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Proline metabolism in Trypanosoma scelopori epimastigotes
, pp.
$M
401 to 409. Pergamon Press. Printed in
IN
TRYPANOSOMA
PORI
'IMASTIGOTES* M. K R A S S N E R and K. B. 1~ gy, University of California, Irvine, C U.S.A.
54,
(Received 1 August 1973) Abstract--1. Oxygen uptake by epimastigotes of Tr:ypanosomz ~s stimulated by L-proline and to a lesser degree by L-asp~artate. 2. L-Proline reversed partially KCN-induced inhibition inhil~ of r~ :I, completely, inhibition caused by malonate. 3. Labeled proline, glutamate, alanine, aspartate and ar cystine d by thin-layer chromatography in the free amino acid p, ~ool from t !d with L-proline-t4C. 4. Labeled tricarboxylic acid intermediates were also al~, found [ pd in extracts from organisms incubated with L-prolir ~roline-14C wh ,,d also pyruvate. )5. Labeled L-proline was not found in epimasti~ ep~mastlgotes incu glucose-14C, glutamate-**C or aspartateJ4C, indicating that the flow of carbon from proline to glutamate and aspartate may not be reversible. re 6. The catabolism of proline and glucose to CO2 is reduced in the presence of the other substrate, indicating that there is physiological physiol interplay between the two substrates. NADP-, 7. Indirect evidence for the presence of an NADP-dependent malic enzyme was obtained by Ochoa's method. 8. All results suggest the presence of a proline~gl[utamate interconversion pathway in T. scelopori epimastigotes. INTRODUCTION PROLINE oxidation has been '.en demonstrated in a variety of hemoflagellate culture forms ranging from the Crithidia (Hutner, personal communication) to the Trypanosoma (Srivastava & Bowman, 1971, 1972; Evans & Brown, 1972). Little work has been done with epimastigote stages of trypanosomes; preliminaryY investigations on epimastig, gotes of Trypanosoma scelopori, a parasite of the western vestern fence lizard Sceloporus occidenta ~talis indicated that this parasite was capable ot :cidentalis, of proline oxidation. A stud,ly was undertaken to see if the pathway in this species ~ecles resembled that found in other ther Trypanosomatidae. A preliminary report of some ot of the findings was given at the 13th Seminar on United Kingdom Trypanosomiasis )mlasls Research (Krassner et al., 1973). * This investigation was supported by Research Grant No. AI 06827 from NIAID U.S. Public Health Service. 401
S. M. KRASSNERAND K. B. MUNSO:
ERIALS AND METHODS ained originally from Dr. J. Chao, Un xmintained in N N N 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 m M sodium phosphate b
alifornia 1968) at sks with
T h e numbers of epimastigotes were determined in a Petroff-Haus~ Pet1 the total protein content was estimated by the Folin t)henol m~ 1).
:hamber
erimental methods Measurements of respiration.
). T h e y aqueous (PBS),
y e t al.,
Oxygen consumption was determ! Gilson [erential Respirometer at 27°C (Dunn & Arditti, 1968). Th ' e proce ed were ilar to those described previously (Krassner, 1969). Measuremen Mc Luptake e converted to standard conditions as suggested by Gregor y & Winte Thin-layer chromatography. After incubation with labeled precursoJ pension centrifuged at 800 g for 10 min; the pellet was resuspend ~ended in 3 m] tool and wed to sit overnight. T h e suspension was transferred to a porcelain tporated [1 dry and the residue dissolved in 50/xl redistilled H20. Twenty/zl '] •ial were :ed onto thin-layer chromatography ( T L C ) plates (200 × x 200 m m coated Ninhydrin-collidine chromageni~ cellulose M N 300 to a thickness of 0'25 mm). Nin enlc & ;ent was used for detection of amino acids after two-dimensional two-dir separation (Jones & ensional Heathcote, 1966). Tricarboxylie acid cycle intermediates, obtained by two-dimensiona ins & Von Brand, 1966). Solvent., ~olvents ~ration, were detected by analine-ribose reagent (Higgins, separation, Labeled compounds were wer{ Resu and developing times used in the separation are given in Results. 2-2 '.cted with the aid of Eastman Kodak BB-54 Medical X-ray X-ra~ film applied to a plate for 2-3 detected weeks. indirectly by the method methoc Malic enzyme determination. T h e malic enzyme was measured meas Ma. )choa (1955) in a Beckman D U - 2 spectrophotometer. of Ochoa !-14 c Measurement of 14CO2 production. 14CO 2 evolved from cells c~ incubated in L-proline-l~C 0-5 ml hyamine hydroxide xide B-glucose-14C was measured in respirometer flasks containing contair and Dn alin in the side arm and the cell suspension (-~ 109 cellsi liv in the center well, 1 ml 10% formalin the trtment. Immediately after the addition of radioisotope to the 3 ml PBS) in the main compartment. lnts. as quickly removed to determine initial radioactivity counts main compartment 100/xl was )ed cubated for 2 hr at 27°C at which time the formalin was tipped Flasks were stoppered and incuba ts transtransinto the main compartment to kill the cells. Hyamine hydroxide in the center well was lter nted in a Nuclear Chicago Unilux I I I scintillation counteJ ferred to scintillation fluid andd counted counte cel (Da Cruz & Krassner, 1971)) to measure dissolved 14CO~ activity. In addition, the cell he The suspension was washed three times by centrifuging at 800 g for 10 min in 3 ml PBS. T unfirst supernatant and subseq~uent washings were counted to determine the activity of unincorporated label outside the cell. Three ml cold 5 % trichloroacetic acid (TCA) was added to the pellet and the mixture chilled for 10 min on ice. T h e mixture was centrifuged at 800 g ion removed. oved. This was repeated three times. After resuspension for 3 rain and the supernatantt remove )ore al was suction filtered on Millipore H A W P 29325 filters (pore in cold 5 % T C A , the material ers d 5% T C A and washed with 10-15 ml cold 5% T C A . Filter~, size 0'45 m) presoaked in cold gs beled protein activity and the initial supernatant and washings were counted to determine labeled ~tracellular .qlular labeled free amino acid activity. were counted to determine intracellu
IN TR YPANOSOMA
SCELOPORI
-rv,J
EPIMAS
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
IABLE
I--KESPIRATION
* AND SUBSTRATE UTILIZATION
Substrate
QO2f
152.2 _+65-9 L-Proline (4)§ Glucose (3) 148.0 + 52.8 L-Proline + glucose (4) 236.3 + 81.3 Alanine (4) -17.4+21.2 Isoleucine (4) 8-7 + 3-2 18-7 _+25.5 Endogenous L-Proline (5) 115.5 _+37.7 D-Proline (5) 46.2 + 53-5 L-Hydroxyproline (5) 20.2 + 20.6 Endogenous 71.6 + 28-1 L-Proline (4) 247-7 + 76.7 Aspartate (4) 99"3 + 64.2 L-Proline +Aspartate (4) 243"7 + 90"8 Endogenous 46.4 + 21-1 -
7". scelopor
BY .
