From the Department of Biochemistry and Cell Biology,
Faculty of Veterinary Medicine, Utrecht University, NL-3508 TD Utrecht,
The Netherlands, the § Molecular Cell Biology Unit,
Institute for Molecular Biology and Biochemistry, Free University
Berlin, Hindenburgdamm 27, D-12203 Berlin, Germany, the
Department Internal Medicine III, University of Heidelberg,
Bergheimer Strasse 58, D-69115 Heidelberg, Germany, and the
** Agrotechnological Research Institute (ATO-DLO),
NL-6700 AA, Wageningen, The Netherlands
Received for publication, December 26, 2002, and in revised form, January 22, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The importance of a functional Krebs
cycle for energy generation in the procyclic stage of Trypanosoma
brucei was investigated under physiological conditions during
logarithmic phase growth of a pleomorphic parasite strain. Wild type
procyclic cells and mutants with targeted deletion of the gene coding
for aconitase were derived by synchronous in vitro
differentiation from wild type and mutant
( Trypanosoma brucei, one of the causative agents of
African trypanosomiasis, is a unicellular eukaryote, which during its
life cycle alternates between the bloodstream of its mammalian host and
the blood-feeding insect vector, the tsetse fly (Glossina spp.) (1). In the mammalian bloodstream, the long slender form of
T. brucei proliferates, until at the peak of the
parasitaemia nonproliferative short stumpy form cells accumulate that
are prepared to differentiate to insect stage (procyclic form)
parasites. In vivo this takes place in the tsetse midgut
shortly after the insect blood meal. In culture this differentiation
process (also referred to as transformation) can be induced by a
temperature shift and the addition of millimolar concentrations of
citrate or cis-aconitate (2), and in fully
differentiation-competent trypanosome strains (termed pleomorphic)
transformation is very rapid and perfectly synchronous (3-5).
Differentiation of T. brucei is accompanied by several
profound structural and biochemical changes, including the glucose metabolism. The long slender bloodstream form depends entirely on
glycolysis for energy generation and excretes pyruvate as the major end
product of carbohydrate metabolism (6-8). In the procyclic stage, the
end product of glycolysis, pyruvate, is not excreted but further
metabolized inside the mitochondrion. Pyruvate is first oxidatively
decarboxylated by pyruvate dehydrogenase to acetyl-CoA, which is
supposed to be further degraded to carbon dioxide by the Krebs cycle.
This presumed flux through the Krebs cycle is, however, only poorly
supported by direct experimental data and is mainly based on the
detected presence of all the enzyme activities involved (6, 7, 9, 10).
As a result of this incomplete data set, many published metabolic
schemes of procyclic T. brucei do not represent main
catabolic fluxes but rather present a survey of all possible pathways
of carbohydrate breakdown, including a complete Krebs cycle (6, 8, 11,
12). The prevailing view is summarized in statements such as:
"procyclic forms depend primarily on Krebs cycle mediated
mitochondrial metabolism" (6, 10, 12-15). However, a large part of
the acetyl-CoA is converted to acetate inside the mitochondrion. In
trypanosomatids this acetate is formed by a mitochondrial
acetate:succinate CoA-transferase (ASCT)1 and involves a
succinate/succinyl-CoA cycle, thereby generating extra ATP (16).
Succinate is also an end product of glucose degradation and is
excreted by procyclic trypanosomes (17). Recently, it was shown that
this succinate is predominantly produced inside the glycosomes (18). In
contrast to bloodstream form T. brucei, procyclic form cells
can also use amino acids, most notably proline, for the generation
of energy (9).
To investigate the contribution of the Krebs cycle to carbon fluxes in
the energy metabolism of procyclic trypanosomes, we studied the
degradation of glucose and proline by a combined genetic and
biochemical approach that was stimulated by our analysis of a targeted
deletion of the single aconitase gene of T. brucei (19, 20).
We noticed that procyclic trypanosomes devoid of any aconitase activity
were viable and able to proliferate.
To assess the Krebs cycle contribution to energy production in the
normal procyclic form trypanosome, ideally a complete but conditional
repression of a key enzyme would be most informative. Conditional
repression of genes by RNA interference has become extremely popular
but suffers from a variable degree of repression and often leakiness,
complicating metabolic studies. Targeted gene deletion of a key enzyme,
on the other side, may lead to selection of compensatory epigenetic
changes. Here we have targeted the aconitase gene in the bloodstream
stage of the parasite where the encoded enzyme is only marginally
expressed and is dispensable for growth (19, 20). To be able to induce
rapid and synchronous differentiation to the procyclic stage, we
knocked out aconitase in a pleomorphic T. brucei strain for
which we recently developed a gene transfer and selection procedure
(21). Thus, we have constructed isogenic strains of procyclic form
trypanosomes with and without expression of aconitase activity.
Our investigations showed that deletion of the aconitase gene had no
effect on differentiation and growth in culture of procyclic T. brucei. Further analysis of the metabolism of glucose and proline by procyclic trypanosomes led us to conclude that under the conditions studied, these substrates were not degraded by Krebs cycle activity. In
addition, no metabolic or growth differences could be observed between
wild type and knockout strains, which confirmed that Krebs cycle
activity is not used for the generation of energy in procyclic T. brucei cells.
Culture and Differentiation of Trypanosomes--
The pleomorphic
strain AnTat1.1 of T. brucei brucei was used in this study
(for references see Ref. 21). Bloodstream form parasites were grown
either on agarose plates (21, 22) or in HMI9 medium supplemented with
15% heat-inactivated fetal calf serum and 1.1% methylcellulose
(Methocel®MC 3000-5500 mPa.s, Fluka) (23). The cells were counted
with a Neubauer hemocytometer (depth, 0.01 mm). In vitro
differentiation to the procyclic stage (transformation) was initiated
as described before (21). Procyclic cultures were maintained in SDM-79
supplemented with 10% (v/v) heat-inactivated fetal bovine serum (2).
Growth was monitored with a CASY TTC cell analyzer (Schärfe
System, Reutlingen, Germany).
