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The pathway of arginine catabolism in Giardia intestinalis
Molecular and Biochemical Parasitology, 51 (1992) 29 36 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
29
MOLBIO 01674
The pathway of arginine catabolism in Giardia intestinalis Philip J. Schofield, M i c h a e l R. E d w a r d s , J a c q u e l i n e M a t t h e w s a n d Justine R. W i l s o n School of Biochemistry, University of New South Wales, Kensington, NSW, Australia (Received 21 June 1991; accepted 2 October 1991)
In Giardia intestinalis, arginine is catabolised by the arginine dihydrolase pathway. The enzymes of the pathway (arginine deiminase, ornithine transcarbamoylase and carbamate kinase) were investigated and their basic kinetic parameters determined. The specific activity of arginine deiminase was 270 _+ 23 nmol min i (mg protein)-~; ornithine transcarbamoylase, in the direction of citrulline utilisation 170 _+ 22 nmol min ~ (mg protein) l, and in the direction of ornithine utilisation 2100 + 100 nmol min i (mg protein) l; and carbamate kinase 2100 + 400 nmol rain i (mg protein) a. The activities of these enzymes are between 10 and 250 fold greater than those reported for the enzymes in Trichomonas vaginalis, the only other parasite in which the arginine dihydrolase pathway has been reported. The flux through the pathway in G. intestinalis, as determined by the liberation of 14C02from 1 mM [laC-guanidino]arginine was 30 nmol min-J (mg p r o t e i n ) - i This flux was not affected by valinomycin (0.1 /~M), nigericin (3/~M), azide (5 mM) or cyanide (1 mM). The flux was only marginally affected by glucose up to 10 mM concentration. Conversely, the flux through glucose metabolism, as determined by the release of 14CO2 from 1 mM [l-14C]glucose was only 2 nmol rain I (mg protein)- t, and was unaffected by arginine concentrations up to 10 mM. These observations suggest that there is no direct metabolic interface between arginine and glucose catabolism. The potential energy yield of ATP from the arginine flux is 7 8-fold greater than that from glucose, providing evidence for the prime importance of arginine in the energy economy of G. intestinalis. It is suggested that the flux through the arginine dihydrolase pathway is the major source of energy production, particularly in anaerobic conditions. Key words: Giardia intestinalis; Arginine metabolism; Arginine dihydrolase pathway; Arginine deiminase; Ornithine transcarbamoylase; Carbamate kinase
Introduction
Although it has been commonly accepted that glucose is the only source of energy for Giardia intestinalis, there is mounting evidence that this is not so. Glucose catabolism cannot account for all the end products of G. intestinalis [1] and it is evident that other carbon sources must be utilised. The most likely of these other sources are amino acids Correspondence address: P.J. Schofield, School of Biochemistry, University of NSW, P.O. Box 1, Kensington, NSW 2033, Australia. Abbreviations: PBS, phosphate-buffered saline; OTC, ornithine transearbamoylase (EC 2.1.3.3); ADI, arginine deiminase (EC 3.5.3.6); CK, carbamate kinase (EC 2.7.2.2).
and/or proteins. Recent observations have indicated that a number of amino acids are involved in the energy metabolism during the proliferation of G. intestinalis in culture [2]. A dramatic feature of the changes in the amino acid profile of the medium is a rapid depletion of arginine [2]. The avid consumption of arginine by G. intestinalis suggested that it had a significant role as an energy source, a view that was consolidated by our observation that the arginine was being utilised by the arginine dihydrolase pathway [3]. This pathway, which converts arginine to ornithine and ammonia, has the biological advantage that it produces ATP directly via substrate level phosphorylation, and eliminates the need for oxygen or for intermediate redox systems. It is thus most appropriate for G. intestinalis whose
30
natural environment is low in oxygen tension and which is generally regarded as being microaerophilic or a facultative anaerobe. Although the arginine dihydrolase pathway occurs in a number of prokaryotes, the only eukaryotic organism in which it has been previously observed is another parasitic protozoan, Trichomonas vaginalis [4]. In a previous short communication we reported activities for the enzymes of the arginine dihydrolase pathway [3]. We now report results demonstrating the biological potential of this pathway in Giardia together with the characterisation of the basic properties of the individual enzymes of the pathway.
Materials and Methods
Organism. G. intestinalis trophozoites (Portland 1 strain) were grown in TYI-S-33 medium as described previously [2]. Materials. [14C-guanidino]arginine and [14Ccarbamoyl]citrulline were obtained from Amersham. All other chemicals were of the highest grade commercially available.
