of Parasites and Host-parasite Relationships (Van den Borsche, H., ed.), pp. 67-80, Elsevier Amsterdam. 23 Andrews, S., Cox, G.B. and Gibson, F. (1977) The anaerobic oxidation of dehydroascorbate by Escherichia coli K-12. Biochim. Biophys. Acta 462, 153-160. 24 Hauber, J. and Singer, T.P. (1969) Studies on succinate dehydrogenase. 14. Intracellular distribution, catalytic properties and regulation of fumarate reductase in yeasts. Eur. J. Biochem. 3, 107-116. 25 Tisdale, H., Hauber, J., Prager, G., Turini, P. and Singer, T.P. (1968) Studies on succinate dehydrogenase. 15. Isolation, molecules properties and isoenzymes of fumarate reductase. Eur. J. Biochem. 4, 472-477. 26 Boiso, J.F. de and Stoppani, A.O.M. (1973) The mechanism of acetate and pyruvate oxidation by Trypanosoma cruzi. J. Protozool. 20, 673-678. 27 Hill, G.C. (1976) Electron transport systems in kinetoplastida. Biochim. Biophys. Acta 456, 149-193. 28 Boveris, A., Sies, H., Martino, E.E., Docampo, R., Turrens, J.F. and Stoppani, A.O.M. (1980) Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi. Biochem. J. 188,643-648. 29 Cannata, J.J.B., Valle, E., Docampo, R. and Cazzulo, J.J. (1982) Subcellular localization of phosphoenol-pyruvate carboxykinase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata. Mol. Biochem. Parasitol. 6, 151-160. 30 Cannata, J.J.B. and Cazzulo, J.J. (1984) Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi. Mol. Biochem. Parasitol. 11, 37-49. 31 Gutteridge, W.E., Cover, B. and" Gaborak, M. (1978) Isolation of blood and intracellular forms of Trypanosoma cruzi from rats and other rodents and preliminary studies of their metabolism. Parasitology 76, 159-176. 32 Kusel, J.P. and Weber, M.M. (1965) Coenzyme Qq (ubiquinone (45)) and ergosterol in Crithidia fasciculata. Biochim. Biophys. Acta 98, 632-639.