Biochem. J. (1982) 204, 191-196 191
Printed in Great Britain
A pathway for the interconversion of hexose and pentose in the parasitic
amoeba Entamoeba histolytica
Brian M. SUSSKIND,*t Lionel G. WARREN*§ and Richard E. REEVESt
*Department of Tropical Medicine and Medical Parasitology, and tDepartment ofBiochemistry, Louisiana
State University Medical Center, New Orleans, LA 70112, U.S.A.
(Received 23 November 1981/Accepted 3 December 1981)
Isotope studies indicate that hexose-to-pentose interconversion by axenic Entamoeba
histolytica conserves the C-1 and C-6 hexose carbon atoms. Transketolase was readily
identified in amoebal extracts, but transaldolase could not be demonstrated. However,
sedoheptulose 7-phosphate is a substrate for the PP,-dependent amoebal phos-
phofructokinase, and sedoheptulose 1,7-bisphosphate is cleaved by amoebal aldolase to
dihydroxyacetone phosphate and erythrose phosphate. Since these three enzymes
catalyse physiologically reversible reactions, a non-oxidative pathway for hexose-
pentose interconversion exists in amoebae in the absence of transaldolase. By using
known amoebal enzymes, the conversion of ribose into fructose was confirmed in vitro.
Some kinetic parameters of amoebal phosphofructokinase, transketolase and aldolase
Nearly one-half of the ribose moieties in amoebal 1968). Penicillin G, 900units/ml, was added. Calci-
AMP are synthesized from glucose (Susskind et al., ferol was omitted from the vitamin mixture.
1980). The predominant pathway of glucose meta-
bolism in Entamoeba histolytica is the Embden- Harvesting amoebae
Meyerhof pathway (Bragg & Reeves, 1962). Since Amoebae were harvested after 72-90h culti-
the parasite lacks the first two enzymes of the vation. Cells were resuspended in the spent growth
oxidative pentose phosphate pathway (Hilker & medium, transferred to a graduated tube and packed
White, 1959; Bragg & Reeves, 1962; Warren et al., by centrifugation for 5 min at 850g. The volume of
1964), it appears unlikely that a hexose mono- packed amoebae was noted. Cells were then thrice
phosphate shunt pathway exists. The present study washed by centrifugation in buffer A [10mM-
shows that C-1 and C-6 on glucose are conserved K2HPO4/20 mM-KCl/O. 5 mM-MgCl2/140mM-NaCl/
during hexose-pentose interconversion, and evi- 0.1 mM-Ca(NO3)2, adjusted to pH 7.0 with HClI.
dence will be presented for a pathway by which three
amoebal enzymes reversibly catalyse this inter- Growth of amoebae with [ 14C]glucose
conversion. E. histolytica trophozoites were grown in 125-ml
Some of the results described here are taken from flasks containing a TP-S-1 medium prepared with
a dissertation (Susskind, 1979) submitted to the one-half the prescribed amount of glucose. A
Graduate Faculty of the Louisiana State University 3-5pCi portion of [14C]glucose was added to each
Medical Center in partial fulfilment of the require- flask. The specific radioactivity of the glucose was
ments of the Doctor of Philosophy degree. based on radioactivity counts and measurements of
total carbohydrate in the medium. The flasks were
Materials and methods inoculated with 1.2 x 106 amoebae per flask, and
incubated at 360C in a sealed chamber.
Axenic Entamoeba histolytica, strain 200:NIH, Cell extracts
was maintained in TP-S-1 medium (Diamond, Buffer A-washed cells were suspended in an equal
t Present address: Department of Immunobiology, volume of ice-cold 20 mM-imidazole/HCl buffer,
Sloan-Kettering Institute for Cancer Research, 145 pH 7.0, and ruptured with 15 strokes of a conical
Boston Road, Rye, NY 10580, U.S.A. ground-glass tissue grinder at 900 rev./min. The
§ To whom correspondence and requests for reprints homogenates were diluted with 2 or 3 vol. of the same
should be addressed. buffer and centrifuged for 30min at 35000g--and
Vol. 204 0306-3283/82/040191-06$01.50/1 (© 1982 The Biochemical Society
192 B. M. Susskind, L. G. Warren and R. E. Reeves
at 40C. The supernatant solutions were dialysed for Standard enzyme assays
20h against 500vol. of buffer at 40C, and then The standard assay for transketolase activity (EC
stored at -200C until used. 220.127.116.11) contained, per ml: 50,umol of imidazole/
HCl, pH 7.0, 2.5,umol of MgCl2, 0.2,umol of thiamin
Extracting andpurifying RNA pyrophosphate, 2.0,umol of ribose 5-phosphate,
Buffer A-washed cells were suspended in 5 vol. of 0.3,umol of xylulose 5-phosphate, 0. 16,umol of
a buffer containing 50mM-Tris/HCl, pH 8, 150mm- NADH, 2 units of glycerol-3-phosphate dehydro-
NaCl and 0.3% sodium dodecyl sulphate. Bentonite genase and 20 units of triosephosphate isomerase.