imania 'ect on ulated ~olized
TE8
QNO2~ 10-19 _+1.91 7.31 + 0.5(. 16.50 + 3.7~ - 0 . 8 0 + 1.5~ 0.34 + 0.4: 1-49 + 1.4! 3.37 _+0.8. 1.19 + 1.4; - 0.90 + 1.0, 2-30 + 1.2! 17.65 + 1.22 6-84 + 1.67 17"00 + 1"28 3"23 + 0.40
• (Tables 1 and 2.) Rates were calculated for the first 90 rain after the addition of substrate. The system consisted of flagellates susp}ended in 2"5 ml sodium phosphate buffered saline (PBS), pH 7-4, in the main compartment, 0"5 ml substrate dissolved in PBS in the side arm and 0-2 ml KOH i in the center well. The concentration of each substrate was 10 raM. ~ (Tables 1 and 2.) QO~ }Oz =/zl O2/109 cells per hr "~Net O3 uptake less the (Tables 1 and 2.) QNO lNO2 =/zl O2/mgN per hr Jendogenous rate. § (Tables 1 and 2.) Number ~lumber her of du duplicate determinations.
by different metabolic pathwa I respiratory activity in the presence of both athways, substrates was followed (Table Fable 1). Oxygen consumption was significantly higher with the combined substrates ates than with either one alone [P = 0-016 in Sign test (Siegel, 1956)]. A similar experiment was performed using proline in combination with aspartate and oxygen1 consumption was not higher with the combined substrates than with proline alone. T o analyze further the possible role of L-proline in epimastigote respiration, we studied the effect of this amino acid on oxygen uptake inhibition by K C N and malonate (Table 2). K C N -I-induced indu, inhibition was reversed only partially, but that
, S . M . KRASSNER AND K . B . M U N S C
rsed completely by the addition
e to the
ALONIC ACID ON RESPIRATION* OF T . $¢
STIGOTES
QO~t )§ L-Proline (2) L-Proline + KCN (2) L-Proline + malonic acid (2)
77"6 _+54.8 2.9 + 14.8 13"3 + 3"6 254"6 _+18"8 19"3 + 7"2 243.9 + 56'4
QN( 3-76 _+ 0"30 _+ 0"48 _+ 10"30+ 0-69 + 10"36_+
:similation of metabolic intermediates T. scelopori epimastigotes were incubated with each ea, of th~
labeled npounds: L-proline-14C (uniformly labeled--UL), D-gluc IL), L tamic acid (UL), L-aspartic acid (UL), L-arginine (UL) ( and ruvate~,(pyruvate-2-1~C). Extracts from the flagellates were w, sepat LC and ir radioactive free amino acids and tricarboxylic acid ac cycle J es were ected by autoradiography (Table 3). Labeled proline, glutamate, alanine, aspartate and cystine were found iitn anisms incubated with L-proline-14C. Pyrroline-5rroline-5-carboxylic acid (PCA), the th~ firstt intermediate in proline oxidation, was not found i;In any plate. Four tricarbo. xylic ic acid cycle intermediates were demonstrated by ~I T L C after incubation of the th, epimastlgc mastigotes with L-proline-t4C. Pyruvate was also del detected. All of the other amino acids studied gave rise to tricarboxylic tri acid cycle inter. lntermeqdiates; this suggests that they are capable of stimulating stimulal T. scelopori respiration lratlon. As~)artate, the only one of these amino acids tested directly, stimulated oxyger en consumption (Table 1). L, To elucidate the originn of labeled alanine from L-proline-14C, extracts fron"t organisms incubated with sodium pyruvate-14C were analyzed by TLC. Labelec ~abeled alanine and possibly aspartate artate were found in these extracts. Although T TLC LC analyses with malic acid-t4C LC were not done, indirect evidence for the presence of t malic enzyme was obtainedt by testing crude cell extracts by Ochoa's method. This Thi~ test gave a positive reactionn for an NADP-dependent malic enzyme. Labeled proline was nott found in chromatograms of extracts from cells incubated ubated in either glutamate-14C or aspartate-l~C (Table 3). Apparently the flow of carbon }on from proline to glutamate and aspartate is not reversible under the conditions Jitions employed in this study. Offf great interest in this regard was the absence of labeled proline in chromatograms of extracts from cells incubated with B-glucose-14C. acose is not incorporated into free amino acid pool LEvidently carbon from glucose proline. The extract from D-glucoseJ4C incubated epimastigotes containedt labeled alanine, glutamate and possibly aspartate.
