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(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. 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In Vitro, pp. 14-15. Blackwell, Oxford. osoma scelopori; proline metabolism; glutamate. OCHOA In 739- SIEGEL SRIVASTAVA soma 981. SRIVASTAVA rhodesiense TAYLOR A. In Cultivation of Parasites Key Word Index--Trypanosoma IN TR YPANOSOMA SCELOPORI EPIMA~ 1971) Assimilatory sulfate reduction t ~zool. 18, 718-722. ~perimental Physiology. Holt, Rineha 2) The utilization of glucose and proli tozool. 19, 686-690. (1965) Data reduction with constant t 519-531. 66) Separation of lactic acid and som Analyt. Biochem. 15, 122-126. 6) The rapid resolution of naturally ot 106-111. rtania tarentolae. E: in the cu Leishmania between pr )g. (In pre Proline metabol J. (1951) P 265-275. lnimals. Chaprr Oanosom 7r~7 agellate n) New e forms .,espiro- le acids lo acids 4, 348- hmania omasti- cose in nosoma rement ondon. b
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