Preparation of extracts. The trophozoites were detached from the vessel wall by cooling in ice, collected by centrifugation and sonicared in 0.25 M sucrose with 5 x 20 s bursts at 80 W. The homogenate was centrifuged for 2 min (Beckman Microfuge) and the supernatant used for enzyme assays. General method jor 14C0 2 collection. Trophozoites or extracts were incubated in conical flasks sealed with rubber seals. The final total volume was 1 ml. The flasks contained an inner small glass vial seated in a larger glass vial. The inner glass vial contained 100 ~tl 1 M hyamine hydroxide in methanol. The incubation was initiated by the addition of cells or extract, the flask immediately sealed, and the reaction allowed to proceed at 37°C. At the end of the appropriate incubation time the reaction was terminated by the injection of 1 ml 1.3 M perchloric acid. Absorption of the liberated
14C0 2 was allowed to proceed for 1 hour in a shaking water bath. The inner well containing the absorbed ~4CO2 in hyamine was then removed, placed in a scintillation vial containing 10 ml scintillant (0.5% 2,5-diphenyloxazole in toluene) and the radioactivity determined in a Packard Tricarb 300 scintillation counter.
Arginine JTux. The flux through the arginine dihydrolase pathway was determined from the 14COz production from [14C-guanidino] arginine. The incubation mixture contained, in a total volume of 1 ml of phosphate buffered saline (PBS)/1.8 mM KH2PO4/5 mM KzHPO4/0.9% (w/v) NaCI pH 7.4/1 mM arginine (0.1 /iCi) and trophozoites (approx. 0.2 m~ protein which is equivalent to approx. 5 x 10°cells). The reaction was initiated by the addition of the cells, and the ~4CO2 release determined. For the determination of the effect of pH on arginine flux, the buffer used was 150 mM NaCI/5 m M KC1/10 mM Mes/10 mM Hepes for the pH range 5-8. Glucose Jlux. The flux through glucose metabolism was determined from the release of 14CO2 from [1-14C]glucose. The incubation mixture contained, in a total volume of 1 ml, PBS pH 7.4, 1 mM [1-14C]glucose (0.1/~Ci) and trophozoites (approx. 5 x 106 cells). The ~4CO2 released was determined as above. Arginine deiminase (EC 3.5.3.6).
The activity was assayed by the colorimetric determination of the product citrulline. The assay is essentially that described by Linstead and Cranshaw [4]. The assay mixture contained 1 mM arginine/40 mM Mes pH 7 and extract (approx. 0.1 mg protein). The incubation time was 20 min at 37°C. The reaction was terminated by the addition of 0.2 ml 6% (w/v) trichloroacetic acid and citrulline was determined by the method of Boyde and Rahmatullah [5].
Ornithine transcarbamoylase ( EC 2.1.3.3) assayed in the direction of citrulline utilisation. The activity was determined by the release of
31
14CO2from L-[~4C-carbamoyl]citrulline in the presence of added ADP and Pi. The incubation mixture contained 40 mM Mes, pH 7/10 mM MgC12/1 mM ADP/10 mM Pi/5 mM citrulline (0.1 #Ci) and extract (approx. 0.1 mg protein). Fhe mixture was incubated at 37°C for 10 min, and the ~4CO2 released determined as described above. Ornithine transcarbamoylase ( EC 2.1.3.3) assayed in the direction of ornithine utilisation. The activity was determined from the formation of citrulline from carbamoyl phosphate and ornithine. The reaction mixture contained l0 mM carbamoyl phosphate/10 mM ornithine/100 mM Mes pH 7 and extract (approx. 0.02 mg protein) in a total volume of 1 ml. After incubation for 5 min at 37°C, the reaction was terminated by the addition of 0.2 ml 10% (w/v) trichloroacetic acid, centrifuged, and the citrulline in the supernatant determined colorimetrically [5].