was added (4 mg/ml of suspension). The cells were After equilibration at 300C, reaction was started by
chilled in ice and broken in a tissue grinder as the addition of the amoebal enzyme. The rate of
described above. The homogenate was diluted with decrease in A340 was monitored at 300C. A cuvette
an equal volume of the suspending buffer. Protein lacking ribose 5-phosphate served as a control.
was precipitated from the homogenate by the The standard assay for aldolase activity (EC
hot-phenol method described by Brawerman (1973), 18.104.22.168) contained, per ml: 50,mol of Tris/HCl,
and the RNA was precipitated with ethanol and pH 8.6, 2.0,umol of fructose 1,6-bisphosphate,
centrifuged. The RNA pellet was dissolved in 0.16,mol of NADH, 2 units of glycerol-3-phos-
50mM-sodium acetate buffer, pH 5.1, and the solu- phate dehydrogenase, and 20 units of triosephos-
tion was clarified by centrifugation for 20min at phate isomerase. Reaction was started by the
390OOg at 40C. The supernatant solution was addition of the amoebal enzyme after equilibration at
precipitated with cetyltrimethylammonium bromide 300C. The rate of decrease in A340 was measured.
to remove traces of remaining polysaccharide Transaldolase was assayed by the methods of
(Ralph & Bellamy, 1964). RNA was quantified by Cooper et al. (1958) and Tsolas & Horecker (1970).
its absorbance at 260nm. Yeast RNA (Sigma, type The assay for the pyrophosphate-requiring 6-
XI) served as the standard. phosphofructokinase (EC 22.214.171.124) was described
by Reeves et al. (1974).
The molar absorption coefficient for NADH and
Substrate assays NADPH was taken as 6.22 x 103 M-1l cm-1.
Ribose and ribose 5-phosphate were assayed with
an orcinol-containing reagent as described by Assayfor ribose 5-phosphate utilization
Schneider (1957). Total carbohydrate was deter- Ribose 5-phosphate utilization in cell extracts was
mined by the phenol/H2SO4 method of Mont- measured at 300C in tubes containing, per ml:
gomery (1957), with glucose as the standard. Protein 50,umol of imidazole/HCl buffer, pH 7.0, 2.5,pmol of
was assayed by the method of Lowry et al. (1951), MgCl2, 0.2,umol of thiamin pyrophosphate and
with bovine serum albumin as the standard. Triose cell-extract protein (2-4 mg/ml). Reaction was star-
phosphate was assayed in a cuvette containing, per ted by the addition of 8,umol of ribose 5-phosphate.
ml: 50,umol of imidazole buffer, pH 7.0, 0.16,umol of A control lacked the added substrate. After 1 h,
NADH, 2 units of glycerol-3-phosphate dehydro- tubes were diluted 5-fold with buffer, capped, heated
genase and 20 units of triosephosphate isomerase. for 1 min at 1000C, centrifuged to remove de-
Decrease in A340 was measured. In some instances natured protein, and the remaining substrate was
'triose phosphate equivalents' were measured. This assayed with the orcinol reagent.
was accomplished by adding 0.25 unit of aldolase to
the preceding assay mixture. This value represents Chemicals
triose phosphate (mol) plus twice the fructose [U-'4C]glucose, [1-'4C]glucose and [6-'4C]glu-
bisphosphate (mol) present. cose were from Amersham Radiochemical Centre,
Fructose 6-phosphate was assayed in a cuvette Amersham, Bucks., U.K. Linking enzymes used in
containing, per ml: 50,umol of imidazole buffer, the assays were from Boehringer and Sons, Mann-
pH 7.0, 2.5 gmol of MgCl2, 0.32,umol of NADP, 1 heim, Germany. Enzyme suspensions in (NH4)2SO4
unit of glucosephosphate isomerase, and 5 units of solution were centrifuged, the pellet was dissolved in
glucose-6-phosphate dehydrogenase. Increase in 1 mM-sodium EDTA, pH 7.0, and dialysed at 40C
A340 was measured. overnight against 500vol. of the EDTA solution.
