Parasitology (1975), 71, 93-107
93
With 3 figures in the text
Carbon dioxide fixation in trypanosomatids R. A. KLEIN, D. J. LINSTEAD and M. V. WHEELERf Medical Research Council, Biochemical Parasitology Unit, Molteno Institute, University of Cambridge, Downing Street, Cambridge CB2 3EE (Received 24 January 1975) SUMMARY
Fixation of carbon dioxide has been demonstrated for extracts from Crithidia fasciculata, Trypanosoma mega and Trypanosoma brucei brucei bloodstream and culture forms. The enzymes involved in this fixation were found to be ADP-stimulated phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32), 'malic' enzyme (E.C. 1.1.138-40) and pyruvate carboxylase (E.C. 6.4.1.1). The subcellular localization of these enzymes has been investigated in all three organisms. Products of short and long term fixation experiments were separated and identified. The importance of carboxylation reactions is discussed in relation to the maintenance of oxidized and reduced coenzyme levels. INTRODUCTION
In recent years considerable interest has centred on the ability of several groups of parasites, in particular the helminths, to utilize exogenous sources of CO2 for metabolic purposes. However, relatively little attention has been paid to the generality or possible significance of CO2 fixation in parasitic protozoa. In helminths, fixation of CO2 and the ultimate appearance of carbon from this source in excreted succinate and volatile acids appears to be a rather general phenomenon (Scheibel & Saz, 1966). In view of the well-known excretion of large amounts of succinate by the culture forms of T. cruzi (Bowman, Tobie & von Brand, 1963) and T. brucei rhodesiense (Ryley, 1962), particularly under anaerobic conditions, it seemed of value to reassess the enzymology and importance of this pathway. The initial fixation of carbon from CO2, and its ultimate fate, have been rather extensively studied in helminths by Saz (1972) and Bryant (1972), who concluded that carboxylation of phosphoenolpyruvate (PEP) by a PEP carboxykinase was quantitatively the most important reaction fixing CO2. Also involved, but quantitatively less significant, was an acetyl CoA stimulated pyruvate carboxylase (Bryant, 1972). The importance of 'malic enzyme' activity (E.C. 1.1.1.38-40) as a source of CO2 fixation, as opposed to pyruvate dismutation, was discounted by Bryant (1972). The subsequent fate of carbon from CO2 was compatible with the pathway oxaloacetate > malate > fumarate > succinate. Information on the enzymology of CO2 fixation in trypanosomatid flagellates is much more scanty; apart from the early report (Raw, 1959) of 'malic enzyme' activity in T. cruzi, the only substantial work is that of Bacchi, Ciaccio, Kaback & Hutner (1970). These workers, using the A.T.C.C. 11745 strain of Crithidia t Present address: Cytogenetics Unit, University of Liverpool, Liverpool L69 3BX.
94
R. A. KLEIN, D. J. LINSTEAD AND M. V. WHEELER
fascicidata, an Anopheline isolate, showed that an ADP-requiring PEP carboxylase (E.C. 4.1.1.32) was quantitatively the most important CO2-fixing enzyme in the organism. However, some caution must be exercised in interpreting the results of these workers. The assay system used involved coupling of the reaction > oxaloacetate PEP + CO2 to the highly active endogenous malate dehydrogenase and measuring the disappearance of added reduced nicotinamide adenine dinucleotide (NADH) at 340 nm. This assay does not directly measure fixed CO2, and account must be taken of other endogenous systems capable of reducing NAD, or oxidizing NADH, particularly in the light of the competing reaction PEP+ADP i=i pyruvate +ATP catalysed by pyruvate kinase (Bacchi et al. 1970). In the light of this objection and the increasing complications induced by attempts to measure both ' malic enzyme' and pyruvate carboxylase activity by an indirect method in such a heterogenous system as a cell homogenate, we preferred the more direct method of measuring incorporation of 14CO2 into acid-stable material.