Carbamate kinase (EC 2.7.2.2). The activity was determined spectrophotometrically at 30°C by coupling to hexokinase and NADdependent glucose 6-phosphate dehydrogenase. Preliminary experiments established that the optimal ADP concentration for the giardial enzyme was 0.2 mM. The reaction mixture contained 1 mM carbamoyl phosphate/10 mM glucose/30 mM MgC12/1 mM NAD/0.2 mM ADP/1 I.U. NAD-dependent glucose 6-phosphate dehydrogenase/10 I.U. hexokinase/20 mM Tris pH 8.3 and extract in a total volume of 3 ml. The reaction was determined from the change in absorption at 340 nm. Effect of inhibitors on arginine flux. The cells were incubated in ! ml PBS, containing 1 mM arginine (0.25/~Ci) and inhibitor. The inhibitors used were 5 mM sodium azide, 1 mM sodium cyanide, 3 ~M nigericin, 0.1 #M valinomycin. The cells were incubated for 30 min at 37°C, and 14CO2 was determined as before. Protein.
For whole cells the method of Lowry et al. [6] was used, and for extracts the method of Bradford [7].
Kinetic analysis.
Data were fitted to the Michaelis-Menten equation using translations into BASIC of the F O R T R A N programs HYPER and HYPERL described by Cleland [8].
Results
Flux through the arginine dihydrolase pathway. When intact trophozoites of G. intestinal& were incubated in PBS with 2.5 mM [14Cguanidino]arginine, 14CO2 was rapidly produced. The rate of production was constant for at least 2 h. In order to confirm that PBS was indeed a suitable alternative to the nondefined and complex growth medium of Diamond's TYI-S-33, trophozoites were similarly incubated in Diamond's TYI-S-33 medium (which contains approximately 2.5 mM arginine) and 50 mM glucose to which trace [14C-guanidino]arginine was added. 14CO2 production in the complex medium was about 20% less than that in PBS, clearly indicating that PBS was an appropriate but simple defined medium in which to measure flux rates over periods of up to 2 h. On the basis of these observations, PBS was used in all subsequent studies unless indicated otherwise. The saturability of the complete pathway was assessed by incubating intact trophozoites with varied arginine concentrations (range 0.25 mM) and the release of 14COz determined. The system was saturable with respect to arginine, and exhibited apparent hyperbolic kinetics with half maximal velocity at an arginine concentration of 0.15 + 0.01 mM and an effective Vmax of approximately 40 nmol min-1 (rag protein)-1 (Fig. 1). Metabolism of arginine via the dihydrolase pathway generates citrulline. To determine whether intact trophozoites can also utilise citrulline, the trophozoites were incubated with 1 mM [14C-carbamoyl]citrulline in the absence of arginine. Citrulline was not significantly metabolised to 14CO2 by intact trophozoites; the rate was 0.9 nmol min-1 (mg protein)-1, less than 3% of the rate with arginine under the same conditions. Furthermore, 1 mM
32
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1/[Argininel (raM) -1 Fig. 1. Double reciprocal plot for the effect of arginine concentration on the liberation of J4CO2 from [tacguanidino]arginine by intact G. intestinalis trophozoites. The trophozoites were incubated in PBS at 37°C containing varied concentrations of arginine, and the liberated ~4CO2 determined as described in Materials and Methods.
unlabelled citrulline had no effect on the conversion of [~4C-guanidino]arginine to 14CO2 The most likely reason for the failure of citrulline to be metabolised to CO2 was that the giardial membrane is impermeable to citrulline.
However, there was no significant effect on the arginine flux (Table I). The arginine flux through the dihydrolase pathway was also independent of external pH. Trophozoites incubated with 1 mM [14C-guanidino]arginine in 150 mM NaC1/5 mM KCI/10 mM Hepes/10 mM Mes buffers over the pH range 5-8 showed no pH-dependent change in ~4CO2 production. Since arginine was clearly of potential importance in the energy economy of Giardia, particularly during the early stages of growth, and glucose is a documented energy source [1], it was pertinent to investigate metabolic interaction between arginine and glucose utilisation. When intact trophozoites were incubated with 1 mM arginine in the presence of varied concentrations of glucose (concentration range 0.1-10 mM), carbon dioxide production from arginine was unaffected, and conversely arginine did not inhibit the glucose flux (Table II).
Enzymes of the arginine dihydrolase pathwa.v. Linstead and Cranshaw [4] reported the arginine dihydrolase pathway in Trichomonas TABLE II Effect of glucose on the arginine flux and vice versa Arg conc. (raM)
Glucose conc. Arg flux rate (mM) nmol rain i (mg protein) i
compounds known to inhibit active membrane transport processes in other systems were tested to determine whether they affected arginine metabolism by intact trophozoites.