IN TR YPANOSOMA
S C E L O P O R I EPIMA8
IATES IDENTIFIED I N EXTRACTS FROM
7rvJ 'IMASTI-
ATED W I T H 14C-LABELED PRECURSORS
Intermediates Amino acids
Tricarboxylic acid cycle
Glutamate Alanine Proline Aspartate Cystine ~rolineb
IIt
31ucose °
I
31ucose a
Succinate Malic acid Isocitrate Citrate Glutamate Alanine Threonine Aspartate (?) Arginine (?) Cysteine
II
fled
Succinate Malic acid Isocitrate Citrate Oxaloacetate e~-Ketoglutarate
Glutamate e
I
Glutamate r
II
Pyruvate g
I
Alanine Aspartate (?)
Two unidentified spots
Aspartate h
I
Alanine Glutamate (?) Aspartate
One unidentified spot
Aspartate i
II
Arginine j
I
Glutamate Alanine Aspartate
Three unidentified spots Succinate (?) Isocitrate c~-Ketoglutarate
Succinate Malic acid (?) Isocitrate Citrate
Two unidentified spots
One unidentified spot Two unidentified spots
S. M. KRASSNER AND K . B. MuNso} TABLE 3
(cont.) Intermediates
Amino acids
Tricarboxylic acid cycle Succinate Malic acid (?) Isocitrate Citrate Fumarate
* Solvents: First dimension--propanol (40 pts) : formic fc ack (10 pts); Second dimension--tert-butanol (25 pts) p : meth: (15 pts) : 0"88 m NHa (5 pts) : H~O (5 pt.' ,ts). t Solvents: First dimension--96% ethanol (50 pts) : 25% NI H~O (2 pts) ; 1 Second dimension--propanol (25 pts) : eucalyptol e, acid (10 pts) : H 2 0 (25 pts).
r
]ed
20 ~ne
~) : nic
a 1 /zCi; sp. act., 2 5 1 . 0 m C i / m M incubation 1 hrl; 20/xl o )m 1"06 x 10 ~ cells. b 1 /xCi; sp. act,, 251"0 m C i / m M incubation 3½ ½hr; hr 20/~1 o )m 2"6 x 107 cells. e 1/zCi; sp. act., 4"8 m C i / m M incubation 3½ hr; 20/z]d of extract from 2'6 x 107 cells. ~1 of extract from 1"06 x 107 d 1 k~Ci; sp. act., 4"8 m C i / m M incubation 5 hr; 20/xl~ cells. e 1/xCi; sp. act., 195'0 m C i / m M incubation 3½ hr; hr': 20/xl of extract from 2"6 x 10 7 cells. f 1 p~Ci; sp. act., 195-0 m C i / m M incubation 3½ hr; 20/xl of extract from 2-6 x 10 7 cells. M of extract from 6"5 x 107 g 1/zCi; sp. act., 3"52 m C i / m M incubation 1 hr; 20/z] cells. h 1 /zCi; sp. act., 150-0 m C i / m M incubation 3~ hr; hrl 20/xl of extract from 2'6 x 107 cells. I 1/zCi; sp. act., 150"0 ) ' 0 m C i / m M incubation 3½hr; 20/xl of extract from 2"6 x 107 cells. 5"0 mCi m C i / m M incubation 3½ hr; 20/zl of extract from J 1/~Ci; sp. act., 305"0 2-6 x 107 cells. 5"0 m C i / m M incubation 3~ hr; 20/zl of extract from k 1/zCi; sp. act., 305"0 2"6 x 107 cells. P h y s i o l o g i c a l i n t e r p l a y o c c u r s b e t w e e n p r o l i n e a n d glucose in L. tarentolae p r o m a s t i g o t e m e t a b o l i s m ( K r a s s n e r & F l o r y , 1973). W e t h e r e f o r e m e a s u r e d t h e m 14CO2 b y e p i m a s t i g o t e s in t h e p r e s e n c e a n d a b s erna c e of of o x i d a t i o n o f L - p r o l i n e J 4 C to t h e o t h e r ( u n l a b e l e d ) s u b s t r a t e . E v o l u t i o n of 14CO2 f r o m e i t h e r s u b s t r a t e was r e d u c e d ( ,,~ 66 p e r cent w i t h p r o l i n e a n d ~ 20 p e r cent w i t h glucose) w h e n t h e o t h e r s u b s t r a t e was p r e s e n t ( T a Lble b l e 4). L i t t l e c h a n g e was f o u n d in t h e p e r c e n t a g e of
I N TR YPANOSOMA SCELOPORI EPIMAS
FIGOTE8
~l DIFFERENT FRACTIONS FROM T . $¢el( H 14C-LABELED PROLINE AND GLUCOSl~
Total 14C-recovered Free
in ,ation dia :ee
amino
roline +"cold" glucose1: (2) ,lucoset (2) ~lucose+ "cold" proline § (2)
a4CO~
acids
Prote
32'3 10-3 32"4 25"9
2"1 3"8 11-3 13"9
0"8 0-9 3"2 1"4
~'7 )'0 ~'5 ~'7
* 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~ )orated into p~ strates were present (Table 4). There was a marked increase mincorporated label found in the medium outside the t[ cells w e incubated in L-proline-14C and unlabeled glucose (Table 4).
A
1 both entage tigotes
DISCUSSION
Proline metabolism The fact that epimastigote culture forms of T. scelop( ~pori, presumably physiologic equivalents ~ivalents of some of the insect stages, oxidize prol ~roline is of interest because proline line oxidation has also been found in the other culture cu forms [promastigotes (Krassner, 'assner. 1969: Krassner & Flory, Florv. 1972) and trypomastigotes trvo (Srivastava & Bowman, 1971, 1972; Evans ans & Brown, 1972)] of higher hemoflagellates. This supports the hypothesis that hat the abundance of proline, primarily as an energy source for flight muscle, m in many insects has favored the presence of a proline oxidase system in hemoflagq;ellate insect stages (see Discussion in Krassner & Flory, 1972). Recent studies on T. brucei su rucei subgroup sub trypomastigotes suggests that there is an inverse correlation between n the ability of trypomastigotes to oxidize proline and infectivity for the vertebrate te host. This is because: (1) the infectivity of a primary culture for the vertebrate host is related to the number of blood stream trypomastigotes present (Mendez Jez & Honigberg, 1972) and (2) bloodstream trypomastigotes do not oxidize proline roline whereas culture forms (presumably physiologically equivalent to the insect mid Lidgut stage) are characterized by a high rate of proline oxidation (Srivastava & Bowman )wman, 1971, 1972; Evans & Brown, 1972).