MATERIALS AND METHODS
Crithidia fasciculata Anopheline isolate was maintained at 25 °C in a complex medium of composition: 20 g Difco proteose peptone, 2 g Difco liver infusion broth, 2 g Difco yeast extract, 50 mg adenine, 5 mg folic acid, 10 g glucose per litre of glass distilled water. The pH of the medium was adjusted to pH 8-0 before autoclaving. This medium was also suitable for growth of Trypanosoma mega. Both organisms were subcultured at intervals of 4 days. The isolate of T. mega used was that originally obtained from M. Steinert (Steinert & Bone, 1956), which has been maintained by routine subculture in complex medium ever since. Trypanosoma brucei brucei strains 427 (monomorphic) and S 42 (pleiomorphic) were maintained as stabilates in liquid nitrogen and passaged in laboratory rats as necessary (Cross & Manning, 1973). Rats (350-400 g) were given an inoculum of 1-2 x 107 organisms by intraperitoneal injection, and the infection allowed to proceed for 3 days. Whole blood was collected by aortic puncture when the parasitaemia reached 1 x 109 trypanosomes per ml. Trypanosomes were isolated from infected blood by the method of Lanham (1968). A culture form of T. b. brucei S 42 was maintained at 25 °C in a modified defined medium after Cross & Manning (1973), lacking both linoleic acid and sodium succinate, and with sodium acetate reduced to 10 mg/1. Cells were subjected to twice weekly subculture in this medium. Acetyl CoA, adenosine diphosphate (ADP) and triphosphate (ATP), phosphoenolpyruvate (PEP), nicotinamide adenine dinucleotide and its reduced form (NAD, NADH), nicotinamide adenine dinucleotide phosphate and its reduced form (NADP, NADPH) and pyruvic acid were obtained from the Sigma Chemical Co., Kingston-Upon-Thames, U.K. Guanosine diphosphate (GDP), inosine diphosphate (IDP) and cytosine diphosphate (CDP) were obtained from Cambrian Biochemicals, Croydon, U.K. NaH14CO3 was obtained from the Radiochemical
Carbon dioxide fixation in trypanosomatids
95
Centre, Amersham, Bucks., U.K. and was diluted with distilled water to a concentration of 100 /^Ci/ml, 1-6 HIM. NaH^Og was obtained from Prochem division of the British Oxygen Company. All other reagents were of analytical grade, and glass-distilled water was used throughout. Incorporation of radioactivity into acid-stable material was measured essentially according to the method of Lane, Chang & Miller (1969). For the measurement of specific activities protein concentrations were measured by the method of Lowry, Rosebrough, Farr & Randall (1951) with bovine serum albumin (Sigma fraction V) as the standard; a correction was made for interference in this assay by components of the suspending buffer. For the investigation of the distribution of radiocarbon, reactions in homogenates were terminated by adding perchloric acid to give a final concentration of 2 N and removing the protein precipitate by centrifugation. The supernatant fluid was taken to neutrality with concentrated KOH in the cold (0-2 °C) and the resulting precipitate again spun down. The neutral supernatant fluid was lyophylized and stored at — 20 °C. For ion-exchange chromatography the lyophilized solid was dissolved in a minimum volume of distilled water and applied to a 0-8 x 26 cm column of Dowex 1-X8 (Sigma) in the formate form. The column was eluted with a water-3 N formic acid gradient, and then with 2 N ammonium formate according to the method of La Noue, Nicklas & Williamson (1970). Compounds were identified by co-chromatography on this column with authentic radioactive material and by co-chromatography on thin layer plates (20 x 20 cm cellulose 300-25, MachereyNagel and Co., 516, Diiren Werkstrasse 6-8, West Germany) in two different solvent systems (solvent 1 diethyl ether:formic acid:water 7:2:1 (v/v); solvent 2 phenol:water:formic acid 150:50:2 (w/v)). For the study of long-term incorporation of 13CO2 and 14CO2, T. mega and T. b. brucei were grown in modified defined medium in a large culture vessel with provision for continuous monitoring of pH and pO2 by autoclavable electrodes, and gas-phase 14CO2 concentration by means of a Gieger-Miiller counter through a thin, autoclavable aluminium foil window (Klein & Wheeler, 1973, unpublished). At the termination of the culture period, 14C- or 13C-labelled acidic material was isolated, purified and characterized as indicated in Fig. 1. Cells were harvested by centrifugation at 5000 g for 10 min, and the pellet washed with 145 mM sodium chloride. The pellet was then extracted with 20 ml chloroform-methanol and 4 ml of water added. Samples were taken from the aqueous and organic layers for the determination of radioactivity. The aqueous supernatant fluid left after harvesting the cells was acidified with concentrated hydrochloric acid to pH 2, and any free CO2 removed by bubbling with nitrogen for 60 min. The treated supernatant fluid was then applied to a 2 x 70 cm column of IRA-400 (British Drug Houses) and the column exhaustively washed with water. Organic acids were eluted with 1-5 M ammonium carbonate. Ammonia was removed from the eluate by heating to 80-90 °C for 1-2 h with stirring and bubbling with nitrogen, until no free ammonia was detectable. This solution was applied to a 3 x 23 cm column of IR-120 (British Drug Houses) in order to separate amino acids from other organic acids. The organic acid fraction 7
PAR 71
96
E. A. KLEIN, D. J. LINSTEAD AND M. V. WHEELER Culture HX12A plus~200 /id NaHl4CO3 and 20 mg cold sodium bicarbonate
Harvest by centrifugation
422±9x10'd.p.m.
1-4x10'cells
399±10x10 6 d.p.m. supernatant N 2 :60min:292±13x10*d.p.m.
Lipid 0-2x10*
Cell sap 1-6x10*
IRA-400 HCCy form
1-5M ammonium carbonate eluant T 6x10*
282±2x10*d.p.m.
N,: 80-90 °C: stir 279±9x10'd.p.m. IR-120H + form
275±10x10'd.p.m.
-,1-5M-NH 4 OH f 2-1x10'
Dry down and methylate (or ethylate) (see text)
GLC
Fig. 1. Diagrammatic representation of the column chromatography of organic acids present in the culture medium. Typical amounts of radioactivity recovered at each stage are shown. 69 % of the added 14CO2 was fixed, and of the activity fixed 94 % was recovered.