Transfection of Bloodstream Form Trypanosomes--
For
disruption of the aconitase in the pleomorphic T. brucei
strain AnTat1.1, promoterless targeting constructs
(pB Southern and Western Blot Analyses--
Genomic DNA was isolated
from transfectants as described previously (22). Southern blot analysis
was performed following standard procedures (24). Approximately 1 µg
of SacII-digested DNA was electrophoresed on a 0.8% agarose
gel, blotted onto a nylon membrane, and hybridized in
QuickHybTM (Stratagene) for 1 h at 68 °C according
to the manufacturer's instructions with ACO-,
NEO-, and HYG-specific probes (a 2435-bp PstI-StuI fragment, a 526-bp
EagI-NcoI fragment, and a 425-bp SacII-NcoI fragment, respectively). The blots
were washed twice under high stringency conditions (0.1× SSC, 0.1%
SDS) for 30 min at 65 °C. The probes were random prime-labeled with
[ Enzymatic Assays--
Krebs cycle enzyme activities in freshly
differentiated (transformed) procyclic T. brucei have
previously been reported to change significantly with time and culture
conditions (10). Therefore, all experiments and comparisons of wild
type and
aco::NEO/aco::HYG clones reported here are based on established cultures of comparable cell density and passage number after in vitro
differentiation. Protein extracts for the determination of enzymatic
activity were essentially prepared as described by Overath et
al. (25). 1 × 109 cells were harvested, washed
twice with ice-cold phosphate-buffered saline (140 mM NaCl,
2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM,
KH2PO4, pH 7.4) or trypanosome dilution buffer
(5 mM KCl, 80 mM NaCl, 1 mM
MgSO4, 20 mM Na2HPO4, 2 mM NaH2PO4, 20 mM
glucose, pH 7.7), resuspended in 0.5 ml of 50 mM Tris/HCl,
pH 7.4, 10 mM DL-isocitrate, and cracked by
three cycles of freeze-thaw lysis. The lysate was homogenized by
several passages through a 0.6-mm syringe and then diluted 1:2 with
stock solutions of cysteine and sucrose (final concentrations, 10 and
250 mM, respectively). The extract was centrifuged
(20,000 × g for 10 min at 4 °C), and the
supernatant containing 1-5 mg/ml protein (26) was used for the
determination of enzymatic activity. Part of the extract was treated 10 min at 4 °C with Fe2+ by adding Fe2+-loading
buffer (0.5 mM MES, 0.5 mM
FeNH4SO4, 0.25 mM dithiothreitol, pH 5.5) prior to the measurement of aconitase activity. Aconitase activity was measured continuously with a spectrophotometer
thermostated at 25 °C according to the UV method (27). The reaction
buffer 50 mM
Na2HPO4/NaH2PO4 (pH
7.4) was supplemented with 30 mM DL-isocitrate as substrate. The reaction was started with protein extract, and the
increase in absorbance at 240 nm was followed for 6 min. The specific
activity was defined as the formation of nmol product min Microscale Determination of Metabolite Concentrations by
HPLC--
Established procyclic form cells in mid-log growth phase
were harvested and washed twice in trypanosome dilution buffer. The pellets were frozen in liquid nitrogen and stored at Metabolic Incubations--
Incubations (5 × 106 cells/ml) were carried out for 17 h at 27 °C in
sealed 25-ml Erlenmeyer flasks, containing 5 ml of SDM-79 incubation
medium. The incubations were performed after the addition of either 5 µCi of D-[6-14C]glucose (2.07 GBq/mmol) or
5 µCi of L-[U-14C]proline (9.47 GBq/mmol)
(both Amersham Biosciences). This implies that all of the incubations
were performed in complete SDM-79 medium containing both glucose and
proline with only one of these two radioactively labeled at a time.
Incubations were terminated by addition of 40 µl of 6 M
HCL to lower the pH to 3.5. Preceding acidification, 0.1 ml of 1 M NaHCO3 was added through the rubber stopper,
and the flasks were placed on ice. Immediately after acidification, the
incubation flasks were flushed with nitrogen for 90 min at 0 °C. In
this way all carbon dioxide is removed, whereas acetate remains in the
incubation medium. The carbon dioxide was trapped in a series of four
scintillation vials, each filled with 1 ml of 0.3 M NaOH
and 15 ml of scintillation fluid (29). The radioactivity in this
fraction was counted in Tritisol modified according to the method
described by Pande (30). After removal of carbon dioxide, the acidified
supernatant was separated from the cells by centrifugation (4 °C for
10 min at 500 × g) and neutralized by the addition of
40 µl of 6 M NaOH. Analysis of the labeled end products
occurred by anion-exchange chromatography on a Dowex 1X8, 100-200 mesh
column (Serva) (60 × 1.1 cm) in chloride form (31). The column
was eluted successively with 200 ml of 5 mM HCl, 130 ml of
0.2 M NaCl, and 130 ml of 0.5 M NaCl. The
fractions were collected and counted with 2 ml of Lumac LCS in a
scintillation counter. All of the values were corrected for blank
incubations. Labeled end products were identified by their
Rf values. The identity of the major end
products produced in the [14C]glucose incubations was
checked by 13C NMR spectroscopy. For that purpose both wild
type and
aco::NEO/aco::HYG procyclic trypanosomes were incubated in the same medium as described above, except now all of the glucose normally present in SDM-79 (10 mM) was replaced by
D-[U-13C]glucose (10 mM) (Sigma).
13C NMR spectra of the incubation media were measured using
a GARP sequence (globally optimized alternating phase rectangular
pulses) for 1H decoupling and a 2-s repetition time
(32). A total of 30,000 scans was acquired using a spectral window of
31250 Hz and 32,000 data points after a 90° pulse of 10 µs.
Chemical shifts were measured with respect to C-1 of Genetic Ablation of Krebs Cycle Activity in Procyclic T. brucei--
Our strategy to investigate the metabolic role of the
Krebs cycle in insect stage T. brucei included targeted
deletion of the single gene encoding aconitase (20), which had been
shown to be nonessential in the bloodstream stage of the parasite (19). Induced differentiation of these aconitase-deficient lines into the
procyclic stage indicated that, in contrast to our expectation, aconitase was dispensable for growth of procyclic trypanosomes (not
shown). However, inefficient differentiation of the monomorphic parental line introduced a selection step and did not provide us with
mutant procyclic long term cultures for biochemical analysis of this
phenotype. Therefore, homozygous deletion of ACO was
performed in bloodstream form T. brucei of the pleomorphic
strain AnTat1.1 that can be triggered to differentiate to the procyclic
form in vitro with high efficiency and fast kinetics.