Sedoheptulose 7-phosphate was assayed in a
cuvette containing, per ml: 50,umol of imidazole Results
buffer, pH 7.0, 40,umol of ATP, 25,umol of phos-
phoenolpyruvate, 2.5,umol of MgCl2, 0.16,umol of Incorporation oflabelled glucose into amoebal RNA
NADH, 4 units of rabbit muscle 6-phosphofructo- Entamoeba histolytica were grown with [U-'4C]-
kinase, 2.5 units of pyruvate kinase, and 2.5 units of glucose, [1-'4C]glucose or [6-14C]glucose. RNA
lactate dehydrogenase. Decrease in A340 was was extracted from the cells after 84h of culti-
measured. vation. Glucose utilized was the total carbohydrate
Hexose pentose interconversion in Entamoeba 193
in TP-S-1 medium at the time of inoculation less the (2) It catalyses the formation of glyceraldehyde
amount in the amoeba-free culture fluid at the time of 3-phosphate from xylulose 5-phosphate in the
harvesting. Data from these experiments are listed in presence of either erythrose 4-phosphate or ribose
Table 1. 5-phosphate.
(3) It catalyses the utilization of glyceraldehyde
Amoebal transketolase 3-phosphate in the presence of fructose 6-phos-
Cell extracts utilized 8.1 ±1.7nmol of ribose phate. The kinetic values for amoebal transketolase
5-phosphate/min per mg of protein in four pre- are summarized in Table 2.
parations. Triose phosphate equivalents and sedo-
heptulose 7-phosphate formed, per mol of ribose Amoebalphosphofructokinase
5-phosphate, were 0.46 + 0.04 and 0.40 + 0.04 res- Sedoheptulose 7-phosphate was assayed by sub-
pectively. No fructose 6-phosphate was formed in stituting it for fructose 6-phosphate in the standard
these experiments. The formation of these products
by cell extracts indicated that amoebae possess
transketolase. These products accounted for 86% of
the pentose utilized. Failure to produce fructose
6-phosphate suggested that the amoebae lack trans-
aldolase. We have repeatedly been unable to detect
transaldolase activity in amoeba.
A freshly prepared cell extract was applied to a
Sephacryl 200-S column, bed volume 100ml, 0.6
equilibrated with 20mM-imidazole/HCl buffer,
pH 7.0. Fractions rich in both phosphofructokinase
and transketolase were eluted with the buffer, ,.r
pooled, and applied to a column of DEAE-cellulose,
bed volume 30ml, which had been equilibrated with 0.4
the buffer. The column was washed with the buffer
until the A280 of the effluent approached zero. The
enzymes were then eluted with buffer containing
0.1 M-NaCl. Transketolase and phosphofructokin-
ase were separated by the second column frac-
tionation. The partially purified enzymes were stored
at -100C after the addition of 30% (v/v) glycerol.
Thiamin pyrophosphate, 0.2 mm, and 1 mM-MgCl2 0 10 20
were also added to the transketolase preparation. Time (min)
The identity of the amoebal transketolase was
established according to the following three criteria Fig. 1. Dependence for activity by the apoenzyme of
(Racker, 1971). amoebal transketolase on added thiamin pyrophosphate
(1) It has a cofactor requirement for thiamin and Mg2+
pyrophosphate and Mg2+ (Fig. 1). The order of the The conditions were those of the standard assay (see
the Materials and methods section), but lacking
addition of the cofactor had an effect on trans- thiamin pyrophosphate and MgCl2. Three such
ketolase. When Mg2+ was followed by the addi- cuvettes were equilibrated with enzyme. After 5 min
tion of thiamin pyrophosphate, there was a lag period (arrow) the following were added: both cofactors
before enzyme activity reached the maximum rate (A); thiamin pyrophosphate only (B); and MgCl2
(Fig. 1). Optimum transketolase activity occurred at only (C). Then, after 10min (arrow), for B and C,
pH7.0. the respective missing cofactor was added.