was concentrated \)y evaporation, and derivatives prepared. Typical amounts of radioactivity recovered at each stage are shown in Fig. 1. A scaled-down version of this procedure was used to isolate fumarate and succinate after interconversion by 'fumarate reductase' in homogenates. The acids were subsequently identified and quantitatively measured as their methyl esters by gas liquid chromatography
Carbon dioxide fixation in trypanosomatids
97
using a polyethylene glycol adipate (PEGA 10%) column at 130 °C in a Pye 104 chromatograph (Pye Instruments, Cambridge, U.K.). For the examination of distribution of U C, labelled acidic material was purified as in Fig. 1, and converted to the ethyl esters by treatment with 5 % (w/v) sulphuric acid in absolute ethanol in a sealed vial under nitrogen at 105 °C for 5 h. Under these conditions no detectable degradation of the succinic acid occurred. Diethyl succinate, which constituted > 90 % of the incorporated label, was purified by preparative gas-liquid chromatography, followed by collection of the material in a glass trap cooled with liquid nitrogen. Mass spectrometric analysis of the purified ester was carried out using an Associated Electrical Industries (U.K.) MS 902 mass spectrometer. Carbon-13 and proton nuclear magnetic resonance data were also used to determine the position of 1SC substitution in the succinic acid molecule. Phospho-enol pyruvate carboxylase, ADP stimulated (E.C. 4.1.1.32), pyruvate carboxylase (E.C. 6.4.1.1), 'malic enzyme' (E.C. 1.1.1.38-40) and 'fumarate reductase' (E.C. 1.3.99.1) were measured as indicated in the text, tables and figures. Experiments were carried out under air. Incubation vessels were small vials or test tubes fitted with self-sealing rubber caps through which injections could be made. Homogenates of the trypanosomatids were prepared by sonicating a concentrated suspension (~ 5x 108/ml) of the organisms in sucrose buffer (0-25 M sucrose, 0-020 M tris-(hydroxymethyl)aminoethane chloride, pH 7-9, at 4 °C) for 10 sec at 4 A using a Dawes Soniprobe type 1130 A (Dawes Instruments, London, U.K.). This sufficed to break over 95 % of organisms without the formation of very small particles. For separation of the particulate and soluble fractions this homogenate was diluted to 5 ml with sucrose buffer and centrifuged at 30 000 £ for 30 min using 10 ml adaptors in the 8 x 50 ml rotor of the MSE High Speed 18 centrifuge (Measuring and Scientific Instruments, London, U.K.). The particulate fraction was washed twice by centrifugation and resuspended in a small volume of sucrose buffer. Both fractions could be stored at — 20 °C for up to a month with less than 20 % loss of activity. RESULTS
The activities of three enzymes possibly concerned with the incorporation of carbon dioxide into organic acids in trypanosomatids were measured. These enzymes were PEP carboxykinase (ADP stimulated), pyruvate carboxylase and 'malic enzyme'. The requirements of the three enzymes as measured in a particulate fraction from T. mega are set out in Tables 1, 2 and 3. The requirements for optimal activity of PEP carboxykinase are given in Table 1. Optimal activity was observed in the presence of PEP, manganous ions and ADP. ATP could be exchanged for ADP, presumably because of the activity of endogenous ATP-ases. A similar phenomenon was observed by Bacchi et al. (1970). Enzyme activity was inhibited to varying degrees by UDP, IDP, CDP and acetyl CoA. The stimulating effect of magnesium ions was much less than that of manganese at equimolar concentrations. Table 2 shows the requirements for optimal ' malic enzyme' activity. Optimal 7-2
98
R. A. KLEIN, D. J. LINSTEAD AND M. V. WHEELER Table 1. Requirements for phosphoenolpyruvate carboxykinase activity in a particulate fraction from Trypanosoma mega (The enzyme was assayed at 25 °C in a total volume of 1-0 ml containing final concentrations of 0-1 M imidazole buffer (Cl~) pH 6-6, 0049 M KHCO3, 2-0 /id NaH14CO3, 000125 M ADP, 0001 M MnSO4, 0-020 at NADH, 000125 M PEP, 0-2 ml enzyme preparation. The reaction was started by the addition of [14C]bicarbonate. The subsequent procedure was that of Lane et al. (1969) except that a Triton-X-100based, water-compatible scintillation fluid was used (methanol: Triton X-100: toluene 1:6:13 by volume containing 5 g/1 2,5-diphenyloxazole, and 0-5 g/1 1,4-di 2-(5-phenyloxazolyl) benzene). Where present MgCl2 was 0-001 M, IDP, UDP, GDP and CDP were 0-00125 M and acetyl CoA was 0-001 at. Results are mean + standard deviation for four determinations. Limit of detection 0-01 nmole/min/mg.)
System No addition + PEP + NADH + Mn+2 + NADH + Mn+2 + PEP +Mn+2 +PEP + NADH + Mn+2 + P E P + NADPH +Mn+2 + PEP + NADH + ADP + Mn+2 + PEP + NADH + ATP + Mn+2 + P E P + NADH+ IDP + Mn+2 +PEP + NADH+ CDP + Mn+2 +PEP + NADH+ UDP + Mn+2 + PEP + NADH + GDP + Mn+2 + PEP + NADH + AcetylCoA + Mg+2 +PEP + NADH
Activity (nmoles 14CO2 incorporated/min/mg protein) 0-06 0-16 ±006 0-46 + 0-02 2-65±0-13 ll-44±0-95 6-33 ±013 14-79±0-75 16-02 ±0-89 6-00 ±0-47 8-47 ±0-47 9-33 ±0-66 8-66 ±0-68 9-31 ± 0-51 0-54 + 0-04
Table 2. 'Malic enzyme' activity in Trypanosoma mega particulate fraction (The enzyme was assayed at 25 °C in a total volume of 1-0 ml containing final concentrations of 01 M imidazole buffer (C1-) pH 6-6, 0-049 M KHCO3, 0-010 M pyruvic acid, 0001 at MnSO4, 00015 at NADPH; 20/tCi NaH14CO3; 0-2 ml enzyme preparation. The reaction was started by the addition of [14C]bicarbonate. The subsequent procedure was as for phosphoenolpyruvate carboxykinase. Where present NADH was 00015 M.) Activity (nmoles 14CO2 incorporSystem ated/ min/mg protein) No addition +Mn+2 + NADH + NADH + Pyruvate + Mn+2 + pyruvate + NADH + Mn+2 + pyruvate + NADPH
004
0-46 ± 002 006
0-29 + 0-01 1-04 ±0-08 2-77 ±0-30
activity required pyruvate, manganous ions and reduced pyridine nucleotide; NADPH was over twice as eifective as NADH in this respect. Pyruvate carboxylase activity was also found in preparations from T. mega. Optimal activity required pyruvate, acetyl CoA, ATP and magnesium ions (Table 3).