Locus-specific targeting vector integration and deletion of both
alleles of ACO in three independent double drug-resistant
lines are documented in Fig. 1
(A and B). Cultures of
aco::NEO/aco::HYG
clones and wild type AnTat1.1 were then subjected to a standard
in vitro differentiation protocol (21). Surprisingly,
deletion of ACO did not at all change the synchrony and
kinetics of differentiation,2
providing a strong argument against selection, and did not change the
growth rate of the resulting procyclic populations (Fig.
2). Absence of aconitase in these
procyclic cultures was confirmed by Western blotting (Fig.
1C) and activity measurements (Table I). Total aconitase activity was
reproducibly below the sensitivity of the spectrophotometric assay in
whole cell extracts of several independent
aco::NEO/aco::HYG
clones (Table I and data not shown). In contrast, enzymatic assays for
activities of isocitrate dehydrogenase, glutamate dehydrogenase, and
Metabolite Concentrations in Wild Type and
aco::NEO/ aco::HYG Procyclic T. brucei--
A
microscale HPLC separation of the metabolome of wild type AnTat1.1 and
two independent
aco::NEO/aco::HYG
clones was used to simultaneously quantitate the intracellular levels
of Krebs cycle intermediates (citrate, isocitrate, End Products of Glucose Breakdown--
To investigate the role of
the Krebs cycle in the glucose metabolism of procyclic T. brucei, we performed radioactive incubations using
[6-14C]glucose. Table III
shows the end product pattern of radioactive glucose breakdown. The
result shows an almost equimolar production of acetate and succinate
for both the wild type and the
aco::NEO/aco::HYG clone. Only a very limited amount of the glucose (~1%) was broken down to labeled CO2. Because we used
[6-14C]glucose, which only results in labeled
CO2 production when pyruvate is further degraded via the
full Krebs cycle but not during the formation of acetate, this
indicates that Krebs cycle activity is negligible. Furthermore, the
very limited amount of labeled carbon dioxide detected upon
[6-14C]glucose incubations with wild type cells was equal
to the amount detected in the incubations with the aconitase knockout
cells, which cannot exhibit regular Krebs cycle activity because of the lack of aconitase. This clearly demonstrates that even this limited amount of carbon dioxide was not produced by complete Krebs cycle activity but, for instance, by a cyclic succinate production or by the
complete degradation of glucose via a cycling pentose phosphate pathway
(see "Discussion").
Although only three labeled end products are mentioned in Table III, it
should be noted that some minor amounts of other metabolites were also
detected by anion-exchange chromatography. The total radioactivity of
these (unidentified) metabolites never exceeded 10% of the total of
acetate and succinate. Therefore, they have been omitted from the table.
We used 13C NMR spectroscopy experiments to confirm the
identity of acetate and succinate as major end products of glucose
degradation in these incubations (Fig.
3). These studies using
[U-13C]glucose showed that apart from acetate and
succinate as major end products, alanine was also produced as a major
end product, because from the 13C NMR spectra it could be
estimated that the amount of alanine produced was comparable with that
of succinate (Fig. 3). The production of alanine could not be
quantified in our studies with radioactively labeled
[14C]glucose, because alanine and glucose are not
separated in the anion-exchange chromatography analysis necessary to
resolve acetate and succinate. Production of alanine from glucose by
procyclic T. brucei is in agreement with earlier studies
(6). The complex resonance signals around 35 ppm stem from the labeled
CH2 groups of succinate (Fig. 3). The observed pattern of
these peaks indicated that a large portion of the succinate was labeled
at carbon atoms 1, 2, and 3, with also a significant amount of
succinate containing 13C only in positions 2 and 3 (the
single central peak at 34.7 ppm). However, the actual pattern of peaks
can only be explained if a small percentage of succinate containing
13C at all four positions in the same molecule of succinate
is also present. Calculations showed that 60%
[1,2,3-13C]succinate, 30%
[2,3-13C]succinate, and 10%
[1,2,3,4-13C]succinate result in NMR spectra very similar
to the spectrum obtained after incubation of both wild type as well as
aconitase knockout trypanosomes with [U-13C]glucose (Fig.
3). Starting with uniformly labeled glucose, succinate labeled in
positions 1, 2, and 3 can only be explained by a pathway where
phosphoenolpyruvate (PEP), which will be uniformly labeled, is
carboxylated to form oxaloacetate by unlabeled carbon dioxide. The
resulting oxaloacetate is then converted into succinate via malate and
fumarate (Fig. 4). This pathway is known
to occur in procyclic T. brucei (7, 8, 18).
Similarly, succinate labeled only in the CH2 groups,
[2,3-13C]succinate, can be explained by formation via the
above described pathway, whereby after the carboxylation of PEP by
unlabeled carbon dioxide, the formed oxaloacetate is equilibrated via
fumarate and again decarboxylated to PEP. Because of the equilibration via the symmetrical fumarate molecule, now half of this PEP will be
labeled only in positions 2 and 3. When this PEP is then carboxylated by unlabeled carbon dioxide via the above described pathway, succinate labeled only in positions 2 and 3 will be formed (Fig. 4). The presence
of [1,2,3,4-13C]succinate can only be explained by
incorporation of 13C-labeled carbon dioxide in PEP, which
is then converted into succinate by the above described pathway (Fig.
4).
It should be noted that the observed label pattern of
succinate (mainly 1,2,3- and 2,3-labeled succinate) cannot be explained via formation of succinate by Krebs cycle activity, neither via incorporation of labeled acetyl-CoA stemming from the labeled glucose
nor via the use of labeled oxaloacetate as starting compound for a
round of Krebs cycle ending in succinate. Furthermore, the observation
that the labeling pattern of succinate formed in the incubations of
wild type and aconitase knockout procyclic T. brucei was
identical excludes the involvement of a functional Krebs cycle because
the latter strain lacks aconitase.
Radioactive End Products of [U-14C]Proline
Breakdown--
Because it has been reported that procyclic T. brucei use proline as a major energy source (9), the breakdown of
proline was also investigated and compared with that of glucose.