1 1 I 1 1
0 0.1 I 5 10
TABLE I
Glucose conc. Arg conc. (mM) (raM)
Inhibition of arginine uptake. A number of
Effect of potential inhibitors on the arginine flux Inhibitor
% control
Sodium azide (5 mM) Nigericin (3 ~M) Valinomycin (0.1 #M) Nigericin (3 pM) + valinomycin (0.1 /~M) Sodium cyanide (1 mM)
97 85 85 91 94
Intact trophozoites were incubated in 5 mM
1 1 1 1 1
[14C-
guanMino]arginine in the presence of potential inhibitors, and the flux determined from the liberated 14CO2 as described in Materials and Methods. The values are representative values from sets of triplicates.
0 0.1 I 5 10
28.9 32.6 27.9 23.9 24.4
+ + + + +
5.4 1.4 1.9 1.8 2.9
% Control
115 96 80 85
Glucose flux rate % Control nmol min i (rag protein) / 1.50 + 0.07 1.83 + 0.33 2.15 + 0.23 1.59 + 0.31
123 146 108
1.77 + 0.17
123
Trophozoites were incubated with 1 mM [14C-guanidino]arginine with varying concentration of glucose, and the flux determined from the liberated 14CO2 as described in Materials and Methods. Similarly, the glucose flux was determined from the liberation of 14CO2 from [1-14C]glucose in the presence of varied arginine concentrations. The values are expressed as mean _+ S.D. (n = 3).
33
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1/[Carbamoylphosphate] (mM) -1
Fig. 2. Lineweaver-Burk plot for ornithine transcarbamoylase (anabolic direction). (A) Ornithine. The assay contained 10 mM carbamoyl phosphate/100 m M Mes pH 7.0/extract (approx 0.02 mg protein), and ornithine (0.1 10 mM) in a total volume of 1 ml. The mixture was incubated for 5 min at 37°C, the reaction terminated, and the citrulline determined colorimetrically as described in Materials and Methods. (B) Carbamoyl phosphate. The assay was as above, except that the ornithine concentration was maintained at 10 mM, and the carbamoyl phosphate concentration varied from 0.15 to 10 mM.
vaginalis and determined the activity and some properties of the individual enzymes. Their methods and the properties of the individual trichomonad enzymes were used as the initial guide to develop appropriate methods for defining the activities and characteristics of the giardial enzymes. Arginine deiminase (ADI) was determined by the formation of citrulline which was assayed colorimetrically. 1 m M arginine was routinely used for the assay, but at a pH of 7 which was optimal for the giardial enzyme. The specific activity was 270 + 23 nmol m i n - l (mg protein)-] (n = 4). Ornithine transcarbamoylase (OTC) was measured in the direction of citrulline utilisation by the release of i4co2 from [14Ccarbamoyl]citrulline. Initially, 0.1 m M citrulline at an assay pH of 6.0 was used, as per Linstead and Cranshaw [4], but this was found to be suboptimal. In addition, since the 14CO2 release is dependent upon the coupling to endogenous carbamate kinase, it was necessary to optimise the conditions to ensure an appropriate A D P concentration for carbamate kinase, and inorganic phosphate concen-
tration for the transcarbamoylase. From a matrix of Pi concentration (range 1-10 mM) and ADP concentration (range 0.01-10 mM), the optimal combination was found to be 10 m M Pi and 1 m M ADP. When these conditions were used, the OTC activity was found to be very much higher than previously reported. However, it was necessary to use a short incubation period (10 rain) as linearity was not retained at longer times, and a protein content of not greater than 0.1 mg in the assay• Under these conditions, the OTC activity was 170 + 22 nmol min-1 (mg protein)-1 (n = 3) which is approximately 80-fold higher than that reported for T. vaginalis and approximately 200-fold higher than reported previously by ourselves for Giardia under suboptimal conditions [3]. The OTC activity in the reverse direction, as determined by the production of citrulline from ornithine plus carbamoyl phosphate, was considerably greater. The activity was 2100 + 100 nmol min -1 (mg protein)-] (n -- 11). The optimal pH was 8.2. The apparent Km for ornithine was 180 + 24 #M (Fig. 2A), and that for carbamoyl phosphate was 3.8 + 0.7 m M (Fig. 2B).