Carbon dioxide fixation in trypanosomatids
99
Table 3. Pyruvate carboxylase activity in a particulate fraction from Trypanosoma mega (The enzyme was assayed at 25 °C, in a total volume of 1-0 ml containing final concentrations of 0-1 M imidazole buffer (Cl~) pH 6-6, 0-010 M pyruvic acid, 0-049 M KHCO3, 0001 M ATP; 0005 M MgCl2, 0-001 M acetyl CoA; 2-0 /tCi NaH14CO3, 0-2 ml enzyme preparation. The reaction was started by the addition of [14C] bicarbonate. The subsequent procedure was as for phosphoenolpyruvate carboxykinase.)
System No addition + Pyruvate + Pyruvate + acetyl CoA + Pyruvate + acetyl CoA 4 ATP + Pyruvate + Mg+2 + pyruvate + acetylCoA + ATP + Mg+2
Activity (nmoles 14CO2 incorpor ated/min/mg protein) 005
0-29 ±001 0-29 ± 004 0-46 ±0-01 0-33 + 0-01 1-08 + 0-14
Table 4 shows the distribution of enzyme activities in soluble and particulate fractions from C. fasciculata, T. mega and T. b. brucei (culture form). PEP carboxykinase activity was present predominantly in the particulate fraction from T. mega, less markedly so for C. fasciculata and about equally distributed in the culture form of T.b. brucei. 'Malic enzyme' was present predominantly in the soluble fraction from both T. mega and T. b. brucei (culture form), but was mainly in the particulate fraction from C. fasciculata. Pyruvate carboxylase was predominantly in the particulate fraction from both T. mega and C. fasciculata; it could not be detected in either fraction from T. b. brucei, whether culture or bloodstream form, under the conditions of assay described. The ADP-stimulated PEP-carboxykinase was the major CO2-fixing enzyme in T. mega and C. fasciculata, and was also highly active in the culture form of T. b. brucei. However, the T. b. brucei enzyme appeared to differ from the enzyme from the other two organisms by its much more stringent requirement for ADP (~ 8-fold stimulation). 'Malic enzyme' activity was markedly less in C'.fasciculata than in the two Trypanosoma species investigated. In particular 'malic enzyme' was the overwhelmingly predominant activity in T. b. brucei culture form. Pyruvate carboxylase activity was much less than either of the other two activities, though it could be readily detected in both C. fasciculata and T. mega. In the bloodstream form of T. b. brucei S 42 there was much less overall 14CO2 fixing activity than in its culture counterpart. Once again pyruvate carboxylase activity could not be detected, but 'malic enzyme' and ADP-stimulated PEP carboxykinase were both present. Carboxylating activities were measured in whole homogenates of bloodstream forms. Results are included in Table 4. For the investigation of the short term distribution of 14C-label whole homogenates from G. fasciculata and T. mega were incubated for 20 min under optimal conditions for the PEP-carboxykinase in the presence of 10 /tCi (about 0-16 /imoles) of NaH14CO3; no additional unlabelled bicarbonate was supplied. Labelled material was isolated and fractionated as described in the methods section. The distribution
Pyruvate carboxylase
'Malic enzyme'
— = not determined.
Organism
T. mega G. fasciculata T. b. brucei (culture) T. b. brucei (bloodstream) T. mega C. fasciculata T. b. brucei (culture) T. b. brucei (bloodstream) T. mega 0. fasciculata T. b. brucei (culture) T. b. brucei (bloodstream)
Enzyme
PEP carboxykinase (ADP stimulated)
Soluble fraction (%) 90 32-5 47-5 — 78-2 12-0 90-5 — 22-5 24-5 Not detectable Not detectable Particulate fraction (%) 91-0 67-5 52-5 — 21-8 88-0 9-5 — 77-5 75-5 — .—
12-29 ±0-24 16-40±l-59 ll-59±l-00 0-67 + 0-05 8-69 ±0-36 4-42 ±0-28 36-20 ±2-87 2-27 + 0-09 l-56±015 1-31 + 0-15 < 0-02 < 0-02
Total activity (nmoles 14CO2/min/mg protein)
(Activities were determined as in Tables 1-3, soluble and particulate fractions were prepared as described in the methods section. Results are expressed as a percentage of total activity for each enzyme.)