Incubations were performed in the same complete SDM-79 medium used for
the [14C]glucose incubations, except that a tracer amount
of 14C-labeled proline was added instead of a tracer amount
of labeled glucose. Analysis of the radioactive end products in the
presence of labeled proline revealed that the major end product of
proline degradation was succinate, with the concomitant release of an equimolar amount of carbon dioxide (Table
IV). No differences in end products could
be detected between the wild type and aconitase knockout strain.
Role of Electron Transport Chain--
To investigate the relative
importance of the two branches of the electron transport chain, we
incubated both strains in the presence of KCN (inhibitor of the
mammalian type respiratory chain) or SHAM (inhibitor of the plant-like
alternative oxidase). The addition of SHAM or KCN individually resulted
in an increase of the doubling time of both strains (Fig.
5). Inhibition of the mammalian type
respiratory chain apparently resulted in a stronger growth reduction
than inhibition of the alternative oxidase. Again no differences could
be detected between the wild type and aconitase knockout procyclic
cells (Fig. 5).
The simultaneous presence of KCN and SHAM resulted within 1 h in
cell death of both the wild type and aconitase knockout procyclic trypanosomes (not shown). In addition, the complete removal of oxygen
from the incubation, (using ascorbate and ascorbate oxidase as was
described previously (31)), also resulted in rapid death of the
organisms (not shown). Apparently, procyclic T. brucei cells
are completely dependent on the use of oxygen by the respiratory chain
for their energy generation and survival.
In this paper we investigated the importance of the Krebs cycle
for energy generation in the procyclic stage of T. brucei. For these studies we compared a wild type pleomorphic strain and several independently derived mutant clones in which the single copy
aconitase gene was deleted by gene targeting. No aconitase activity
could be detected in the
aco::NEO/aco::HYG
clones, confirming that T. brucei does not express
significant amounts of any other enzyme able to perform the aconitase
reaction. On the other hand, as expected, no differences in the
activities of isocitrate dehydrogenase, glutamate dehydrogenase, and
In an independent biochemical approach, the unimportance of the Krebs
cycle for energy generation in procyclic T. brucei was confirmed by our studies using labeled substrates. To investigate whether the absence of aconitase activity results in altered pathways of energy generation, we performed radioactive incubations using [6-14C]glucose. If the degradation of acetyl-CoA inside
the mitochondrion occurred via multiple pathways, i.e. via
acetate formation caused by ASCT activity, as well as via oxidation in
the Krebs cycle, one would expect the acetate production to increase
when Krebs cycle activity was blocked by the absence of aconitase.
However, the knockout cells did not show an increased production of
acetate compared with the wild type control. Both wild type and
knockout procyclic T. brucei metabolize
[6-14C]glucose to acetate and succinate as major end
products. No significant amount of 14C-labeled
CO2 could be detected, indicating that a normally
functioning Krebs cycle is not involved in energy production in wild
type as well as in knockout cells. These results thus demonstrated the
absence of oxidation of glucose via a complete Krebs cycle in procyclic
T. brucei. NMR studies with [U-13C]glucose
were performed to confirm the identities of acetate and succinate
produced during glucose breakdown.
The pathway of succinate production by procyclic T. brucei
is not yet completely resolved. Earlier reports suggested a
mitochondrial origin of this succinate production involving the
activity of a soluble fumarate reductase (35). Recently, however,
Besteiro et al. (18) showed that succinate production occurs
mainly inside the glycosomes by a soluble glycosomal NADH:fumarate
reductase, and our analysis of the position of the 13C
labels in succinate can provide information on the route by which this
succinate was formed. The NMR spectra demonstrated that PEP
carboxykinase was involved in the production of succinate; PEP is
carboxylated to oxaloacetate, which is subsequently reduced via malate
to succinate. Close inspection of the resonance pattern combined with
computer simulations showed that this succinate was produced by three
variations on this pathway (Fig. 4); directly ([1,2,3-13C]succinate), via cycling of the formed
fumarate ([2,3-13C]succinate), or including the
incorporation of 13C-labeled carbon dioxide
([1,2,3,4-13C]succinate). This last variation seemed
enigmatic, because the source of the 13C-labeled carbon
dioxide was unknown, whereas unlabeled carbon dioxide is abundantly
present in the incubation medium. It is known, however, that a
significant part of the glucose degradation inside the glycosomes
occurs via the pentose phosphate pathway, which results in the
formation of (labeled) carbon dioxide (36), and it was also shown
recently that the major part of succinate production from glucose
occurs inside the glycosome (18). The subcellular
co-compartmentalization of these two pathways could explain the
observed incorporation of labeled carbon dioxide in a fraction (10%)
of the succinate formed. It should be noted that these variations of
succinate production exclude the involvement of Krebs cycle activity in
the forward direction but are in other aspects in full agreement with
the pathway of succinate production suggested by Besteiro et
al. (18).
Earlier studies that have characterized the amino acid uptake from the
environment by the insect stage of T. brucei show that L-proline is a main source of energy and carbon (9, 37). In
our incubations performed to study [14C]proline
breakdown, labeled succinate was found to be a major end product. This
was accompanied by the expected production of 1 mol of labeled
CO2/mol of labeled succinate formed during the conversion
of We showed that procyclic T. brucei, either wild type or
knockout mutants without aconitase activity, are equally unable to survive under anaerobic conditions. Furthermore, simultaneous inhibition of both the mammalian type respiratory chain and the plant-like alternative oxidase resulted in cell death within 1 h.
On the other hand, survival and even growth continued when only one of
the two terminal oxidases was inhibited. A stronger effect on growth
(likely to be due to inhibition of energy production) was found upon
KCN inhibition of the mammalian type respiratory chain as compared with
SHAM inhibition of the plant-like alternative oxidase. Again, no
differences could be detected between the wild type and knockout
procyclic T. brucei. These results confirm that both
branches of the respiratory chain simultaneously play a role in the
normal functioning of procyclic cells (38), but apparently, the
activity of only one of the two branches of the respiratory chain is
sufficient for survival and reduced growth.