34
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1/[Carbamoylphosphatc] (mM) 1
Fig. 3. Lineweaver-Burk plot for carbamate kinase. (A) ADP. The assay contained 1 mM carbamoyl phosphate/10 mM glucose/30 mM MgCI2/1 mM NAD/1 U NAD-dependent glucose 6-phosphate dehydrogenase/10 U hexokinase/20 mM Tris pH 8.3/ADP (0.01 to 1 mM) in a total volume of 2.4 ml, The initial velocity was determined spectrophotometrically from the change in absorption at 340 nm at 30°C. (B) Carbamoyl phosphate. The assay was as above, except that the carbamoyl phosphate concentration was varied from 0.05 to I mM, at a constant ADP concentration of 0.2 raM.
Carbamate kinase (CK) activity was assayed via a continuous spectrophotometric method by coupling the ATP production to hexokinase and glucose 6-phosphate dehydrogenase. It was necessary to optimise the ADP and magnesium concentrations. The most suitable combination was 0.2 mM ADP and 30 mM MgC12. Under these conditions, the measured CK activity at pH 7.5 was 2100 _+ 400 nmol m i n - ' (mg protein) i (n = 7). The apparent Km for ADP was 170 _+ 15 #M (Fig. 3A) and that for carbamoyl phosphate, 200 ___ 29 #M (Fig. 3B).
Discussion
Our previous observations had indicated that when trophozoites of G. intestinalis were grown in Diamond's TYI-S-33 medium over a period of days, the arginine content of the medium was depleted within the first 2 days with the concurrent production of ornithine and ammonia, suggesting the operation of the arginine dihydrolase pathway. From these
observations only a gross estimate, at best, could be obtained as to the possible flux through the pathway, and ultimately the capacity of the pathway to generate energy. In order to quantify the potential flux a more defined system and a shorter time frame were used. In this context [~4C-guanidino]arginine is the most appropriate substrate for determining the flux through the pathway, since its metabolism via the dihydrolase pathway produces ~4CO2. In G. intestinalis this ~4CO2 is a specific determinant of the dihydrolase pathway since there is no evidence that it can be generated from the arginine guanidino group in any other way. Arginase is absent [3] and hence CO2 cannot be generated from the guanidino group by the concerted action of arginase and urease. The individual enzymes of the giardial dihydrolase pathway show a number of distinctive features, the foremost of which is their high activity. The activities of the 3 enzymes are between 10- and 250-fold higher than the activities reported for T. vaginalis. Indeed, the high flux rates of arginine in the
35 intact trophozoites are a reflection of these high enzyme activities, with the maximum rate of carbon dioxide release from arginine being approximately one-quarter of the activity of OTC, the least active of the 3 enzymes. The initial enzyme of the pathway, ADI, had a relatively high specific activity particularly vis-a-vis the overall flux of arginine through the complete pathway. It is thus unlikely to be the rate-controlling step of the pathway. The OTC activity in the direction of citrulline utilisation was considerably higher than that previously reported [3], and this is due to optimisation of the assay procedures. However, with an activity of approximately 170 nmol min-I (mg protein) - / i n the catabolic direction it is the most likely candidate as the rate controlling step in the overall pathway. In this assay the endogenous carbamate kinase is used as a coupling enzyme. However, since the carbamate kinase activity is approx. 10 times higher than that determined for the OTC under these assay conditions, the activity of carbamate kinase will not be limiting and the release of 14CO2 from [14C-carbamoyl]citrulline is thus a valid method for determining OTC activity. The activity in this direction is considerably less than that for citrulline production, the activity in this latter case being about 12-fold higher. Despite this imbalance, the flux through this step in vivo will favour the catabolic direction due to the overall irreversibility of the arginine dihydrolase pathway. The enzymatic properties of this pathway have been analysed in a number of bacteria [9]. The arginine deiminase is an essentially irreversible reaction, and the equilibrium of the final reaction, carbamate kinase, favours ATP synthesis [10]. These properties, together with the relatively high Km of 2.5 mM for carbamoyl phosphate for ornithine transcarbamoylase in the anabolic direction, all suggest a net flow from arginine through to ammonia and carbon dioxide. The final enzyme of the pathway, CK, has one of the highest activities ever recorded for a giardial enzyme, and this high activity together with the high affinities for ADP and carbamoyl phosphate may further function to maintain