Table 4. The occurrence and distribution of enzyme activities fixing C02 in Trypanosoma mega, Crithidia fasciculata, Trypanosoma brucei culture form S 42 and T. b. brucei bloodstream form S 42
F
o
Carbon dioxide fixation in trypanosomatids
101
Table 5. Distribution of label following incubation of whole homogenates o/Trypanosoma mega and Crithidia fasciculata with [14O] bicarbonate for 20 min at 25 °C (Conditions were optimal for P E P carboxykinase (ADP-stimulated) as described in Table 1: 0-1 M imidazole buffer (Cl") p H 6-6; 10/
93
With 3 figures in the text
Carbon dioxide fixation in trypanosomatids R. A. KLEIN, D. J. LINSTEAD and M. V. WHEELERf Medical Research Council, Biochemical Parasitology Unit, Molteno Institute, University of Cambridge, Downing Street, Cambridge CB2 3EE (Received 24 January 1975) SUMMARY
Fixation of carbon dioxide has been demonstrated for extracts from Crithidia fasciculata, Trypanosoma mega and Trypanosoma brucei brucei bloodstream and culture forms. The enzymes involved in this fixation were found to be ADP-stimulated phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32), 'malic' enzyme (E.C. 1.1.138-40) and pyruvate carboxylase (E.C. 6.4.1.1). The subcellular localization of these enzymes has been investigated in all three organisms. Products of short and long term fixation experiments were separated and identified. The importance of carboxylation reactions is discussed in relation to the maintenance of oxidized and reduced coenzyme levels. INTRODUCTION
In recent years considerable interest has centred on the ability of several groups of parasites, in particular the helminths, to utilize exogenous sources of CO2 for metabolic purposes. However, relatively little attention has been paid to the generality or possible significance of CO2 fixation in parasitic protozoa. In helminths, fixation of CO2 and the ultimate appearance of carbon from this source in excreted succinate and volatile acids appears to be a rather general phenomenon (Scheibel & Saz, 1966). In view of the well-known excretion of large amounts of succinate by the culture forms of T. cruzi (Bowman, Tobie & von Brand, 1963) and T. brucei rhodesiense (Ryley, 1962), particularly under anaerobic conditions, it seemed of value to reassess the enzymology and importance of this pathway. The initial fixation of carbon from CO2, and its ultimate fate, have been rather extensively studied in helminths by Saz (1972) and Bryant (1972), who concluded that carboxylation of phosphoenolpyruvate (PEP) by a PEP carboxykinase was quantitatively the most important reaction fixing CO2. Also involved, but quantitatively less significant, was an acetyl CoA stimulated pyruvate carboxylase (Bryant, 1972). The importance of 'malic enzyme' activity (E.C. 1.1.1.38-40) as a source of CO2 fixation, as opposed to pyruvate dismutation, was discounted by Bryant (1972). The subsequent fate of carbon from CO2 was compatible with the pathway oxaloacetate > malate > fumarate > succinate. Information on the enzymology of CO2 fixation in trypanosomatid flagellates is much more scanty; apart from the early report (Raw, 1959) of 'malic enzyme' activity in T. cruzi, the only substantial work is that of Bacchi, Ciaccio, Kaback & Hutner (1970). These workers, using the A.T.C.C. 11745 strain of Crithidia t Present address: Cytogenetics Unit, University of Liverpool, Liverpool L69 3BX.
94
R. A. KLEIN, D. J. LINSTEAD AND M. V. WHEELER
fascicidata, an Anopheline isolate, showed that an ADP-requiring PEP carboxylase (E.C. 4.1.1.32) was quantitatively the most important CO2-fixing enzyme in the organism. However, some caution must be exercised in interpreting the results of these workers. The assay system used involved coupling of the reaction > oxaloacetate PEP + CO2 to the highly active endogenous malate dehydrogenase and measuring the disappearance of added reduced nicotinamide adenine dinucleotide (NADH) at 340 nm. This assay does not directly measure fixed CO2, and account must be taken of other endogenous systems capable of reducing NAD, or oxidizing NADH, particularly in the light of the competing reaction PEP+ADP i=i pyruvate +ATP catalysed by pyruvate kinase (Bacchi et al. 1970). In the light of this objection and the increasing complications induced by attempts to measure both ' malic enzyme' and pyruvate carboxylase activity by an indirect method in such a heterogenous system as a cell homogenate, we preferred the more direct method of measuring incorporation of 14CO2 into acid-stable material.
