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Prévia do material em texto

(Received 1 August 1973) 
Abstract--1. Oxygen uptake by epimastigotes of Tr: 
stimulated by L-proline and to a lesser degree by L-asp 
2. L-Proline reversed partially KCN-induced inhil~ 
completely, inhibition caused by malonate. 
3. Labeled proline, glutamate, alanine, aspartate ar 
by thin-layer chromatography in the free amino acid p, 
with L-proline-t4C. 
4. Labeled tricarboxylic acid intermediates were al~, 
in extracts from organisms incubated with L-prolir 
also pyruvate. 
5. Labeled L-proline was not found in epimasti~ 
glucose-14C, glutamate-**C or aspartateJ4C, indicating 
from proline to glutamate and aspartate may not be re 
6. The catabolism of proline and glucose to CO2 is 
of the other substrate, indicating that there is physiol 
Indirect evidence for the presence of an NADP-, 
was obtained by Ochoa's method. 
8. All results suggest the presence of a proline~gl 
pathway in T. scelopori epimastigotes. 
that the flow of carbon 
is reduced in the presence 
physiological interplay between 
NADP-dependent malic enzyme 
[utamate interconversion 
'.en demonstrated in a variety of hemoflagellate culture 
Crithidia (Hutner, communication) to the 
& Bowman, 1971, 1972; Evans & Brown, 1972). 
epimastigote of trypanosomes; preliminary 
gotes of Trypanosoma scelopori, a parasite of the western 
:cidentalis, indicated that this parasite was capable ot 
ly was undertaken to see if the pathway in this species 
ther Trypanosomatidae. A preliminary report of some ot 
the 13th Seminar on United Kingdom Trypanosomiasis 
supported by Research Grant No. AI 06827 from NIAID 
401 
the two substrates. 
7. 
PROLINE oxidation has been 
forms ranging from the 
Trypanosoma (Srivastava & 
work has been done with 
investigations on epimastig, 
fence lizard Sceloporus occidenta 
proline oxidation. A stud, 
resembled that found in other 
the findings was given at the 
Research (Krassner et al., 1973). 
* This investigation was 
U.S. Public Health Service. 
, pp. 401 to 409. Pergamon Press. Printed in 
$M IN TRYPANOSOMA 
' IMAST IGOTES* 
M. KRASSNER and K. B. 1~ 
gy, University of California, Irvine, C 
U.S.A. 
ypanosomz 
~artate. 
inhibition of r~ 
and cystine 
~ool from t 
also found [ 
~roline-14C wh 
ep~mastlgotes incu 
reversible. 
INTRODUCTION 
personal 
stages 
~talis 
PORI 
54, 
~s 
:I, 
d 
!d 
pd 
,,d 
) - 
the 
Little 
Y 
vestern 
of 
~ecles 
of 
)mlasls 
The numbers of epimastigotes were determined in a Pet1 
the total protein content was estimated by the Folin t 
1). 
erimental methods 
Measurements of respiration. 
[erential Respirometer at 27°C (Dunn & Arditti, 1968). ' 
ilar to those described previously (Krassner, 1969). Mc 
e converted to standard conditions as suggested by Gregor 
Thin-layer chromatography. After incubation with labeled 
centrifuged at 800 g for 10 min; the pellet was resuspend 
wed to sit overnight. The suspension was transferred to a 
[1 dry and the residue dissolved in 50/xl redistilled H20. '] 
:ed onto thin-layer chromatography (TLC) plates (200 x 
cellulose MN 300 to a thickness Nin 
;ent was used for detection of amino acids after two-dir 
Tricarboxylie acid cycle intermediates, 
~ration, were detected by analine-ribose reagent (Higgins, 
developing times used in the separation are given in Resu 
'.cted with the aid of Eastman Kodak BB-54 Medical X-ra~ 
Malic enzyme determination. The malic enzyme was meas 
)choa (1955) in a Beckman DU-2 spectrophotometer. 