net flux through ornithine transcarbamoylase in the direction of citrulline utilisation. The potential arginine flux, with its subsequent energy production via the substrate level phosphorylation at CK, is much greater than the potential glucose flux. The arginine flux rate at 1 mM arginine is approximately 30 nmol min -I (mg protein) -I, whereas the glucose flux, as measured by direct glucose U4Pctake(data not shown) or by the liberation of 02 from 1 mM [1-14C]glucose, is approximately 2 nmol min-1 (mg protein)-1. Thus at equimolar concentrations the potential flux through arginine in G. intestinalis is approximately 15-fold greater than the potential flux through glucose, which has previously been assumed to be the major energy source in Giardia. Under these circumstances the ATP yield from arginine (1 mM) is approximately 30 nmol min-1 (mg protein)-i, whereas that from 1 mM glucose (based on a metabolic flux rate of 2 nmol m i n - I (mg protein)-1 and a stoichiometry of 2 mol ATP per mol glucose) is of the order of only 4 nmol min -1 (mg protein) -1. Hence the potential energy yield from arginine is therefore some 7-8-fold greater than that from glucose. Clearly there is the capability in Giardia for using arginine as an energy source in preference to glucose, consistent with our previous observations that proliferating trophozoites rapidly deplete the arginine in the growth medium before the glucose is significantly utilised, suggesting that glucose is a minor source of energy during the rapid proliferative stage. The maximal arginine flux, approximately 40 nmol min -1 (mg protein) -I, is approximately 80-fold higher than the reported maximal flux of 0.46 nmol min - ~ (mg protein) -1 through the arginine dihydrolase pathway in T. vaginalis which is the only other protozoan parasite in which the pathway has been demonstrated. In T. vaginalis, it has been suggested that the pathway may make a significant contribution to the energy balance for the parasite in its natural habitat [4], even though the maximal flux through the pathway is relatively low and glycolysis is usually considered to be the major source of ATP in
36
T. vaginalis. Even at physiological ranges of arginine concentrations, the potential energy yield from arginine in G. intestinalis still greatly exceeds that from glucose. The arginine concentration of duodenal juice can be up to 0.2-0.4 mM (unpublished data). At 0.3 mM arginine concentration, which is the midpoint of this physiological concentration range, the measured arginine flux and hence ATP yield, was approximately 21 nmol rain ~ (mg protein) ~. This still represents an energy yield some 3-4-fold greater than that from the concentrations of glucose likely to be encountered in vivo, and it suggests that even within the natural habitat, rather than the artificial conditions of growth media, arginine is the major energy source for G. intestinalis. The release of 14CO2 from [14Cguanidino]arginine provides a quantitative determinant of the overall arginine metabolism which, in turn, incorporates the component of the initial transport and/or uptake of arginine. Although the CO2 liberation cannot provide direct information as to the mechanism of transport or uptake, it can nevertheless provide a substantial guide to the possible types of mechanisms present. On the basis of arginine fluxes, the arginine uptake appears quite specific. Whereas arginine is readily metabolised, [~4C-carbamoyl]citrulline is metabolised only very slightly to 1aco2 by intact trophozoites, the rate being less than 3% of that of arginine, suggesting that the giardial membrane is impermeable to citrulline. Ionophores and inhibitors such as valinomycin, nigericin, azide and cyanide at concentrations which severely inhibit energy-dependent transport processes in a number of protozoan parasites had no significant effect on arginine fluxes. Although these effects cannot be regarded as conclusive with respect to the possible mechanisms of arginine transport, they suggest that the transport is not energy dependent. From a phenomenological aspect, an energy dependent transport system for arginine would be unlikely; it would be
biologically wasteful, since the energy yield I¥om arginine metabolism is only one tool ATP per tool arginine utilised. Hence a transport system that does not require the direct intervention of ATP but which still confers specificity by means of a selective carrier is more likely. We are currently addressing the question of the nature of this arginine transport.
Acknowledgements This work was supported by the National Health and Medical Research Council.
References 1 Schofield, P.J., Edwards, M.R. and Kranz, P. (1991) Glucose metabolism in Giardia intestinalis. Mol. Biochem. Parasitol. 45, 39-48. 2 Edwards, M.R., Gilroy, F.V., Jimenez, B.M. and O'Sullivan, W.J. (1989) Alanine is a major end product of metabolism by Giardia lamblia: a proton nuclear magnetic resonance study. Mol. Biochem. Parasitol. 37, 19-26.