S. M. KRASSNERAND 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
~tes are • Evans tdroxy-
celopori
mediates ed in cell extracts shortly afte
opori to L-proline-14C. PCA was not detected in il chrom
of T. ~erhaps ~ecause )f T L C version Lway in
ause it was present in concentrations too low for detection de b ms too labile to survive the technique employed. In light of 1 arations of labeled tricarboxylic acid cycle intermediates, intermec th glutamate provides evidence for the existence of a proline c ~celopori epimastigotes. ~artate-14C il An interesting result was the presence of aspart; ~f cells ause this amino acid is capable of stimulating T. scel, partate scelopori rest y be formed by transamination from oxaloacetic ack acid which ( om the arboxylic acid cycle (Luckner, 1972). Although aspartat :red in :acts of cells incubated in L-proline-14C, labeled proline und in :acts from cells incubated in aspartate-14C as occur., "urs in T. g~ happell l., 1972). The flow of carbon from proline to glutamate and aspartate appears not to be b( r show a reversible flOW. flow rev,ersible; perhaps cells grown in proline free media may We do not know at this time whether proline is syntt rnthesized by T. scelopori epi)lmastigotes. ~ori There is a physiological interplay between proline and glucose in T. scelopor in the presence of the suchh that the catabolism of either substrate to CO~ is reduced re¢ otes other substrate. A similar result has been found in L. tarentolae promastigote, )lex assner & Flory, 1973), lending support to the idea that tl~ proline plays a comple~ (Krassner role in hemoflagellate metabolism abolism ism (Krassner & Flory, 1971). Alanine-14C was foundd in extracts of cells incubated in L-proline-14C and )roline 7or transaminases, it is reasonable to postulate a proline although we did not test for othm metabolic pathway in T. scelopori epimastigotes similar to that found in other hemoflagellate culture stages. Whether proline oxidation in T. scelopori is as Lte energy metabolism as seems to be the case for insects important for hemoflagellate remains to be determined. re grateful to Dr. S. Kuwahara, University of California at Acknowledgements--We are ~arbara tudy and acknowledge the technical assistance of Mrs. Barbara Irvine, for his advice in this stud Flory. REFERENCES CHAPPELL L, H., SOUTHWORTTH H G. C. & READ C. P. (1972) Short-interval absorption and 75-387• no acids in Trypanosoma garnbieme. Parasitol. 64, 375-387 metabolism of some amino
IN TR Y P A N O S O M A S C E L O P O R I EPIMA~
7r~7
1971) Assimilatory sulfate reduction t ~zool. 18, 718-722. ~perimental Physiology. Holt, Rineha
agellate n) N e w
2) T h e utilization of glucose and proli e forms tozool. 19, 686-690. (1965) Data reduction with constant t .,espiro519-531. 66) Separation of lactic acid and som le acids Analyt. Biochem. 15, 122-126. 6) The rapid resolution of naturally ot lo acids by thin layer chromatography, ft. Chromatogr. 24, 106-111. 106-11: ~SSNERS. M. (1969) Proline metabolism in Leishmania tare rtania tarentolae. E: 4, 348363. ~SSNER S. M. & FLORY B. (1971) Essential amino acids in the cu hmania tarentolae. .7. Parasit. 57, 917-920. ~SSNER S. M. & FLORY B. (1972) Proline metabolism in Leishmania Z omastigotes..7. Protozool. 19, 682-685. ~SSNER S. M. & FLORY B. (1973) Physiological interplay between b pr cose in Leishmania tarentolae metabolism. 4th Int. Protozool. Con,)g. (In pre ~SSN~RS. M., SYLWSTER D. & MUNSON K. B. (1973) Prolir Proline metabol nosoma ~celopori. Trans. R. Soc. Trop. Med. Hyg. 67, 258. wY O. H., ROSEBROUGHN. J., FARE A. L. & RANDALLR. JJ. (1951) P rement with the Folin phenol reagent..7, biol. Chem. 193, 265-275. 265-271 :KNERM. (1972) Secondary Metabolism in Plants and.4nima lnimals. Chaprr ondon. ~DEZ Y. & HONIGBEEG B. M. (1972) Infectivity of TJ,ypanosoma Oanosom Orucet--subgroup b flagellates maintained in culture..7. Parasit. 58, 1122-1136. 1122-113t OCHOA tOA S. (1955) "Malic" enzyme. A "malic" enzyme from pigeon liver and wheat germ. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 1, pp. 739739-748. Academic Press, New York. SIEGEL ~EL S. (1956) Nonparametric Statistics. McGraw-Hill, New Ne York. SRIVASTAVA CASTAVAH. K. & BOWMANI. B. R. (1971) Adaptation )tation in oxk oxidative metabolism of Trypanos~oma o m a rhodesiense during transformation in culture. Comp. Biochem. Physiol. 40B, 973981. SRIVASTAVA CASTAVAH. K. & BOWMAN I. B. R. (1972) Metabolic transformation trs of Trypanosoma rhodesiense ,hodesiense in culture. Nature (New Biology) 235, 152-153. 152-15: TAYLOR "LOR A. E. R. & BAKERJ. R. (1968) N N N medium (WEN~ (WENYON, 1926, slightly modified). In Cultivation of Parasites In Vitro, pp. 14-15. Blackwell, Oxford.
Key Word Index--Trypanosoma osoma scelopori; proline metabolism; glutamate.