The role of aconitase is still unresolved, but no matter what role
aconitase plays in procyclic trypanosomes, it is clearly not in the
generation of energy via the Krebs cycle. The end products of the
incubations of the wild type strain with labeled glucose and proline
demonstrated already that Krebs cycle activity does not play a
significant role in the degradation of these substrates. The fact that
no metabolic differences could be detected between procyclic wild type
and procyclic aconitase knockout clones is solid confirmation of the
unimportance of Krebs cycle activity for energy generation in the
procyclic life cycle stage. On the other hand, the metabolism of
procyclic T. brucei is not purely fermentative, because
oxygen is required as a final electron acceptor for survival and
growth. The earlier described absence of a growth phenotype upon RNA
interference-mediated ablation of succinate dehydrogenase or
Mitochondria of African trypanosomes are unique in many aspects
including the structure of the mitochondrial genome and the extensive
RNA editing during mitochondrial gene expression, the fact that each
cell contains a single mitochondrion whose division is closely linked
to the cell cycle, and the developmental regulation of their
morphological structure and function in the life cycle. From a
bioenergetical point of view, it is surprising that mitochondria of
procyclic form T. brucei ferment substrates to mainly
acetate and succinate, whereas they contain the entire set of Krebs
cycle enzymes and a functional respiratory chain including an
alternative oxidase, which is otherwise only present in plants and
fungi. The presence of a complete aerobic machinery together with the incomplete oxidation of substrates places these mitochondria in a
special category in between classical aerobic mitochondria and the
fully anaerobically functioning mitochondria of, for instance, adult
parasitic helminths (39). This is also reflected by the presence of
ASCT, an enzyme absent in classical type aerobic mitochondria.
Although our results demonstrate that apparently an intact Krebs cycle
is not used for complete oxidation of glucose or proline, it should be
realized that part of the Krebs cycle is used for the degradation of
proline to succinate. The enzyme succinyl-CoA synthetase of this part
of the Krebs cycle is also functioning in the ASCT cycle (16) and has
been shown to be essential for substrate phosphorylation in isolated
mitochondrial vesicles and for growth of procyclic trypanosomes (13).
It should also be noted that the Krebs cycle (or at least the first two
reactions) is probably active in the wild type strain, because citrate
levels were dramatically increased in the aconitase knockout strain. At
the moment, the possible function of aconitase and other Krebs cycle
enzymes in procyclic trypanosomes is unknown, but no matter what the
role is, it is clearly not related to the generation of energy in the
procyclic stage. The most appealing interpretation is that the Krebs
cycle plays a role in energy generation in other stages of the life
cycle of the parasite in the tsetse fly (e.g. in the
salivary gland) or under other environmental conditions, for instance
when substrate availability is limited.
Even though clearly not all of the details are known yet, a combination
of older and recently published studies (13, 18) with our experiments,
in which we studied for the first time the glucose and proline
metabolism of intact procyclic T. brucei of a pleomorphic
strain under growing conditions, results in a model of the main
pathways of glucose degradation as shown in Fig.
6. This metabolic scheme shows the
fermentation pathways coupled to oxidative phosphorylation in these
unique mitochondria of procyclic T. brucei and accounts for
the degradation of glucose to acetate, succinate, and alanine, the
degradation of proline to succinate via part of the Krebs cycle, and
the essential role of the branched respiratory chain in the oxidation
of NADH formed in these fermentation pathways.
aco::NEO/
aco::HYG)
bloodstream stage parasites, respectively, where aconitase is not
expressed and is dispensable. No differences in intracellular levels of
glycolytic and Krebs cycle intermediates were found in procyclic wild
type and mutant cells, except for citrate that accumulated up to
90-fold in the mutants, confirming the absence of aconitase activity.
Surprisingly, deletion of aconitase did not change differentiation nor
the growth rate or the intracellular ATP/ADP ratio in those cells.
Metabolic studies using radioactively labeled substrates and NMR
analysis demonstrated that glucose and proline were not degraded via
the Krebs cycle to CO2. Instead, glucose was degraded to
acetate, succinate, and alanine, whereas proline was degraded to
succinate. Importantly, there was absolutely no difference in the
metabolic products released by wild type and aconitase knockout
parasites, and both were for survival strictly dependent on respiration
via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not
use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
aconeo and pB
acohyg) described in detail
by Fast et al. (19) were used. Trypanosomes were isolated
from the blood of irradiated Wistar rats 2.5 days after infection at
2-5 × 107 cells/ml of blood. Electroporation and
selection was done as described by Vassella et al. (23).
Briefly, 2 × 107 buffy coat trypanosomes were
transfected with 10 µg of SalI-MluI-linearized target construct using a BTX Electro Cell Manipulator set at 1.2 kV, 25 microfarads, and 186
. After the electroporation cells were
transferred to 10 ml of prewarmed HMI9 medium and incubated for 1 h at 37 °C. The cultures were spun down, and 8 ml of the supernatant
were discarded. The cell pellets were resuspended in the remaining 2 ml
of HMI9 and transferred to 50 ml of prewarmed methylcellulose medium
and incubated for 18 h at 37 °C. The culture volume was
adjusted to 200 ml of the same medium, and transfectants were selected
with 0.8 µg ml
1 G418 or a combination of 0.8 µg
ml
1 G418 and 0.8 µg ml
1 hygromycin B. During selection the cell density was strictly kept below 5 × 105 cells ml
1. Double-resistant clonal cell
lines were generated by limiting dilution.
-32P]dCTP using the PrimeIT random labeling kit
(Stratagene). Lysates for Western blot analysis were prepared,
subjected to SDS-PAGE on a 10% gel, and blotted onto a polyvinylidene
difluoride membrane following standard procedures (24). The blot was
probed with an affinity-purified anti-TbACO rabbit serum
(20) diluted 1:1000 in Tris-buffered saline plus Tween 20 and 5% dried
milk powder. The blot was reprobed with an antibody against the
mitochondrial protein Hsp60 from Synechococcus sp.
(StressGen) diluted 1:2000 in Tris-buffered saline plus Tween 20 and
5% dried milk powder. The signals were visualized by ECL (Amersham
Biosciences).
1 mg
1 protein using an absorption
coefficient of 3.4 mM
1 cm
1
(27).
-Ketoglutarate dehydrogenase activity was determined by
monitoring the initial rate of NAD+ reduction at 340 nm.