14CO 2 evolved from c~ 
B-glucose-14C was measured in respirometer flasks contair 
Ninhydrin-collidine chromageni~ 
two-dimensional separation (Jones & 
obtained by two-dimensiona 
ins & Von Brand, 1966). Solvent., 
Results. Labeled compounds wer{ 
X-ray film applied to a plate for 2-2 
measured indirectly by the methoc 
cells incubated in L-proline-l~C 
containing 0-5 ml hyamine hydroxide 
formalin in the side arm and the cell suspension (-~ 109 cells iv 
trtment. Immediately after the addition of radioisotope to the 
as quickly removed to determine initial radioactivity counts 
cubated for 2 hr at 27°C at which time the formalin was tipped 
kill the cells. Hyamine hydroxide in the center well was trans- 
d counted in a Nuclear Chicago Unilux I I I scintillation counteJ 
) to measure dissolved 14CO~ activity. In addition, the cel 
times by centrifuging at 800 g for 10 min in 3 ml PBS. The 
uent washings were counted to determine the activity of un- 
cell. Three ml cold 5 % trichloroacetic acid (TCA) was added 
chilled for 10 min on ice. The mixture was centrifuged at 800 g 
t removed. This was repeated three times. After resuspension 
al was suction filtered on Millipore HAWP 29325 filters (pore 
d 5% TCA and washed with 10-15 ml cold 5% TCA. Filter~, 
beled protein activity and the initial supernatant and washings 
~tracellular labeled free amino acid activity. 
Heathcote, 1966). 
separation, 
and 
detected with 
weeks. 
Ma. 
of Ochoa 
Measurement of 14CO2 production. 
and D- 
in the center well, 1 ml 10% formalin 
3 ml PBS) in the main compartment. 
main compartment 100/xl was 
Flasks were stoppered and incuba 
into the main compartment to kill 
ferred to scintillation fluid and counte 
(Da Cruz & Krassner, 1971) 
suspension was washed three times 
first supernatant and subseq~ 
incorporated label outside the 
to the pellet and the mixture 
for 3 rain and the supernatant remove 
in cold 5 % TCA, the material 
size 0'45 m) presoaked in cold 
were counted to determine labeled 
were counted to determine intracellu 
S. M. KRASSNER AND K. B. MUNSO: 
ERIALS AND METHODS 
ained originally from Dr. J. Chao, Un 
xmintained in NNN medium (Tayk 
al analysis were grown in 125-ml Er 
]e medium and 50 ml for the overlay. 
sted during the exponential growth ] 
tion at 800 g, 10 min each, in cold 
nded in 10 mM sodium phosphate b 
Petroff-Haus~ 
)henol m~ 
Oxygen consumption was determ! 
The proce 
Measuremen 
y & Winte 
with labeled precursoJ 
~ended in 3 m] 
porcelain 
Twenty/zl 
× 200 mm 
of 0'25 mm). 
alin in 
nted 
oved. 
.qlular 
alifornia 
1968) at 
sks with 
). They 
aqueous 
(PBS), 
:hamber 
yet al., 
Gilson 
ed were 
L uptake 
pension 
tool and 
tporated 
• ial were 
coated 
enlc 
& 
ensional 
~olvents 
were 
2-3 
method 
!-14 c 
xide 
i l n 
the 
lnts. 
)ed 
ts trans- 
lter 
cell 
The 
un- 
added 
at 800 g 
i on 
)ore 
ers 
gs 
IABLE I - -KESP IRAT ION * AND SUBSTRATE UT IL IZAT ION BY . 
Substrate 
L-Proline (4)§ 
Glucose (3) 
L-Proline + glucose (4) 
Alanine (4) 
Isoleucine (4) 
Endogenous 
L-Proline (5) 
D-Proline (5) 
L-Hydroxyproline (5) 
Endogenous 
L-Proline (4) 
Aspartate (4) 99"3 
L-Proline +Aspartate (4) 243"7 + 90"8 
• (Tables 1 and 2.) Rates were calculated for the first 
The system consisted of flagellates susp 
saline (PBS), 
substrate dissolved in PBS in the side arm and 0-2 ml i 
The concentration of each substrate was 10 raM. 
247-7 + 76.7 17.65 + 1.22 
+ 64.2 6-84 + 1.67 
17"00 + 1"28 
3"23 + 0.40 
90 rain after the addition 
}ended in 2"5 ml sodium 
pH 7-4, in the main compartment, 0"5 ml 
KOH in the center well. 
}Oz =/zl O2/109 cells per hr "~Net O3 uptake less the 
lNO2 =/zl O2/mgN per hr Jendogenous rate. 