Table 1. Incorporation Of D-[ 14C]glucose into amoebal RNA
Percentage of radioactivity
Position 10-6 x Glucose Glucose utilized 10- x Radioactivity in utilized
of label radioactivity (d.p.m./mmol) (mmol) recovered RNA (d.p.m.) recovered in RNA
U 4.8 4.1 4.3 0.022
U 8.8 8.2 19.3 0.027
1 4.4 6.0 6.4 0.024
1 2.0 8.1 3.8 0.024
6 5.5 4.6 3.5 0.021
6 2.5 5.0 2.6 0.021
194 B. M. Susskind, L. G. Warren and R. E. Reeves
Table 2. Km of amoebal transketolasefor various substrates
Apparent Km (mM)
Varied substrate Co-substrate (mM) of varied substrate
Ribose 5-phosphate Xylulose 5-phosphate (0.3) 0.027*
Xylulose 5-phosphate Ribose 5-phosphate (2.0) 0.23*
Glyceraldehyde 3-phosphate Fructose 6-phosphate (12.0) 1.60t
Erythrose 4-phosphate Xylulose 5-phosphate (0.1) 0.005:
* Determined by changing the substrate in the standard transketolase assay.
t Determined by measuring the disappearance of glyceraldehyde 3-phosphate after 10min incubation at 300C in a
tube containing MgCl2, thiamin pyrophosphate, the co-substrate and enzyme.
4 Determined spectrophotometrically by the rate of formation of glyceraldehyde 3-phosphate.
assay for the latter. That the product of the reaction 0.25
with sedoheptulose 7-phosphate was the corres-
ponding 1,7-bisphosphate was indicated by paper
chromatography of the product of a reaction
conducted in the absence of added aldolase. The 0.20 I
paper-chromatographic technique employed the
ammonium acetate/ethanol solvent and the period-
ate/benzidine staining technique of Wawszkiewicz A
(1961). The Km of sedoheptulose 7-phosphate for 0
amoebal phosphofructokinase is 64pM, which com- * 0.115
pares with a reported Km value of 38juM for fructose
6-phosphate (Reeves et al., 1974). The Km for PP1
was about 10pUM with either co-substrate.
Amoebal aldolase 0.1l0-
I. 1/ z/
Aldolase was purified from a cell extract frac-
tionated on a Sephacryl 200-S column, 300ml bed
volume, equilibrated with 20mM-imidazole/HCl, --L
pH 7.0. Aldolase was eluted from this column after I.
phosphofructokinase, and our preparation was 0 20 40
slightly contaminated by this enzyme. The kinetic Time (min)
constants of amoebal aldolase for sedoheptulose Fig. 2. Demonstration in vitro of a proposed amoebal
1,7-bisphosphate and fructose 1,6-bisphosphate pentose phosphatepathway
were determined in cuvettes having a 5 cm light-path. The complete reaction system contained, in a
The Km for the former substrate was 13 uM; for the volume of 1 ml: 50mpl of imidazole/HCI, pH 7.0,
latter, 3,UM. The relative Vm.. for the former is 0.lOjumol of thiamin pyrophosphate, 0.O5umol of
one-half that for the latter. Triosephosphate iso- MgCl2, 0.lOjmaol of PP1, 0.62,umol of xylulose
5-phosphate, 2.0,umol of ribose 5-phosphate,
merase was not required in the assay system when 0.32pAnol of NADP+, 1 unit of yeast phospho-
sedoheptulose bisphosphate was the substrate, since glucose isomerase, 5 units of yeast glucose 6-
the products of its cleavage by aldolase are phosphate dehydrogenase, 0.07 unit of amoebal
dihydroxyacetone phosphate and erythrose 4-phos- aldolase (except B) and 0.15 unit of amoebal
phate. The addition of triosephosphate isomerase to phosphofructokinase. After equilibration at 30°C,
the cuvette did not alter the rate of this reaction. the reaction was started by the addition of 0.03 unit
of amoebal transketolase (arrow at left). A340 was
Demonstration ofpentose-to-hexose interconversion monitored spectrophotometrically. A, complete sys-
A reaction mixture devised for the intercon- tem; B, contained 70munits of yeast aldolase in
version of pentose and hexose in vitro contained place of the amoebal aldolase, and phosphofructo-
kinase was not added until 35 min (arrow on right);
enzymes that had been partially purified from C, minus amoebal aldolase; D, minus PP,; E, minus
amoebal cell extracts. The formation of fructose transketolase; F, minus glucose 6-phosphate de-
6-phosphate from xylulose 5-phosphate and ribose hydrogenase.