The reaction mixture contained 150 mM Tris/HCl, pH 7.4, 3 mM cysteine, 0.2 mM coenzyme A, 10 mM
-ketoglutarate, 4 mM NAD+,
and protein extract. The reaction was started by adding the protein
extract and NAD+ simultaneously. Isocitrate dehydrogenase
activity was determined by monitoring the rate of NADP+
reduction at 340 nm. The reaction mixture contained 50 mM
Tris/HCl, pH 7.4, 1.4 mM MnSO4, 5 mM DL-isocitrate, 0.5 mM
NADP+, and protein extract. The reaction was started by
adding protein extract and NADP+ simultaneously. Glutamate
dehydrogenase activity was measured by monitoring the initial rate of
NADH oxidation at 340 nm. The reaction mixture contained 50 mM Tris/HCl, pH 7.4, 50 mM NH4Cl, 10 mM
-ketoglutarate, 0.2 mM NADH, and
protein extract. The reaction was started by adding the protein extract
and NADH simultaneously. Nonspecific NADH oxidase activity was not
detected in the same assay and time period without NH4Cl
and
-ketoglutarate, and hence correction was not required.
80 °C.
Metabolite contents were determined using a HPLC method employing
anion-exchange columns (Dionex AS-11 and AG-11) and dual (conductivity
and UV) detection (28). Briefly, the trypanosomes were homogenized in 0.5 ml of 60% acetonitril (v/v) using a Braun Dismembrator (B. Braun,
Melsungen, Germany) cooled with liquid nitrogen. After thawing, the
homogenate was centrifuged twice for 10 min at 10,000 × g in a cooled Eppendorf centrifuge at 0 °C. From the
supernatant, 30 µl were lyophilized using a vacuum centrifuge and
resuspended in 300 ml of ice-cold water with a specific resistance of
18 m
or greater. After filtration through 0.22-µm syringe filters
(Gelman Acrodisc), the analyte was injected into the HPLC system after appropriate dilution. The metabolite contents were normalized to total
cellular protein content that was assessed from the redissolved pellet
(in 90 °C NaOH) according to standard methods (26).
-glucose at
96.6 ppm relative to tetramethylsilane at 0 ppm. 13C NMR
spectra of succinate were also simulated, using the computer program
NMRSIM of the Aurelia software package of Bruker. Glucose was assayed
enzymatically using a standard procedure (33), and the protein level
was determined with the Lowry method, using bovine serum albumin
obtained from Roche Molecular Biochemicals, defatted with active
carbon, and dialyzed before use, as standard (34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ketoglutarate dehydrogenase did not reveal any significant
difference between wild type procyclic trypanosomes and two independent
aco::NEO/aco::HYG
clones (Table I).
View larger version (30K):
[in a new window]
Fig. 1.
Gene disruption of TbACO in
the pleomorphic T. brucei strain AnTat1.1.
A, schematic drawing of the ACO locus before and
after the integration of the NEO and HYG
replacement cassettes, respectively. The position of the
SacII (S) restriction sites, the genomic fragment
sizes relevant for Southern blot analysis, and the respective probe
fragments are indicated. ACO coding sequence present in the
two targeting constructs is highlighted in gray.
B, Southern blot analysis of AnTat1.1 wild type
(wt) and double (G418/hygromycin) drug-resistant
(aco::NEO/aco::HYG)
recombinant lines. Lane 1, clone 121; lane 2,
clone 311; lane 3, clone 313. SacII-digested
genomic DNA blots (1 µg/lane) were hybridized with ACO-,
NEO-, and HYG-specific probes. The size marker is
in kilobase pairs. C, Western blot analysis of AnTat1.1 wild
type (lane wt) and
aco::NEO/aco::HYG
clones 1-3. Procyclic whole cell lysates (2 × 106
cell equivalent/lane) were separated on a 10% SDS-polyacrylamide gel
and probed with an affinity-purified antibody against TbACO
(20). As an internal control the filters were reprobed with an
antibody (purchased from StressGen) detecting mitochondrial HSP60
protein. The molecular mass of the single band is indicated in
kDa.
View larger version (16K):
[in a new window]
Fig. 2.
Growth of procyclic AnTat1.1 clones devoid of
aconitase activity. The cells were counted every 24 h using a
CASY (Schärfe System) cell analyzer and then diluted to 2 × 106 cells/ml to maintain cultures in the log phase of
growth and counted again. The values represent the means of two
measurements. Circle, wild type AnTat1.1, 11.6-h population
doubling time; rectangle,
aco::NEO/aco::HYG
clone 121, 11.1-h population doubling time;
triangle,
aco::NEO/aco::HYG
clone 311, 12.9-h population doubling time.
Enzyme activities in procyclic T. brucei
, not determined. NAD+-dependent
isocitrate dehydrogenase activity was not detectable in the same
extracts.
-ketoglutarate,
succinate, fumarate, and malate), of glycolytic intermediates (glucose
1-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose
1,6-bisphosphate, fructose 2,6-bisphosphate, glyceraldehyde
3-phosphate, 2,3 bisphosphoglycerate, 2,3-phosphoglycerate, and
phosphoenolpyruvate), and of ATP, ADP, AMP, and Pi (Table
II). The citrate concentration was
greatly increased (60-90-fold) in the aconitase knockout clones (Table II), confirming a complete block of the aconitase reaction. In contrast, the concentrations of all other Krebs cycle metabolites and
glycolytic intermediates were not significantly different in wild type
procyclic cells and
aco::NEO/aco::HYG
clones.
Intracellular metabolite concentrations in procyclic T. brucei
Radioactive end products of [6-14C]glucose breakdown by
procyclic T. brucei
View larger version (10K):
[in a new window]
Fig. 3.
13C NMR spectra.
A, 13C NMR spectrum of excreted end products of
[U-13C]glucose metabolism by procyclic T. brucei. Wild type and aconitase knockout organisms were incubated
for 17 or 72 h, after which the incubation medium was analyzed by
13C NMR. All of the incubations resulted in identical
patterns of the spectra. The spectrum of the 72-h incubation of the
aco::NEO/aco::HYG
clone 121 is shown. The asterisks indicate peaks that are
also present in the blank incubations. B, close up of
A (34-36 ppm). C, calculated spectrum of
succinate (mixture of 60% [1,2,3-13C]succinate, 30%
[2,3-13C]succinate, and 10%
[1,2,3,4-13C]succinate).