$M
401 to 409. Pergamon Press. Printed in
IN
TRYPANOSOMA
PORI
'IMASTIGOTES* M. K R A S S N E R and K. B. 1~ gy, University of California, Irvine, C U.S.A.
54,
(Received 1 August 1973) Abstract--1. Oxygen uptake by epimastigotes of Tr:ypanosomz ~s stimulated by L-proline and to a lesser degree by L-asp~artate. 2. L-Proline reversed partially KCN-induced inhibition inhil~ of r~ :I, completely, inhibition caused by malonate. 3. Labeled proline, glutamate, alanine, aspartate and ar cystine d by thin-layer chromatography in the free amino acid p, ~ool from t !d with L-proline-t4C. 4. Labeled tricarboxylic acid intermediates were also al~, found [ pd in extracts from organisms incubated with L-prolir ~roline-14C wh ,,d also pyruvate. )5. Labeled L-proline was not found in epimasti~ ep~mastlgotes incu glucose-14C, glutamate-**C or aspartateJ4C, indicating that the flow of carbon from proline to glutamate and aspartate may not be reversible. re 6. The catabolism of proline and glucose to CO2 is reduced in the presence of the other substrate, indicating that there is physiological physiol interplay between the two substrates. NADP-, 7. Indirect evidence for the presence of an NADP-dependent malic enzyme was obtained by Ochoa's method. 8. All results suggest the presence of a proline~gl[utamate interconversion pathway in T. scelopori epimastigotes. INTRODUCTION PROLINE oxidation has been '.en demonstrated in a variety of hemoflagellate culture forms ranging from the Crithidia (Hutner, personal communication) to the Trypanosoma (Srivastava & Bowman, 1971, 1972; Evans & Brown, 1972). Little work has been done with epimastigote stages of trypanosomes; preliminaryY investigations on epimastig, gotes of Trypanosoma scelopori, a parasite of the western vestern fence lizard Sceloporus occidenta ~talis indicated that this parasite was capable ot :cidentalis, of proline oxidation. A stud,ly was undertaken to see if the pathway in this species ~ecles resembled that found in other ther Trypanosomatidae. A preliminary report of some ot of the findings was given at the 13th Seminar on United Kingdom Trypanosomiasis )mlasls Research (Krassner et al., 1973). * This investigation was supported by Research Grant No. AI 06827 from NIAID U.S. Public Health Service. 401
S. M. KRASSNERAND K. B. MUNSO:
ERIALS AND METHODS ained originally from Dr. J. Chao, Un xmintained in N N N 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 m M sodium phosphate b
alifornia 1968) at sks with
T h e numbers of epimastigotes were determined in a Petroff-Haus~ Pet1 the total protein content was estimated by the Folin t)henol m~ 1).
:hamber
erimental methods Measurements of respiration.
). T h e y aqueous (PBS),
y e t al.,
Oxygen consumption was determ! Gilson [erential Respirometer at 27°C (Dunn & Arditti, 1968). Th ' e proce ed were ilar to those described previously (Krassner, 1969). Measuremen Mc Luptake e converted to standard conditions as suggested by Gregor y & Winte Thin-layer chromatography. After incubation with labeled precursoJ pension centrifuged at 800 g for 10 min; the pellet was resuspend ~ended in 3 m] tool and wed to sit overnight. T h e suspension was transferred to a porcelain tporated [1 dry and the residue dissolved in 50/xl redistilled H20. Twenty/zl '] •ial were :ed onto thin-layer chromatography ( T L C ) plates (200 × x 200 m m coated Ninhydrin-collidine chromageni~ cellulose M N 300 to a thickness of 0'25 mm). Nin enlc & ;ent was used for detection of amino acids after two-dimensional two-dir separation (Jones & ensional Heathcote, 1966). Tricarboxylie acid cycle intermediates, obtained by two-dimensiona ins & Von Brand, 1966). Solvent., ~olvents ~ration, were detected by analine-ribose reagent (Higgins, separation, Labeled compounds were wer{ Resu and developing times used in the separation are given in Results. 2-2 '.cted with the aid of Eastman Kodak BB-54 Medical X-ray X-ra~ film applied to a plate for 2-3 detected weeks. indirectly by the method methoc Malic enzyme determination. T h e malic enzyme was measured meas Ma. )choa (1955) in a Beckman D U - 2 spectrophotometer. of Ochoa !-14 c Measurement of 14CO2 production. 14CO 2 evolved from cells c~ incubated in L-proline-l~C 0-5 ml hyamine hydroxide xide B-glucose-14C was measured in respirometer flasks containing contair and Dn alin in the side arm and the cell suspension (-~ 109 cellsi liv in the center well, 1 ml 10% formalin the trtment. Immediately after the addition of radioisotope to the 3 ml PBS) in the main compartment. lnts. as quickly removed to determine initial radioactivity counts main compartment 100/xl was )ed cubated for 2 hr at 27°C at which time the formalin was tipped Flasks were stoppered and incuba ts transtransinto the main compartment to kill the cells. Hyamine hydroxide in the center well was lter nted in a Nuclear Chicago Unilux I I I scintillation counteJ ferred to scintillation fluid andd counted counte cel (Da Cruz & Krassner, 1971)) to measure dissolved 14CO~ activity. In addition, the cell he The suspension was washed three times by centrifuging at 800 g for 10 min in 3 ml PBS. T unfirst supernatant and subseq~uent washings were counted to determine the activity of unincorporated label outside the cell. Three ml cold 5 % trichloroacetic acid (TCA) was added to the pellet and the mixture chilled for 10 min on ice. T h e mixture was centrifuged at 800 g ion removed. oved. This was repeated three times. After resuspension for 3 rain and the supernatantt remove )ore al was suction filtered on Millipore H A W P 29325 filters (pore in cold 5 % T C A , the material ers d 5% T C A and washed with 10-15 ml cold 5% T C A . Filter~, size 0'45 m) presoaked in cold gs beled protein activity and the initial supernatant and washings were counted to determine labeled ~tracellular .qlular labeled free amino acid activity. were counted to determine intracellu
IN TR YPANOSOMA
SCELOPORI
-rv,J
EPIMAS
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
IABLE
I--KESPIRATION
* AND SUBSTRATE UTILIZATION
Substrate
QO2f
152.2 _+65-9 L-Proline (4)§ Glucose (3) 148.0 + 52.8 L-Proline + glucose (4) 236.3 + 81.3 Alanine (4) -17.4+21.2 Isoleucine (4) 8-7 + 3-2 18-7 _+25.5 Endogenous L-Proline (5) 115.5 _+37.7 D-Proline (5) 46.2 + 53-5 L-Hydroxyproline (5) 20.2 + 20.6 Endogenous 71.6 + 28-1 L-Proline (4) 247-7 + 76.7 Aspartate (4) 99"3 + 64.2 L-Proline +Aspartate (4) 243"7 + 90"8 Endogenous 46.4 + 21-1 -
7". scelopor
BY .