5-phosphate was measured. The hexose phosphate
was formed only when the three amoebal enzymes
transketolase, aldolase and phosphofructokinase Discussion
were present (Fig. 2). Its formation was dependent Ribose phosphate is necessary for synthesis of
on added PP1. nucleotides de novo or their formation by the salvage
Hexose w pentose interconversion in Entamoeba 195
of free bases. Glucose carbon has been shown to be sidered and is shown in Scheme 1. We have demon-
one source of nucleotide ribose, and the glucose strated that E. histolytica possesses transketolase,
incorporated into amoebal ribonucleotides is that amoebal aldolase utilizes sedoheptulose bis-
recovered only in the ribose moiety (Susskind et al., phosphate as a substrate, and that the reversible
1980). The results reported here show that labelled amoebal phosphofructokinase catalyses conversion
ribonucleotides were recovered when the amoebae of sedoheptulose bisphosphate into sedoheptulose
were grown with [1-'4C]glucose or [6-'4C]glucose. 7-phosphate. We have reconstituted this system in
Thus neither the hexose monophosphate nor the vitro by using partially purified amoebal enzymes
glucuronic acid pathway appear to be operating in and demonstrated that it functions in the pentose-to-
E. histolytica. The percentage of labelled glucose hexose direction. Since each reaction involved is
carbon recovered in RNA was the same when E. readily reversible, it is reasonable to suppose that the
histolytica was cultivated with [U-'4C]glucose, [1- system can function in the opposite direction. That it
"4C]glucose, or [6-14C]glucose, confirming that the does so in vivo is likely in view of our inability to
amoebal pentose-hexose interconversion pathway is demonstrate transaldolase in amoebal extracts.
characterized by the conservation of glucose car- The two predominant pathways in Nature for the
bon. synthesis of pentose from hexose are the hexose
The usual mechanism for the non-oxidative monophosphate shunt and the transketolase-trans-
interconversion of hexose and pentose is the trans- aldolase pathway. Despite the widespread distri-
ketolase-transaldolase pathway (Horecker et al., bution of aldolase (Horecker et al., 1972) and
1954; Racker, 1955; Marks & Feigelson, 1957; sedoheptulose bisphosphatase activity (Pontremoli &
Horecker, 1965; Rognstad & Katz, 1974). How- Horecker, 1971) in plants, animals and micro-
ever, in the Calvin cycle a series of reactions organisms, there is little indication that the alternate
catalysed by transketolase, aldolase and sedoheptul- pathway plays an important role in net pentose
ose bisphosphatase are involved in the regeneration synthesis. Gibbs & Horecker (1954) found a small
of pentose from hexose (Racker & Schroeder, 1958; amount of label from [1-14Clribose incorporated into
Bassham, 1971). The latter two enzymes replace C-4 and C-6 of hexose by pea (Pisum sativum)
transaldolase in the formation of sedoheptulose leaf preparations. These results, and the labelling
7-phosphate, a key intermediate. Aldolase catalyses pattern in pentose from [1-14C]glucose obtained by
a condensation, yielding sedoheptulose 1,7-bisphos- Plaut & Broberg (1956) with Ashbya gossyppi would
phate (Horecker & Smyrnitotis, 1952), and its be consistent with the operation of the alternate
dephosphorylation at C-1 leaves sedoheptulose transketolase pathway. However, Plaut & Broberg
7-phosphate. In many organisms, sedoheptulose (1956) also obtained data using [6-14C]glucose that
bisphosphatase activity is a property of fructose indicated the presence of a hexose monophosphate
bisphosphatase (EC 126.96.36.199) (Racker & Schroeder, shunt, and the data of Gibbs & Horecker (1954)
1958; Pontremoli & Horecker, 1971). indicated that the major pathway in pea leaves and
Since E. histolytica lacks transaldolase, an alter- roots was the transketolase-transaldolase pathway.
native pentose-hexose interconversion scheme Both the hexose monophosphate shunt and the
similar to that involved in the Calvin cycle was con- transketolase-transaldolase pathways are energy-
Fructose 6-phosphate Xylulose 5-phosphate
Glyceraldehyde 3-phosphate Erythrose 4-phosphate
*eaoneptulose I1, ,7-bisphosphate
Dihydroxyacetone phosphate Orthophosphate
Sedoheptulose 7-phosphate (a)
Scheme 1. A schematic pathwayfor hexose-pentose interconversion in E. histolytica
Amoebal enzymes comprising the enzyme system are: (a) transketolase; (b) aldolase; (c) phosphofructokinase (PP1).