View larger version (21K):
[in a new window]
Fig. 4.
Pathways of labeled succinate formation
from [U-13C]glucose by procyclic T. brucei. The asterisks indicate
13C atoms.
Radioactive end products of [U-14C]proline breakdown by
procyclic T. brucei
View larger version (13K):
[in a new window]
Fig. 5.
Growth curves of established procyclic
trypanosomes in the presence of KCN (1 mM) or SHAM (0.5 mM). Growth was started at a concentration of 2 × 106 cells/ml in a total volume of 5 ml of SDM-79. 50 µl of 100 mM KCN or 50 µl of 50 mM SHAM
were added separately to incubations of both cultures. Growth was
monitored during 24 h. The cell numbers were determined by
counting with a Bürker counter. The values represent the means of
three independent incubations. WT, wild type; KO,
knockout.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ketoglutarate dehydrogenase were found between wild type and
aconitase knockout trypanosomes. In contrast to our previous study on
iron uptake regulation (19), here we constructed the aconitase deletion
in a fully differentiation competent, tsetse fly passagable strain
background (AnTat 1.1) to exclude any bias of long term culture
adaptation of the procyclic form parasites. Bloodstream form parasites
devoid of aconitase were fully viable as noticed before (19) and were
efficiently transformed into the procyclic stage with normal kinetics.
The growth rate of the resulting wild type and
aco::NEO/aco::HYG
mutant procyclic T. brucei was, very surprisingly, identical
(Figs. 2 and 5). Apparently, the energy metabolism of the
aconitase-deficient procyclic T. brucei is not notably
disturbed, which is particularly obvious from the perfectly normal
ATP/ADP ratio (Table II). No significant differences were detected in
intracellular levels of glycolytic- and Krebs cycle intermediates
between both lines, except for citrate levels, which were up to 90-fold
increased in the knockout clones, because of a lack of aconitase
activity. It is unknown whether citrate accumulated in the cytosol or
inside the mitochondrion, because aconitase is located in both cellular compartments in T. brucei (20).
-ketoglutarate to succinyl-CoA. Again, no differences could be
detected between the wild type and knockout cells. This result also
demonstrated that proline is only degraded to succinate and not further
degraded via the Krebs cycle because this would have resulted in a
higher CO2/succinate ratio. The incubations in which we
studied the glucose and proline degradation were performed in both
cases in the same complete medium (SDM-79), the only difference being
the tracer amount of 14C label added. This means that we
studied for both substrates their degradation in the presence of the
other substrate and that the observed rates of degradation and patterns
of end products of glucose and proline occurred at the same time.
Comparison of the total amount of end products produced from each
substrate in complete medium (SDM-79) showed that in the simultaneous
presence of glucose and proline, glucose is the preferred substrate.
Under these conditions, the rate of glucose degradation was around
three times as high as that of proline. It should be realized, however, that proline could well be the preferred substrate in vivo
inside the insect gut where glucose concentrations are probably much lower.
-ketoglutarate dehydrogenase in procyclic T. brucei (13)
is in agreement with our observation that the Krebs cycle is not
involved in energy generation in this developmental stage. The observed
differences in the kinetics of induction of Krebs cycle enzymes during
bloodstream to procyclic differentiation (Ref. 10 and references there
in) are also compatible with a nonessential role of the Krebs cycle in
the procyclic stage.
View larger version (36K):
[in a new window]
Fig. 6.
The energy metabolism of procyclic T. brucei. Shown are the main pathways used by procyclic
T. brucei for the degradation of glucose to the main end
products, acetate, succinate, and alanine, for the degradation of
proline to succinate via part of the Krebs cycle, and the essential
role of the branched respiratory chain in the oxidation of NADH formed
in these fermentation pathways. Degradation of glucose by the pentose
phosphate pathway is not included in this scheme. Furthermore, it
should be realized that, next to the transaminase reaction shown, the
conversion of glutamate into -ketoglutarate can also be catalyzed by
glutamate dehydrogenase. AA, amino acid; AcCoA,
acetyl-CoA; AO, alternative oxidase; 1,3BPGA,
1,3-bisphosphoglycerate; c, cytochrome c;
Citr, citrate; DHAP, dihydroxyacetone phosphate;
FBP, fructose 1,6-bisphosphate; Fum, fumarate;
GAP, glyceraldehyde 3-phosphate; Glu, glutamate;
G-3-P, glycerol 3-phosphate;
KG,
-ketoglutarate; Mal, malate; OA, 2-oxoacid;
Oxac, oxaloacetate; 3-PGA, 3-phosphoglycerate;
Pyr, pyruvate; Succ, succinate;
Succ-CoA, succinyl-CoA; UQ, ubiquinone.
![]() |
ACKNOWLEDGEMENT |
---|
Cordula Ackermann is gratefully thanked for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by funds from the Earth and Life Science Foundation (ALW) and the Netherlands Organisation of Scientific Research (NWO) (to A. G. M. T.) and by Bundesministerium für Bildung, Wissenschaft und Forschung Grant 0311092 and Fonds der Chemi-schen Industrie (to M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Bayer AG, PH-R AI I, D-42096 Wuppertal, Germany.
Present address: Aventis Pharma Deutschland GmbH, Disease Group
Osteoarthritis, D-65926 Frankfurt, Germany.
§§ To whom correspondence may be addressed: Dept. of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80176, NL-3508 TD Utrecht, The Netherlands. Tel.: 31-30-2535380; Fax: 31-30-2535492; E-mail: tielens@biochem.vet.uu.nl.