~lumber of duplicate determinations. 
athways, in the presence of both 
Fable 1). Oxygen consumption was significantly higher 
ates than with either one alone [P = 0-016 in Sign test 
experiment was performed using proline in combination 
1 consumption was not higher with the combined sub- 
possible role of L-proline in epimastigote respiration, we 
amino acid on oxygen uptake inhibition by KCN and 
I-induced inhibitionwas reversed only partially, but that 
Endogenous 
of substrate. 
phosphate buffered 
~ (Tables 1 and 2.) QO~ 
(Tables 1 and 2.) QNO 
§ (Tables 1 and 2.) Number 
by different metabolic pathwa I 
substrates was followed (Table 
with the combined substrates 
(Siegel, 1956)]. A similar 
with aspartate and oxygen 
strates than with proline alone. 
To analyze further the 
studied the effect of this 
malonate (Table 2). KCN-indu, 
IN TR YPANOSOMA SCELOPORI EP IMAS 
RESULTS 
:ls shown to either stimulate or i 
sner & Flory, 1972) were tested J 
)le 1). Glucose, L-proline and as l 
mine whether glucose and L-proli 
7". scelopor 
QO2f QNO2~ 
152.2 _+ 65-9 10-19 _+ 1.91 
148.0 + 52.8 7.31 + 0.5(. 
236.3 + 81.3 16.50 + 3.7~ 
-17.4+21.2 -0.80+ 1.5~ 
8-7 + 3-2 0.34 + 0.4: 
18-7 _+ 25.5 1-49 + 1.4! 
115.5 _+ 37.7 3.37 _+ 0.8. 
46.2 + 53-5 1.19 + 1.4; 
- 20.2 + 20.6 - 0.90 + 1.0, 
71.6 + 28-1 2-30 + 1.2! 
46.4 + 21-1 
her of du 
respiratory activity 
-rv,J 
imania 
'ect on 
ulated 
~olized 
TE8 
L-Proline (2) 
L-Proline + KCN (2) 
L-Proline + malonic acid (2) 
:similation of metabolic intermediates 
T. scelopori epimastigotes were incubated with ea, 
npounds: L-proline-14C (uniformly 
tamic acid (UL), L-aspartic acid (UL), L-arginine ( 
~, (pyruvate-2-1~C). Extracts from the flagellates w, 
ir radioactive free amino acids and tricarboxylic ac 
ected by autoradiography (Table 3). 
Labeled proline, glutamate, 
anisms incubated with L-proline-14C. Pyrroline-5- 
t intermediate in proline oxidation, was not found i; 
ic acid cycle intermediates were demonstrated by ~I 
mastigotes with L-proline-t4C. Pyruvate was also del 
All of the other amino acids studied gave rise to tri 
diates; this suggests that they are capable of stimulal 
)artate, the only one of these amino acids tested 
To elucidate the origin of labeled alanine from L, 
cystine were found it 
rroline-5-carboxylic acid (PCA), th~ 
In any plate. Four tricarbo. 
TLC after incubation of th, 
detected. 
tricarboxylic acid cycle inter. 
of stimulating T. scelopori respiration 
directly, stimulated oxyger 
n of labeled alanine from L-proline-14C, extracts fron" 
sodium pyruvate-14C were analyzed by TLC. Labelec 
artate were found in these extracts. Although TLC 
LC were not done, indirect evidence for the presence of 
t by testing crude cell extracts by Ochoa's method. Thi~ 
n for an NADP-dependent malic enzyme. 
t found in chromatograms of extracts from cells incubated 
aspartate-l~C (Table 3). Apparently the flow of carbon 
and aspartate is not reversible under the conditions 
ff great interest in this regard was the absence of labeled 
of extracts from cells incubated with B-glucose-14C. 
acose is not incorporated into free amino acid pool L- 
epimastigotes contained 
and possibly aspartate. 
first 
xylic 
epimastlgc 
meq 
As~ 
consumption (Table 1). 