imania 'ect on ulated ~olized
TE8
QNO2~ 10-19 _+1.91 7.31 + 0.5(. 16.50 + 3.7~ - 0 . 8 0 + 1.5~ 0.34 + 0.4: 1-49 + 1.4! 3.37 _+0.8. 1.19 + 1.4; - 0.90 + 1.0, 2-30 + 1.2! 17.65 + 1.22 6-84 + 1.67 17"00 + 1"28 3"23 + 0.40
• (Tables 1 and 2.) Rates were calculated for the first 90 rain after the addition of substrate. The system consisted of flagellates susp}ended in 2"5 ml sodium phosphate buffered saline (PBS), pH 7-4, in the main compartment, 0"5 ml substrate dissolved in PBS in the side arm and 0-2 ml KOH i in the center well. The concentration of each substrate was 10 raM. ~ (Tables 1 and 2.) QO~ }Oz =/zl O2/109 cells per hr "~Net O3 uptake less the (Tables 1 and 2.) QNO lNO2 =/zl O2/mgN per hr Jendogenous rate. § (Tables 1 and 2.) Number ~lumber her of du duplicate determinations.
by different metabolic pathwa I respiratory activity in the presence of both athways, substrates was followed (Table Fable 1). Oxygen consumption was significantly higher with the combined substrates ates than with either one alone [P = 0-016 in Sign test (Siegel, 1956)]. A similar experiment was performed using proline in combination with aspartate and oxygen1 consumption was not higher with the combined substrates than with proline alone. T o analyze further the possible role of L-proline in epimastigote respiration, we studied the effect of this amino acid on oxygen uptake inhibition by K C N and malonate (Table 2). K C N -I-induced indu, inhibition was reversed only partially, but that
, S . M . KRASSNER AND K . B . M U N S C
rsed completely by the addition
e to the
ALONIC ACID ON RESPIRATION* OF T . $¢
STIGOTES
QO~t )§ L-Proline (2) L-Proline + KCN (2) L-Proline + malonic acid (2)
77"6 _+54.8 2.9 + 14.8 13"3 + 3"6 254"6 _+18"8 19"3 + 7"2 243.9 + 56'4
QN( 3-76 _+ 0"30 _+ 0"48 _+ 10"30+ 0-69 + 10"36_+
:similation of metabolic intermediates T. scelopori epimastigotes were incubated with each ea, of th~
labeled npounds: L-proline-14C (uniformly labeled--UL), D-gluc IL), L tamic acid (UL), L-aspartic acid (UL), L-arginine (UL) ( and ruvate~,(pyruvate-2-1~C). Extracts from the flagellates were w, sepat LC and ir radioactive free amino acids and tricarboxylic acid ac cycle J es were ected by autoradiography (Table 3). Labeled proline, glutamate, alanine, aspartate and cystine were found iitn anisms incubated with L-proline-14C. Pyrroline-5rroline-5-carboxylic acid (PCA), the th~ firstt intermediate in proline oxidation, was not found i;In any plate. Four tricarbo. xylic ic acid cycle intermediates were demonstrated by ~I T L C after incubation of the th, epimastlgc mastigotes with L-proline-t4C. Pyruvate was also del detected. All of the other amino acids studied gave rise to tricarboxylic tri acid cycle inter. lntermeqdiates; this suggests that they are capable of stimulating stimulal T. scelopori respiration lratlon. As~)artate, the only one of these amino acids tested directly, stimulated oxyger en consumption (Table 1). L, To elucidate the originn of labeled alanine from L-proline-14C, extracts fron"t organisms incubated with sodium pyruvate-14C were analyzed by TLC. Labelec ~abeled alanine and possibly aspartate artate were found in these extracts. Although T TLC LC analyses with malic acid-t4C LC were not done, indirect evidence for the presence of t malic enzyme was obtainedt by testing crude cell extracts by Ochoa's method. This Thi~ test gave a positive reactionn for an NADP-dependent malic enzyme. Labeled proline was nott found in chromatograms of extracts from cells incubated ubated in either glutamate-14C or aspartate-l~C (Table 3). Apparently the flow of carbon }on from proline to glutamate and aspartate is not reversible under the conditions Jitions employed in this study. Offf great interest in this regard was the absence of labeled proline in chromatograms of extracts from cells incubated with B-glucose-14C. acose is not incorporated into free amino acid pool LEvidently carbon from glucose proline. The extract from D-glucoseJ4C incubated epimastigotes containedt labeled alanine, glutamate and possibly aspartate.