¶¶ To whom correspondence may be addressed: Dept. Biology I, Genetics, University of Munich, Maria-Ward-Strasse 1a, D-80638 München, Germany. Tel.: 49-89-2180-6155; Fax: 49-89-2180-63853; E-mail: boshart@lmu.de.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M213190200
2 B. Fast and M. Boshart, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ASCT, acetaat:succinaat CoA-transferase; PEP, phosphoenolpyruvate; SHAM, salicylhydroxamic acid; MES, 4-morpholinoethanesulfonic acid; HPLC, high performance liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Vickerman, K. (1985) Br. Med. Bull. 41, 105-114[Medline] [Order article via Infotrieve] |
2. | Brun, R., and Schönenberger, M. (1981) Z. Parasitenk. 66, 17-24[Medline] [Order article via Infotrieve] |
3. | Ziegelbauer, K., Quinten, M., Schwarz, H., Pearson, T. W., and Overath, P. (1990) Eur. J. Biochem. 192, 373-378[Abstract] |
4. | Rolin, S., Paindavoine, P., Hanocq-Quertier, J., Hanocq, F., Claes, Y., Le Ray, D., Overath, P., and Pays, E. (1993) Mol. Biochem. Parasitol. 61, 115-125[CrossRef][Medline] [Order article via Infotrieve] |
5. | Matthews, K. R., and Gull, K. (1994) J. Cell Biol. 125, 1147-1156[Abstract] |
6. | Fairlamb, A. H., and Opperdoes, F. R. (1986) in Carbohydrate Metabolism in Cultured Cells (Morgan, M. J., ed) , pp. 183-224, Plenum Publishing Corp., New York |
7. | Opperdoes, F. R. (1987) Annu. Rev. Microbiol. 41, 127-151[CrossRef][Medline] [Order article via Infotrieve] |
8. | Clayton, C., and Michels, P. (1996) Parasitol. Today 12, 465-471[CrossRef] |
9. | Evans, D. E., and Brown, R. C. (1972) J. Protozool. 19, 686-690[Medline] [Order article via Infotrieve] |
10. | Durieux, P. O., Schütz, P., Brun, R., and Köhler, P. (1991) Mol. Biochem. Parasitol. 45, 19-28[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Cazzulo, J. J.
(1992)
FASEB J.
6,
3153-3161 |
12. | Tielens, A. G. M., and Van Hellemond, J. J. (1998) Parasitol. Today 14, 265-271[CrossRef] |
13. |
Bochud-Allemann, A.,
and Schneider, A.
(2002)
J. Biol. Chem.
277,
32849-32854 |
14. | Priest, J. W., and Hajduk, S. L. (1994) J. Bioenerg. Biomembr. 26, 179-191[Medline] [Order article via Infotrieve] |
15. | Mutomba, M. C., and Wang, C. C. (1998) Mol. Biochem. Parasitol. 93, 11-22[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Van Hellemond, J. J.,
Opperdoes, F. R.,
and Tielens, A. G. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3036-3041 |
17. | Cross, G. A., Klein, R. A., and Linstead, D. J. (1975) Parasitology 71, 311-326[Medline] [Order article via Infotrieve] |
18. |
Besteiro, S.,
Biran, M.,
Biteau, N.,
Coustou, V.,
Baltz, T.,
Canioni, P.,
and Bringaud, F.
(2002)
J. Biol. Chem.
277,
38001-38012 |
19. | Fast, B., Kremp, K., Boshart, M., and Steverding, D. (1999) Biochem. J. 342, 691-696[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Saas, J.,
Ziegelbauer, K.,
Von Haesler, A.,
Fast, B.,
and Boshart, M.
(2000)
J. Biol. Chem.
275,
2745-2755 |
21. | Vassella, E., and Boshart, M. (1996) Mol. Biochem. Parasitol. 82, 91-105[CrossRef][Medline] [Order article via Infotrieve] |
22. | Carruthers, V. B., van der Ploeg, L. H. T., and Cross, G. A. M. (1993) Nucleic Acids Res. 21, 2537-2538[Medline] [Order article via Infotrieve] |
23. | Vassella, E., Kramer, R., Turner, C. M., Wankell, M., Modes, C., van den Bogaard, M., and Boshart, M. (2001) Mol. Microbiol. 41, 33-46[CrossRef][Medline] [Order article via Infotrieve] |
24. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987) in Current Protocols in Molecular Biology (Janssen, K., ed) , John Wiley & Sons, Inc., New York |
25. | Overath, P., Chichos, J., and Haas, C. (1986) Eur. J. Biochem. 160, 175-182[Abstract] |
26. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Henson, C. P.,
and Cleland, W. W.
(1967)
J. Biol. Chem.
242,
3833-3838 |
28. | Vogt, A. M., Ackermann, C., Noe, T., Jensen, D., and Kubler, W. (1998) Biochem. Biophys. Res. Commun. 248, 527-532[CrossRef][Medline] [Order article via Infotrieve] |
29. | Horemans, A. M. C., Tielens, A. G. M., and Van den Bergh, S. G. (1990) Parasitology 102, 259-265 |
30. | Pande, S. V. (1976) Anal. Biochem. 74, 25-34[Medline] [Order article via Infotrieve] |
31. | Tielens, A. G. M., Van der Meer, P., and Van den Bergh, S. G. (1981) Parasitology 3, 205-214 |
32. | Tielens, A. G. M., Horemans, A. M. C., Dunnewijk, R., Van der Meer, P., and Van den Bergh. (1992) Mol. Biochem. Parasitol. 56, 49-58[Medline] [Order article via Infotrieve] |
33. | Bergmeyer, H. U., Bernt, E., Schmidt, F., and Stork, H. (1970) in Methoden der Enzymatischen Analyse (Bergmeyer, H. U., ed) , pp. 1163-1165, Verlag Chemie, Weinheim, Germany |
34. | Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241-250[Medline] [Order article via Infotrieve] |
35. | Mracek, J., Snyder, S. J., Chavez, U. B., and Turrens, J. F. (1991) J. Protozool. 38, 554-558[Medline] [Order article via Infotrieve] |
36. | Cronin, C. N., Nolan, D. P., and Voorheis, H. P. (1989) FEBS Lett. 224, 26-30[CrossRef] |
37. | Ter Kuile, B. H. (1997) J. Bacteriol. 179, 4699-4705[Abstract] |
38. | Njogu, R. M., Whittaker, C. J., and Hill, G. C. (1980) Mol. Biochem. Parasitol. 1, 13-29[CrossRef][Medline] [Order article via Infotrieve] |
39. | Tielens, A. G. M., Rotte, C., Van Hellemond, J. J., and Martin, W. (2002) Trends Biochem. Sci. 11, 564-572[CrossRef] |