To elucidate the origin 
organisms incubated with 
alanine and possibly aspartate 
analyses with malic acid-t4C 
malic enzyme was obtained 
test gave a positive reaction 
Labeled proline was not 
in either glutamate-14C or 
from proline to glutamate 
employed in this study. Of 
proline in chromatograms 
Evidently carbon from glucose 
proline. The extract from 
labeled alanine, glutamate 
, S . M . KRASSNER AND K . B . MUNSC 
rsed completely by the addition 
ALONIC ACID ON RESPIRATION* OF T. $¢ 
QO~t QN( 
) § 77"6 _+ 54.8 3-76 _+ 
2.9 + 14.8 0"30 _+ 
13"3 + 3"6 0"48 _+ 
254"6 _+ 18"8 10"30 + 
19"3 + 7"2 0-69 + 
243.9 + 56'4 10"36 _+ 
each of th~ 
labeled--UL), D-gluc 
(UL) and 
were sepat 
acid cycle J 
alanine, aspartate and 
D-glucoseJ4C incubated 
e to the 
STIGOTES 
labeled 
IL), L - 
ruvate- 
LC and 
es were 
i n 
the 
the 
lnter- 
lratlon. 
en 
t 
~abeled 
TLC 
t 
This 
ubated 
}on 
Jitions 
labeled 
t 
~roline b I I t 
31ucose ° I 
31ucose a I I 
Three 
unidentified 
spots 
Two 
unidentified 
spots 
Two 
unidentified 
spots 
One 
unidentified 
spot 
One 
unidentified 
spot 
Two 
unidentified 
spots 
Glutamate e I 
Glutamate r I I 
Pyruvate g I 
Aspartate h I 
Aspartate i I I 
Arginine j I 
IN TR YPANOSOMA SCELOPORI EPIMA8 
IATES IDENTIFIED IN EXTRACTS FROM 
ATED WITH 14C-LABELED PRECURSORS 
Intermediates 
Tricarboxylic 
Amino acids acid cycle 
Glutamate 
Alanine 
Proline 
Aspartate 
Cystine 
Succinate 
Malic acid 
Isocitrate 
Citrate 
Glutamate 
Alanine 
Threonine 
Aspartate (?) 
Arginine (?) 
Cysteine 
Succinate 
Malic acid 
Isocitrate 
Citrate 
Oxaloacetate 
e~-Ketoglutarate 
Glutamate 
Alanine 
Aspartate 
Succinate (?) 
Isocitrate 
c~-Ketoglutarate 
Alanine 
Aspartate (?) 
Alanine 
Glutamate (?) 
Aspartate 
Succinate 
Malic acid (?) 
Isocitrate 
Citrate 
7rvJ 
' IMASTI- 
fled 
* Solvents: First dimension--propanol (40 pts) : fc 
(10 pts); 
Second dimension--tert-butanol (25 p 
(15 pts) : 0"88 m NHa (5 pts) : H~O (5 pt.' 
t Solvents: First dimension--96% ethanol (50 pts) 
H~O (2 pts) ; 
Second dimension--propanol (25 pts) : e, 
acid (10 pts) : H20 (25 pts). 
a 1 /zCi; sp. act., 251.0mCi/mM incubation 1 hrl 
1"06 x 10 ~ cells. 
b 1 /xCi; sp. act,, 251"0 mCi/mM incubation 3½ hr 
2"6 x 107 cells. 
e 1/zCi; sp. act., 4"8 mCi/mM incubation 3½ hr; 20/z] 
d 1 k~Ci; sp. act., 4"8 mCi/mM incubation 5 hr; 20/xl~ 
e 1/xCi; sp. act., 195'0 mCi/mM incubation 3½ hr': 
f 1 p~Ci; sp. act., 195-0 mCi/mM incubation 3½ hr; 
g 1/zCi; sp. act., 3"52 mCi/mM incubation 1 hr; 20/z] 
h 1 /zCi; sp. act., 150-0 mCi/mM incubation 3~ hrl 
d of extract from 2'6 x 107 
~1 of extract from 1"06 x 107 
hr; 20/xl of extract from 
20/xl of extract from 
M of extract from 6"5 x 107 
hr; 20/xl of extract from 
) '0mCi /mM incubation 3½hr; 20/xl of extract from 
5"0 mCi/mM incubation 3½ hr; 20/zl of extract from 
5"0 mCi/mM incubation 3~ hr; 20/zl of extract from 
occurs between prol ine and glucose in L. tarentolae 
(Krassner & Flory, 1973). We therefore measured the 
m 14CO2 by epimast igotes in the presence and absence of 
Evolut ion of 14CO2 from either substrate was 
h prol ine and ~ 20 per cent with glucose) when the other 
Lble 4). L i t t le change was found in the percentage of 
cells. 
cells. 