IN TR YPANOSOMA
S C E L O P O R I EPIMA8
IATES IDENTIFIED I N EXTRACTS FROM
7rvJ 'IMASTI-
ATED W I T H 14C-LABELED PRECURSORS
Intermediates Amino acids
Tricarboxylic acid cycle
Glutamate Alanine Proline Aspartate Cystine ~rolineb
IIt
31ucose °
I
31ucose a
Succinate Malic acid Isocitrate Citrate Glutamate Alanine Threonine Aspartate (?) Arginine (?) Cysteine
II
fled
Succinate Malic acid Isocitrate Citrate Oxaloacetate e~-Ketoglutarate
Glutamate e
I
Glutamate r
II
Pyruvate g
I
Alanine Aspartate (?)
Two unidentified spots
Aspartate h
I
Alanine Glutamate (?) Aspartate
One unidentified spot
Aspartate i
II
Arginine j
I
Glutamate Alanine Aspartate
Three unidentified spots Succinate (?) Isocitrate c~-Ketoglutarate
Succinate Malic acid (?) Isocitrate Citrate
Two unidentified spots
One unidentified spot Two unidentified spots
S. M. KRASSNER AND K . B. MuNso} TABLE 3
(cont.) Intermediates
Amino acids
Tricarboxylic acid cycle Succinate Malic acid (?) Isocitrate Citrate Fumarate
* Solvents: First dimension--propanol (40 pts) : formic fc ack (10 pts); Second dimension--tert-butanol (25 pts) p : meth: (15 pts) : 0"88 m NHa (5 pts) : H~O (5 pt.' ,ts). t Solvents: First dimension--96% ethanol (50 pts) : 25% NI H~O (2 pts) ; 1 Second dimension--propanol (25 pts) : eucalyptol e, acid (10 pts) : H 2 0 (25 pts).
r
]ed
20 ~ne
~) : nic
a 1 /zCi; sp. act., 2 5 1 . 0 m C i / m M incubation 1 hrl; 20/xl o )m 1"06 x 10 ~ cells. b 1 /xCi; sp. act,, 251"0 m C i / m M incubation 3½ ½hr; hr 20/~1 o )m 2"6 x 107 cells. e 1/zCi; sp. act., 4"8 m C i / m M incubation 3½ hr; 20/z]d of extract from 2'6 x 107 cells. ~1 of extract from 1"06 x 107 d 1 k~Ci; sp. act., 4"8 m C i / m M incubation 5 hr; 20/xl~ cells. e 1/xCi; sp. act., 195'0 m C i / m M incubation 3½ hr; hr': 20/xl of extract from 2"6 x 10 7 cells. f 1 p~Ci; sp. act., 195-0 m C i / m M incubation 3½ hr; 20/xl of extract from 2-6 x 10 7 cells. M of extract from 6"5 x 107 g 1/zCi; sp. act., 3"52 m C i / m M incubation 1 hr; 20/z] cells. h 1 /zCi; sp. act., 150-0 m C i / m M incubation 3~ hr; hrl 20/xl of extract from 2'6 x 107 cells. I 1/zCi; sp. act., 150"0 ) ' 0 m C i / m M incubation 3½hr; 20/xl of extract from 2"6 x 107 cells. 5"0 mCi m C i / m M incubation 3½ hr; 20/zl of extract from J 1/~Ci; sp. act., 305"0 2-6 x 107 cells. 5"0 m C i / m M incubation 3~ hr; 20/zl of extract from k 1/zCi; sp. act., 305"0 2"6 x 107 cells. P h y s i o l o g i c a l i n t e r p l a y o c c u r s b e t w e e n p r o l i n e a n d glucose in L. tarentolae p r o m a s t i g o t e m e t a b o l i s m ( K r a s s n e r & F l o r y , 1973). W e t h e r e f o r e m e a s u r e d t h e m 14CO2 b y e p i m a s t i g o t e s in t h e p r e s e n c e a n d a b s erna c e of of o x i d a t i o n o f L - p r o l i n e J 4 C to t h e o t h e r ( u n l a b e l e d ) s u b s t r a t e . E v o l u t i o n of 14CO2 f r o m e i t h e r s u b s t r a t e was r e d u c e d ( ,,~ 66 p e r cent w i t h p r o l i n e a n d ~ 20 p e r cent w i t h glucose) w h e n t h e o t h e r s u b s t r a t e was p r e s e n t ( T a Lble b l e 4). L i t t l e c h a n g e was f o u n d in t h e p e r c e n t a g e of
I N TR YPANOSOMA SCELOPORI EPIMAS
FIGOTE8
~l DIFFERENT FRACTIONS FROM T . $¢el( H 14C-LABELED PROLINE AND GLUCOSl~
Total 14C-recovered Free
in ,ation dia :ee
amino
roline +"cold" glucose1: (2) ,lucoset (2) ~lucose+ "cold" proline § (2)
a4CO~
acids
Prote
32'3 10-3 32"4 25"9
2"1 3"8 11-3 13"9
0"8 0-9 3"2 1"4
~'7 )'0 ~'5 ~'7
* 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~ )orated into p~ strates were present (Table 4). There was a marked increase mincorporated label found in the medium outside the t[ cells w e incubated in L-proline-14C and unlabeled glucose (Table 4).
A
1 both entage tigotes
DISCUSSION
Proline metabolism The fact that epimastigote culture forms of T. scelop( ~pori, presumably physiologic equivalents ~ivalents of some of the insect stages, oxidize prol ~roline is of interest because proline line oxidation has also been found in the other culture cu forms [promastigotes (Krassner, 'assner. 1969: Krassner & Flory, Florv. 1972) and trypomastigotes trvo (Srivastava & Bowman, 1971, 1972; Evans ans & Brown, 1972)] of higher hemoflagellates. This supports the hypothesis that hat the abundance of proline, primarily as an energy source for flight muscle, m in many insects has favored the presence of a proline oxidase system in hemoflagq;ellate insect stages (see Discussion in Krassner & Flory, 1972). Recent studies on T. brucei su rucei subgroup sub trypomastigotes suggests that there is an inverse correlation between n the ability of trypomastigotes to oxidize proline and infectivity for the vertebrate te host. This is because: (1) the infectivity of a primary culture for the vertebrate host is related to the number of blood stream trypomastigotes present (Mendez Jez & Honigberg, 1972) and (2) bloodstream trypomastigotes do not oxidize proline roline whereas culture forms (presumably physiologically equivalent to the insect mid Lidgut stage) are characterized by a high rate of proline oxidation (Srivastava & Bowman )wman, 1971, 1972; Evans & Brown, 1972).