MATERIALS AND METHODS
Crithidia fasciculata Anopheline isolate was maintained at 25 °C in a complex medium of composition: 20 g Difco proteose peptone, 2 g Difco liver infusion broth, 2 g Difco yeast extract, 50 mg adenine, 5 mg folic acid, 10 g glucose per litre of glass distilled water. The pH of the medium was adjusted to pH 8-0 before autoclaving. This medium was also suitable for growth of Trypanosoma mega. Both organisms were subcultured at intervals of 4 days. The isolate of T. mega used was that originally obtained from M. Steinert (Steinert & Bone, 1956), which has been maintained by routine subculture in complex medium ever since. Trypanosoma brucei brucei strains 427 (monomorphic) and S 42 (pleiomorphic) were maintained as stabilates in liquid nitrogen and passaged in laboratory rats as necessary (Cross & Manning, 1973). Rats (350-400 g) were given an inoculum of 1-2 x 107 organisms by intraperitoneal injection, and the infection allowed to proceed for 3 days. Whole blood was collected by aortic puncture when the parasitaemia reached 1 x 109 trypanosomes per ml. Trypanosomes were isolated from infected blood by the method of Lanham (1968). A culture form of T. b. brucei S 42 was maintained at 25 °C in a modified defined medium after Cross & Manning (1973), lacking both linoleic acid and sodium succinate, and with sodium acetate reduced to 10 mg/1. Cells were subjected to twice weekly subculture in this medium. Acetyl CoA, adenosine diphosphate (ADP) and triphosphate (ATP), phosphoenolpyruvate (PEP), nicotinamide adenine dinucleotide and its reduced form (NAD, NADH), nicotinamide adenine dinucleotide phosphate and its reduced form (NADP, NADPH) and pyruvic acid were obtained from the Sigma Chemical Co., Kingston-Upon-Thames, U.K. Guanosine diphosphate (GDP), inosine diphosphate (IDP) and cytosine diphosphate (CDP) were obtained from Cambrian Biochemicals, Croydon, U.K. NaH14CO3 was obtained from the Radiochemical
Carbon dioxide fixation in trypanosomatids
95
Centre, Amersham, Bucks., U.K. and was diluted with distilled water to a concentration of 100 /^Ci/ml, 1-6 HIM. NaH^Og was obtained from Prochem division of the British Oxygen Company. All other reagents were of analytical grade, and glass-distilled water was used throughout. Incorporation of radioactivity into acid-stable material was measured essentially according to the method of Lane, Chang & Miller (1969). For the measurement of specific activities protein concentrations were measured by the method of Lowry, Rosebrough, Farr & Randall (1951) with bovine serum albumin (Sigma fraction V) as the standard; a correction was made for interference in this assay by components of the suspending buffer. For the investigation of the distribution of radiocarbon, reactions in homogenates were terminated by adding perchloric acid to give a final concentration of 2 N and removing the protein precipitate by centrifugation. The supernatant fluid was taken to neutrality with concentrated KOH in the cold (0-2 °C) and the resulting precipitate again spun down. The neutral supernatant fluid was lyophylized and stored at — 20 °C. For ion-exchange chromatography the lyophilized solid was dissolved in a minimum volume of distilled water and applied to a 0-8 x 26 cm column of Dowex 1-X8 (Sigma) in the formate form. The column was eluted with a water-3 N formic acid gradient, and then with 2 N ammonium formate according to the method of La Noue, Nicklas & Williamson (1970). Compounds were identified by co-chromatography on this column with authentic radioactive material and by co-chromatography on thin layer plates (20 x 20 cm cellulose 300-25, MachereyNagel and Co., 516, Diiren Werkstrasse 6-8, West Germany) in two different solvent systems (solvent 1 diethyl ether:formic acid:water 7:2:1 (v/v); solvent 2 phenol:water:formic acid 150:50:2 (w/v)). For the study of long-term incorporation of 13CO2 and 14CO2, T. mega and T. b. brucei were grown in modified defined medium in a large culture vessel with provision for continuous monitoring of pH and pO2 by autoclavable electrodes, and gas-phase 14CO2 concentration by means of a Gieger-Miiller counter through a thin, autoclavable aluminium foil window (Klein & Wheeler, 1973, unpublished). At the termination of the culture period, 14C- or 13C-labelled acidic material was isolated, purified and characterized as indicated in Fig. 1. Cells were harvested by centrifugation at 5000 g for 10 min, and the pellet washed with 145 mM sodium chloride. The pellet was then extracted with 20 ml chloroform-methanol and 4 ml of water added. Samples were taken from the aqueous and organic layers for the determination of radioactivity. The aqueous supernatant fluid left after harvesting the cells was acidified with concentrated hydrochloric acid to pH 2, and any free CO2 removed by bubbling with nitrogen for 60 min. The treated supernatant fluid was then applied to a 2 x 70 cm column of IRA-400 (British Drug Houses) and the column exhaustively washed with water. Organic acids were eluted with 1-5 M ammonium carbonate. Ammonia was removed from the eluate by heating to 80-90 °C for 1-2 h with stirring and bubbling with nitrogen, until no free ammonia was detectable. This solution was applied to a 3 x 23 cm column of IR-120 (British Drug Houses) in order to separate amino acids from other organic acids. The organic acid fraction 7
PAR 71
96
E. A. KLEIN, D. J. LINSTEAD AND M. V. WHEELER Culture HX12A plus~200 /id NaHl4CO3 and 20 mg cold sodium bicarbonate
Harvest by centrifugation
422±9x10'd.p.m.
1-4x10'cells
399±10x10 6 d.p.m. supernatant N 2 :60min:292±13x10*d.p.m.
Lipid 0-2x10*
Cell sap 1-6x10*
IRA-400 HCCy form
1-5M ammonium carbonate eluant T 6x10*
282±2x10*d.p.m.
N,: 80-90 °C: stir 279±9x10'd.p.m. IR-120H + form
275±10x10'd.p.m.
-,1-5M-NH 4 OH f 2-1x10'
Dry down and methylate (or ethylate) (see text)
GLC
Fig. 1. Diagrammatic representation of the column chromatography of organic acids present in the culture medium. Typical amounts of radioactivity recovered at each stage are shown. 69 % of the added 14CO2 was fixed, and of the activity fixed 94 % was recovered.