196 B. M. Susskind, L. G. Warren and R. E. Reeves
conserving in terms of retaining phosphate ester Horecker, B. L. (1965) J. Chem. Educ. 42, 244-253
bonds. The proposed pathway of pentose formation Horecker, B. L. & Smyrniotis, P. Z. (1952) J. Am. Chem.
in E. histolytica is also energy-conserving. In most Soc. 74, 2123
organisms, sedoheptulose bisphosphatase activity Horecker, B. L., Gibbs, M., Klenow, H. & Smyrniotis, P.
results in the hydrolysis of a phosphate ester Z. (1954) J. Biol. Chem. 207, 393-403
(Pontremoli & Horecker, 1971) to orthophosphate. Horecker, R. L., Tsolas, 0. & Lai, C. Y. (1972) Enzymes
However, with the amoebal phosphofructokinase 3rdEd. 8,213-258
this reaction is accompanied by the esterification of Kalra, I. S., Dutta, G. P. & Mohan Pao, V. K. (1969)
orthophosphate to form PP1, which is a source of Exp. Parasitol. 24, 26-31
Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall,
energy for this organism (Reeves, 1976). Our results R. J. (195 1) J. BioL Chem. 193, 265-275
indicate that, in E. histolytica, pentose phosphate is Marks, P. A. & Feigelson, P. (1957) J. Biol. Chem. 226,
formed by a series of C2-plus-C3 condensations 1001-1009
catalysed by transketolase, aldolase, phospho- Montgomery, R. (1957) Arch. Biochem. Biophys. 67,
fructokinase (PP1) and transketolase again. 368-386
Glucose carbon and all phosphate bonds are con- Plaut, G. W. E. & Broberg, P. L. (1956) J. Biol. Chem.
served. This pathway of hexose-pentose inter- 219, 131-138
conversion differs significantly from that of the Pontremoli, S. & Horecker, B. L. (1971) Enzymes 3rd
human host, and also from the interconversion Ed. 4, 612-644
Racker, E. (1955) Nature (London) 175, 249-251
occurring in the Calvin cycle. Racker, E. (1971) Enzymes 2nd Ed. 5, 397-406
Racker, E. & Schroeder, E. A. R. (1958) Arch. Biochem.
This work was supported in part by grants AI-0295 1, Biophys. 74, 326-344
AI-15090 and GM-14023 from the U.S. National Ralph, B. K. & Bellamy, A. R. (1964) Biochim. Biophys.
Institutes of Health. Acta 87, 9-16
Reeves, R. E. (1976) Trends Biochem. Sci. 1, 53-63
Reeves, R. E., South, D. J., Blytt, H. J. & Warren, L. G.
References (1974) J. Biol. Chem. 249, 7737-7741
Reeves, R. E., Serrano, R. & South, D. J. (1976) J. Biol.
Bassham, J. A. (1971) Science 172, 526-534 Chem. 251, 2958-2962
Bragg, P. D. & Reeves, R. E. (1962) Exp. Parasitol. 12, Rognstad, R. & Katz, J. (1974) Biochem. Biophys. Res.
393-400 Commun. 61, 774-780
Brawerman, G. (1973) in Methods in Cell Biology Schneider, W. D. (1957) Methods Enzymol. 3, 680-684
(Prescott, D., ed.), pp. 2-22, Academic Press, New Susskind, B. M. (1979) Ph.D. Dissertation, Louisiana
York and London State University
Cooper, J., Srere, P. A., Tabachnick, M. & Racker, E. Susskind, B. M., Warren, L. G. & Reeves, R. E. (1980) J.
(1958) Arch. Biochem. Biophys. 74, 306-314 Parasitol. 66, 759-764
Diamond, L. S. (1968)J. Parasitol. 54, 1047-1056 Tsolas, 0. & Horecker, B. L. (1970) Arch. Biochem.
Gibbs, M. & Horecker, B. L. (1954) J. Biol. Chem. 208, Biophys. 136, 287-302
813-820 Warren, L. G., Tonn, R. J. & Reeves, R. E. (1964) J.
Hilker, D. M. & White, A. G. C. (1959) Exp. Parasitol. 8, Protozool. 2 (Suppl.), 22-23
539-548 Wawszkiewicz, E. J. (1961) Anal. Chem. 33, 252-254