S. M. KRASSNERAND 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
~tes are • Evans tdroxy-
celopori
mediates ed in cell extracts shortly afte
opori to L-proline-14C. PCA was not detected in il chrom
of T. ~erhaps ~ecause )f T L C version Lway in
ause it was present in concentrations too low for detection de b ms too labile to survive the technique employed. In light of 1 arations of labeled tricarboxylic acid cycle intermediates, intermec th glutamate provides evidence for the existence of a proline c ~celopori epimastigotes. ~artate-14C il An interesting result was the presence of aspart; ~f cells ause this amino acid is capable of stimulating T. scel, partate scelopori rest y be formed by transamination from oxaloacetic ack acid which ( om the arboxylic acid cycle (Luckner, 1972). Although aspartat :red in :acts of cells incubated in L-proline-14C, labeled proline und in :acts from cells incubated in aspartate-14C as occur., "urs in T. g~ happell l., 1972). The flow of carbon from proline to glutamate and aspartate appears not to be b( r show a reversible flOW. flow rev,ersible; perhaps cells grown in proline free media may We do not know at this time whether proline is syntt rnthesized by T. scelopori epi)lmastigotes. ~ori There is a physiological interplay between proline and glucose in T. scelopor in the presence of the suchh that the catabolism of either substrate to CO~ is reduced re¢ otes other substrate. A similar result has been found in L. tarentolae promastigote, )lex assner & Flory, 1973), lending support to the idea that tl~ proline plays a comple~ (Krassner role in hemoflagellate metabolism abolism ism (Krassner & Flory, 1971). Alanine-14C was foundd in extracts of cells incubated in L-proline-14C and )roline 7or transaminases, it is reasonable to postulate a proline although we did not test for othm metabolic pathway in T. scelopori epimastigotes similar to that found in other hemoflagellate culture stages. Whether proline oxidation in T. scelopori is as Lte energy metabolism as seems to be the case for insects important for hemoflagellate remains to be determined. re grateful to Dr. S. Kuwahara, University of California at Acknowledgements--We are ~arbara tudy and acknowledge the technical assistance of Mrs. Barbara Irvine, for his advice in this stud Flory. REFERENCES CHAPPELL L, H., SOUTHWORTTH H G. C. & READ C. P. (1972) Short-interval absorption and 75-387• no acids in Trypanosoma garnbieme. Parasitol. 64, 375-387 metabolism of some amino
IN TR Y P A N O S O M A S C E L O P O R I EPIMA~
7r~7
1971) Assimilatory sulfate reduction t ~zool. 18, 718-722. ~perimental Physiology. Holt, Rineha
agellate n) N e w
2) T h e utilization of glucose and proli e forms tozool. 19, 686-690. (1965) Data reduction with constant t .,espiro519-531. 66) Separation of lactic acid and som le acids Analyt. Biochem. 15, 122-126. 6) The rapid resolution of naturally ot lo acids by thin layer chromatography, ft. Chromatogr. 24, 106-111. 106-11: ~SSNERS. M. (1969) Proline metabolism in Leishmania tare rtania tarentolae. E: 4, 348363. ~SSNER S. M. & FLORY B. (1971) Essential amino acids in the cu hmania tarentolae. .7. Parasit. 57, 917-920. ~SSNER S. M. & FLORY B. (1972) Proline metabolism in Leishmania Z omastigotes..7. Protozool. 19, 682-685. ~SSNER S. M. & FLORY B. (1973) Physiological interplay between b pr cose in Leishmania tarentolae metabolism. 4th Int. Protozool. Con,)g. (In pre ~SSN~RS. M., SYLWSTER D. & MUNSON K. B. (1973) Prolir Proline metabol nosoma ~celopori. Trans. R. Soc. Trop. Med. Hyg. 67, 258. wY O. H., ROSEBROUGHN. J., FARE A. L. & RANDALLR. JJ. (1951) P rement with the Folin phenol reagent..7, biol. Chem. 193, 265-275. 265-271 :KNERM. (1972) Secondary Metabolism in Plants and.4nima lnimals. Chaprr ondon. ~DEZ Y. & HONIGBEEG B. M. (1972) Infectivity of TJ,ypanosoma Oanosom Orucet--subgroup b flagellates maintained in culture..7. Parasit. 58, 1122-1136. 1122-113t OCHOA tOA S. (1955) "Malic" enzyme. A "malic" enzyme from pigeon liver and wheat germ. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 1, pp. 739739-748. Academic Press, New York. SIEGEL ~EL S. (1956) Nonparametric Statistics. McGraw-Hill, New Ne York. SRIVASTAVA CASTAVAH. K. & BOWMANI. B. R. (1971) Adaptation )tation in oxk oxidative metabolism of Trypanos~oma o m a rhodesiense during transformation in culture. Comp. Biochem. Physiol. 40B, 973981. SRIVASTAVA CASTAVAH. K. & BOWMAN I. B. R. (1972) Metabolic transformation trs of Trypanosoma rhodesiense ,hodesiense in culture. Nature (New Biology) 235, 152-153. 152-15: TAYLOR "LOR A. E. R. & BAKERJ. R. (1968) N N N medium (WEN~ (WENYON, 1926, slightly modified). In Cultivation of Parasites In Vitro, pp. 14-15. Blackwell, Oxford.
Key Word Index--Trypanosoma osoma scelopori; proline metabolism; glutamate.