was concentrated \)y evaporation, and derivatives prepared. Typical amounts of radioactivity recovered at each stage are shown in Fig. 1. A scaled-down version of this procedure was used to isolate fumarate and succinate after interconversion by 'fumarate reductase' in homogenates. The acids were subsequently identified and quantitatively measured as their methyl esters by gas liquid chromatography
Carbon dioxide fixation in trypanosomatids
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using a polyethylene glycol adipate (PEGA 10%) column at 130 °C in a Pye 104 chromatograph (Pye Instruments, Cambridge, U.K.). For the examination of distribution of U C, labelled acidic material was purified as in Fig. 1, and converted to the ethyl esters by treatment with 5 % (w/v) sulphuric acid in absolute ethanol in a sealed vial under nitrogen at 105 °C for 5 h. Under these conditions no detectable degradation of the succinic acid occurred. Diethyl succinate, which constituted > 90 % of the incorporated label, was purified by preparative gas-liquid chromatography, followed by collection of the material in a glass trap cooled with liquid nitrogen. Mass spectrometric analysis of the purified ester was carried out using an Associated Electrical Industries (U.K.) MS 902 mass spectrometer. Carbon-13 and proton nuclear magnetic resonance data were also used to determine the position of 1SC substitution in the succinic acid molecule. Phospho-enol pyruvate carboxylase, ADP stimulated (E.C. 4.1.1.32), pyruvate carboxylase (E.C. 6.4.1.1), 'malic enzyme' (E.C. 1.1.1.38-40) and 'fumarate reductase' (E.C. 1.3.99.1) were measured as indicated in the text, tables and figures. Experiments were carried out under air. Incubation vessels were small vials or test tubes fitted with self-sealing rubber caps through which injections could be made. Homogenates of the trypanosomatids were prepared by sonicating a concentrated suspension (~ 5x 108/ml) of the organisms in sucrose buffer (0-25 M sucrose, 0-020 M tris-(hydroxymethyl)aminoethane chloride, pH 7-9, at 4 °C) for 10 sec at 4 A using a Dawes Soniprobe type 1130 A (Dawes Instruments, London, U.K.). This sufficed to break over 95 % of organisms without the formation of very small particles. For separation of the particulate and soluble fractions this homogenate was diluted to 5 ml with sucrose buffer and centrifuged at 30 000 £ for 30 min using 10 ml adaptors in the 8 x 50 ml rotor of the MSE High Speed 18 centrifuge (Measuring and Scientific Instruments, London, U.K.). The particulate fraction was washed twice by centrifugation and resuspended in a small volume of sucrose buffer. Both fractions could be stored at — 20 °C for up to a month with less than 20 % loss of activity. RESULTS
The activities of three enzymes possibly concerned with the incorporation of carbon dioxide into organic acids in trypanosomatids were measured. These enzymes were PEP carboxykinase (ADP stimulated), pyruvate carboxylase and 'malic enzyme'. The requirements of the three enzymes as measured in a particulate fraction from T. mega are set out in Tables 1, 2 and 3. The requirements for optimal activity of PEP carboxykinase are given in Table 1. Optimal activity was observed in the presence of PEP, manganous ions and ADP. ATP could be exchanged for ADP, presumably because of the activity of endogenous ATP-ases. A similar phenomenon was observed by Bacchi et al. (1970). Enzyme activity was inhibited to varying degrees by UDP, IDP, CDP and acetyl CoA. The stimulating effect of magnesium ions was much less than that of manganese at equimolar concentrations. Table 2 shows the requirements for optimal ' malic enzyme' activity. Optimal 7-2
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R. A. KLEIN, D. J. LINSTEAD AND M. V. WHEELER Table 1. Requirements for phosphoenolpyruvate carboxykinase activity in a particulate fraction from Trypanosoma mega (The enzyme was assayed at 25 °C in a total volume of 1-0 ml containing final concentrations of 0-1 M imidazole buffer (Cl~) pH 6-6, 0049 M KHCO3, 2-0 /id NaH14CO3, 000125 M ADP, 0001 M MnSO4, 0-020 at NADH, 000125 M PEP, 0-2 ml enzyme preparation. The reaction was started by the addition of [14C]bicarbonate. The subsequent procedure was that of Lane et al. (1969) except that a Triton-X-100based, water-compatible scintillation fluid was used (methanol: Triton X-100: toluene 1:6:13 by volume containing 5 g/1 2,5-diphenyloxazole, and 0-5 g/1 1,4-di 2-(5-phenyloxazolyl) benzene). Where present MgCl2 was 0-001 M, IDP, UDP, GDP and CDP were 0-00125 M and acetyl CoA was 0-001 at. Results are mean + standard deviation for four determinations. Limit of detection 0-01 nmole/min/mg.)
System No addition + PEP + NADH + Mn+2 + NADH + Mn+2 + PEP +Mn+2 +PEP + NADH + Mn+2 + P E P + NADPH +Mn+2 + PEP + NADH + ADP + Mn+2 + PEP + NADH + ATP + Mn+2 + P E P + NADH+ IDP + Mn+2 +PEP + NADH+ CDP + Mn+2 +PEP + NADH+ UDP + Mn+2 + PEP + NADH + GDP + Mn+2 + PEP + NADH + AcetylCoA + Mg+2 +PEP + NADH
Activity (nmoles 14CO2 incorporated/min/mg protein) 0-06 0-16 ±006 0-46 + 0-02 2-65±0-13 ll-44±0-95 6-33 ±013 14-79±0-75 16-02 ±0-89 6-00 ±0-47 8-47 ±0-47 9-33 ±0-66 8-66 ±0-68 9-31 ± 0-51 0-54 + 0-04
Table 2. 'Malic enzyme' activity in Trypanosoma mega particulate fraction (The enzyme was assayed at 25 °C in a total volume of 1-0 ml containing final concentrations of 01 M imidazole buffer (C1-) pH 6-6, 0-049 M KHCO3, 0-010 M pyruvic acid, 0001 at MnSO4, 00015 at NADPH; 20/tCi NaH14CO3; 0-2 ml enzyme preparation. The reaction was started by the addition of [14C]bicarbonate. The subsequent procedure was as for phosphoenolpyruvate carboxykinase. Where present NADH was 00015 M.) Activity (nmoles 14CO2 incorporSystem ated/ min/mg protein) No addition +Mn+2 + NADH + NADH + Pyruvate + Mn+2 + pyruvate + NADH + Mn+2 + pyruvate + NADPH
004
0-46 ± 002 006
0-29 + 0-01 1-04 ±0-08 2-77 ±0-30
activity required pyruvate, manganous ions and reduced pyridine nucleotide; NADPH was over twice as eifective as NADH in this respect. Pyruvate carboxylase activity was also found in preparations from T. mega. Optimal activity required pyruvate, acetyl CoA, ATP and magnesium ions (Table 3).