2"6 x 10 7 cells. 
2-6 x 10 7 cells. 
cells. 
sp. 
2'6 x 107 cells. 
I 1/zCi; sp. act., 150"0 
2"6 x 107 cells. 
J 1/~Ci; sp. act., 305"0 mCi 
2-6 x 107 cells. 
k 1/zCi; sp. act., 305"0 
2"6 x 107 cells. 
Physiological interplay 
promast igote metabo l i sm ( 
oxidat ion of L -pro l ine J4C to 
the other (unlabeled) substrate. 
reduced ( ,,~ 66 per cent w i th 
substrate was present (Tab le 
S. M. KRASSNER AND K. B. MuNso} 
TABLE 3 (cont.) 
Intermediates 
Tricarboxylic 
Amino acids acid cycle 
Succinate 
Malic acid (?) 
Isocitrate 
Citrate 
Fumarate 
formic ack 
pts) : meth: 
,ts). 
: 25% NI 
eucalyptol 1 
; 20/xl o 
½hr; 20/~1 o 
r 
]ed 
20 
~ne 
~) : 
nic 
)m 
)m 
race of 
of 
roline +"cold" glucose1: (2) 
,lucoset (2) 
~lucose + "cold" proline § (2) 
* 1/~Ci; sp. act., 251-0 mCi/mM incubation 2 hr. 
t 1/~Ci, sp. act., 4"8 mCi/mM incubation 2 hr. 
$10-2 M. 
§ 10 -~ M. 
[I (), Number of duplicate determinations. 
.q in intracellular free amino acids or incorporate~ 
strates were present (Table 4). There was a marked 
mincorporated label found in the medium outside t[ 
e incubated in L-proline-14C and unlabeled glucose 
The fact that epimastigote culture forms of T. scelop( 
~ivalents of some of the insect stages, oxidize prol 
line oxidation has also been found in the other cu 
'assner. 1969: Krassner &Florv. 1972) and trvo 
A 
(Table 4). 
~pori, presumably physiologic 
~roline is of interest because 
culture forms [promastigotes 
Flory, 1972) and trypomastigotes (Srivastava & 
ans & Brown, 1972)] of higher hemoflagellates. 
hat the abundance of proline, primarily as an energy 
in many insects has favored the presence of a proline 
;ellate insect stages (see Discussion in Krassner & Flory, 
rucei subgroup trypomastigotes suggests that there is an 
n the ability of trypomastigotes to oxidize proline and 
te host. This is because: (1) the infectivity of a primary 
host is related to the number of blood stream trypo- 
Jez & Honigberg, 1972) and (2) bloodstream trypo- 
roline whereas culture forms (presumably physiologically 
Lidgut stage) are characterized by a high rate of proline 
)wman, 1971, 1972; Evans & Brown, 1972). 
Proline metabolism 
equivalents 
proline 
(Krassner, 
Bowman, 1971, 1972; Evans 
supports the hypothesis that 
source for flight muscle, m 
oxidase system in hemoflagq 
1972). 
Recent studies on T. brucei su 
inverse correlation between 
infectivity for the vertebrate 
culture for the vertebrate 
mastigotes present (Mendez 
mastigotes do not oxidize proline 
equivalent to the insect mid 
oxidation (Srivastava & Bowman 
IN TR YPANOSOMA SCELOPORI EPIMAS 
~l DIFFERENT FRACTIONS FROM T. $¢el( 
H 14C-LABELED PROLINE AND GLUCOSl~ 
Total 14C- recovered 
Free 
amino 
a4CO ~ acids Prote 
32'3 2"1 0"8 
10-3 3"8 0-9 
32"4 11-3 3"2 
25"9 13"9 1"4 
)orated into p~ 
marked increase 
outside the cells w 
DISCUSSION 
sub 
FIGOTE8 
:ee in 
,ation 
dia 
~'7 
)'0 
~'5 
~'7 
1 both 
entage 
tigotes 
This 
opori to L-proline-14C. PCA was not detected il 
ause it was present in concentrations too low for de 
ms too labile to survive the technique employed. In 
arations of labeled tricarboxylic acid cycle intermec 
glutamate provides evidence for the existence of a 
~celopori epimastigotes. 