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Table 3. Pyruvate carboxylase activity in a particulate fraction from Trypanosoma mega (The enzyme was assayed at 25 °C, in a total volume of 1-0 ml containing final concentrations of 0-1 M imidazole buffer (Cl~) pH 6-6, 0-010 M pyruvic acid, 0-049 M KHCO3, 0001 M ATP; 0005 M MgCl2, 0-001 M acetyl CoA; 2-0 /tCi NaH14CO3, 0-2 ml enzyme preparation. The reaction was started by the addition of [14C] bicarbonate. The subsequent procedure was as for phosphoenolpyruvate carboxykinase.)
System No addition + Pyruvate + Pyruvate + acetyl CoA + Pyruvate + acetyl CoA 4 ATP + Pyruvate + Mg+2 + pyruvate + acetylCoA + ATP + Mg+2
Activity (nmoles 14CO2 incorpor ated/min/mg protein) 005
0-29 ±001 0-29 ± 004 0-46 ±0-01 0-33 + 0-01 1-08 + 0-14
Table 4 shows the distribution of enzyme activities in soluble and particulate fractions from C. fasciculata, T. mega and T. b. brucei (culture form). PEP carboxykinase activity was present predominantly in the particulate fraction from T. mega, less markedly so for C. fasciculata and about equally distributed in the culture form of T.b. brucei. 'Malic enzyme' was present predominantly in the soluble fraction from both T. mega and T. b. brucei (culture form), but was mainly in the particulate fraction from C. fasciculata. Pyruvate carboxylase was predominantly in the particulate fraction from both T. mega and C. fasciculata; it could not be detected in either fraction from T. b. brucei, whether culture or bloodstream form, under the conditions of assay described. The ADP-stimulated PEP-carboxykinase was the major CO2-fixing enzyme in T. mega and C. fasciculata, and was also highly active in the culture form of T. b. brucei. However, the T. b. brucei enzyme appeared to differ from the enzyme from the other two organisms by its much more stringent requirement for ADP (~ 8-fold stimulation). 'Malic enzyme' activity was markedly less in C'.fasciculata than in the two Trypanosoma species investigated. In particular 'malic enzyme' was the overwhelmingly predominant activity in T. b. brucei culture form. Pyruvate carboxylase activity was much less than either of the other two activities, though it could be readily detected in both C. fasciculata and T. mega. In the bloodstream form of T. b. brucei S 42 there was much less overall 14CO2 fixing activity than in its culture counterpart. Once again pyruvate carboxylase activity could not be detected, but 'malic enzyme' and ADP-stimulated PEP carboxykinase were both present. Carboxylating activities were measured in whole homogenates of bloodstream forms. Results are included in Table 4. For the investigation of the short term distribution of 14C-label whole homogenates from G. fasciculata and T. mega were incubated for 20 min under optimal conditions for the PEP-carboxykinase in the presence of 10 /tCi (about 0-16 /imoles) of NaH14CO3; no additional unlabelled bicarbonate was supplied. Labelled material was isolated and fractionated as described in the methods section. The distribution
Pyruvate carboxylase
'Malic enzyme'
— = not determined.
Organism
T. mega G. fasciculata T. b. brucei (culture) T. b. brucei (bloodstream) T. mega C. fasciculata T. b. brucei (culture) T. b. brucei (bloodstream) T. mega 0. fasciculata T. b. brucei (culture) T. b. brucei (bloodstream)
Enzyme
PEP carboxykinase (ADP stimulated)
Soluble fraction (%) 90 32-5 47-5 — 78-2 12-0 90-5 — 22-5 24-5 Not detectable Not detectable Particulate fraction (%) 91-0 67-5 52-5 — 21-8 88-0 9-5 — 77-5 75-5 — .—
12-29 ±0-24 16-40±l-59 ll-59±l-00 0-67 + 0-05 8-69 ±0-36 4-42 ±0-28 36-20 ±2-87 2-27 + 0-09 l-56±015 1-31 + 0-15 < 0-02 < 0-02
Total activity (nmoles 14CO2/min/mg protein)
(Activities were determined as in Tables 1-3, soluble and particulate fractions were prepared as described in the methods section. Results are expressed as a percentage of total activity for each enzyme.)
Table 4. The occurrence and distribution of enzyme activities fixing C02 in Trypanosoma mega, Crithidia fasciculata, Trypanosoma brucei culture form S 42 and T. b. brucei bloodstream form S 42
F
o
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Table 5. Distribution of label following incubation of whole homogenates o/Trypanosoma mega and Crithidia fasciculata with [14O] bicarbonate for 20 min at 25 °C (Conditions were optimal for P E P carboxykinase (ADP-stimulated) as described in Table 1: 0-1 M imidazole buffer (Cl") p H 6-6; 10/