An interesting result was the presence of aspart; 
ause this amino acid is capable of stimulating T. scel, 
y be formed by transamination from oxaloacetic ack 
arboxylic acid cycle (Luckner, 1972). Although 
:acts of cells incubated in L-proline-14C, labeled 
:acts from cells incubated in aspartate-14C as occur., 
l., 1972). 
The flow of carbon from proline to glutamate and 
ersible; perhaps cells grown in proline free media r 
do not know at this time whether proline is syntt 
There is a physiological interplay between proline 
h that the catabolism of either substrate to CO~ is re¢ 
A similar result has been found in 
assner & Flory, 1973), lending support to the idea tl~ 
aspartate appears not to b( 
may show a reversible flow 
rnthesized by T. scelopori epi- 
and glucose in T. scelopor 
reduced in the presence of the 
L. tarentolae promastigote, 
that proline plays a comple~ 
abolism (Krassner & Flory, 1971). 
d in extracts of cells incubated in L-proline-14C and 
7or transaminases, it is reasonable to postulate a proline 
scelopori epimastigotes similar to that found in othm 
Whether proline oxidation in T. scelopori is as 
Lte energy metabolism as seems to be the case for insects 
re grateful to Dr. S. Kuwahara, University of California at 
tudy and acknowledge the technical assistance of Mrs. Barbara 
TH G. C. & READ C. P. (1972) Short-interval absorption and 
no acids in Trypanosoma garnbieme. Parasitol. 64, 375-387 
rev, 
We 
mastigotes. 
such that 
other substrate. 
(Krassner & 
role in hemoflagellate metabolism 
Alanine-14C was found 
although we did not test for 
metabolic pathway in T. 
hemoflagellate culture stages. 
important for hemoflagellate 
remains to be determined. 
Acknowledgements--We are 
Irvine, for his advice in this stud 
Flory. 
CHAPPELL L, H., SOUTHWORTH 
metabolism of some amino 
S. M. KRASSNER AND K. B. MUNSO: 
d other amino acids on T. scelopo~ 
sly in other hemoflagellate cultur~ 
ory, 1972). As is true for most or 
ot suitable substitutes for L-prol 
mediates 
ed in cell extracts shortly afte 
in chrom 
detection b 
light of 1 
intermediates, th 
proline c 
~artate-14C il 
scelopori rest 
acid which ( 
aspartat 
proline 
"urs in T. g~ 
ism 
REFERENCES 
~tes are 
• Evans 
tdroxy- 
celopori 
of T. 
~erhaps 
~ecause 
)f TLC 
version 
Lway in 
~f cells 
partate 
om the 
:red in 
und in 
happell 
be 
flOW. 
)l- 
~ori 
of the 
otes 
)lex 
)roline 
other 
as 
insects 
at 
~arbara 
and 
75-387• 
by thin layer chromatography, ft. Chromatogr. 24, 106-11: 
~SSNER S. M. (1969) Proline metabolism in Leishmania tare 
363. 
~SSNER S. M. & FLORY B. (1971) Essential amino acids 
tarentolae. .7. Parasit. 57, 917-920. 
~SSNER S. M. & FLORY B. (1972) Proline metabolism in Z 
gotes..7. Protozool. 19, 682-685. 
~SSNER S. M. & FLORY B. (1973) Physiological interplay b 
Leishmania tarentolae metabolism. 4th Int. Protozool. Con, 
~SSN~R S. M., SYLWSTER D. & MUNSON K. B. (1973) Prolir 
~celopori. Trans. R. Soc. Trop. Med. Hyg. 67, 258. 
wY O. H., ROSEBROUGH N. J., FARE A. L. & RANDALL R. J 
with the Folin phenol reagent..7, biol. Chem. 193, 265-271 
:KNER M. (1972) Secondary Metabolism in Plants and.4nima 
~DEZ Y. & HONIGBEEG B. M. (1972) Infectivity of TJ 
flagellates maintained in culture..7. Parasit. 58, 1122-113t 
tOA S. (1955) "Malic" enzyme. A "malic" enzyme from 
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