3 Schofield, P.J., Costello, M., Edwards, M.R. and O'Sullivan, W.J. (1990) The arginine dihydrolase pathway is present in Giardia intestinalis. Int. J. Parasitol. 20, 697 699. 4 Linstead, D. and Cranshaw, M.R. (1983) The pathway of arginine catabolism in the parasitic flagellate Trichomonas vaginalis. Mol. Biochem. Parasitol. 8, 241-252. 5 Boyde, T.R.C. and Rahmatullah, M. (1980) Optimisation of conditions for the colorimetric determination of citrulline, using diacetyl monoxime. Anal. Biochem. 107, 424431. 6 Lowry, O.H., Rosebrough, N.H., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 7 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248-254. 8 Cleland, W.W. (1979) Statistical analysis of enzyme kinetic data. Methods Enzymol. 63, 103--138. 9 Poolmam B., Driessen, A.J.M. and Konings, W.N. (1987) Regulation of arginine-ornithine exchange and the arginine deiminase pathway in Streptococcus lactis. J. Bacteriol. 169, 5597-5604. 10 Thauer, R.K., Jungermann, K. and Decker, K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41, 100 180.
29
MOLBIO 01674
The pathway of arginine catabolism in Giardia intestinalis Philip J. Schofield, M i c h a e l R. E d w a r d s , J a c q u e l i n e M a t t h e w s a n d Justine R. W i l s o n School of Biochemistry, University of New South Wales, Kensington, NSW, Australia (Received 21 June 1991; accepted 2 October 1991)
In Giardia intestinalis, arginine is catabolised by the arginine dihydrolase pathway. The enzymes of the pathway (arginine deiminase, ornithine transcarbamoylase and carbamate kinase) were investigated and their basic kinetic parameters determined. The specific activity of arginine deiminase was 270 _+ 23 nmol min i (mg protein)-~; ornithine transcarbamoylase, in the direction of citrulline utilisation 170 _+ 22 nmol min ~ (mg protein) l, and in the direction of ornithine utilisation 2100 + 100 nmol min i (mg protein) l; and carbamate kinase 2100 + 400 nmol rain i (mg protein) a. The activities of these enzymes are between 10 and 250 fold greater than those reported for the enzymes in Trichomonas vaginalis, the only other parasite in which the arginine dihydrolase pathway has been reported. The flux through the pathway in G. intestinalis, as determined by the liberation of 14C02from 1 mM [laC-guanidino]arginine was 30 nmol min-J (mg p r o t e i n ) - i This flux was not affected by valinomycin (0.1 /~M), nigericin (3/~M), azide (5 mM) or cyanide (1 mM). The flux was only marginally affected by glucose up to 10 mM concentration. Conversely, the flux through glucose metabolism, as determined by the release of 14CO2 from 1 mM [l-14C]glucose was only 2 nmol rain I (mg protein)- t, and was unaffected by arginine concentrations up to 10 mM. These observations suggest that there is no direct metabolic interface between arginine and glucose catabolism. The potential energy yield of ATP from the arginine flux is 7 8-fold greater than that from glucose, providing evidence for the prime importance of arginine in the energy economy of G. intestinalis. It is suggested that the flux through the arginine dihydrolase pathway is the major source of energy production, particularly in anaerobic conditions. Key words: Giardia intestinalis; Arginine metabolism; Arginine dihydrolase pathway; Arginine deiminase; Ornithine transcarbamoylase; Carbamate kinase
Introduction
Although it has been commonly accepted that glucose is the only source of energy for Giardia intestinalis, there is mounting evidence that this is not so. Glucose catabolism cannot account for all the end products of G. intestinalis [1] and it is evident that other carbon sources must be utilised. The most likely of these other sources are amino acids Correspondence address: P.J. Schofield, School of Biochemistry, University of NSW, P.O. Box 1, Kensington, NSW 2033, Australia. Abbreviations: PBS, phosphate-buffered saline; OTC, ornithine transearbamoylase (EC 2.1.3.3); ADI, arginine deiminase (EC 3.5.3.6); CK, carbamate kinase (EC 2.7.2.2).
and/or proteins. Recent observations have indicated that a number of amino acids are involved in the energy metabolism during the proliferation of G. intestinalis in culture [2]. A dramatic feature of the changes in the amino acid profile of the medium is a rapid depletion of arginine [2]. The avid consumption of arginine by G. intestinalis suggested that it had a significant role as an energy source, a view that was consolidated by our observation that the arginine was being utilised by the arginine dihydrolase pathway [3]. This pathway, which converts arginine to ornithine and ammonia, has the biological advantage that it produces ATP directly via substrate level phosphorylation, and eliminates the need for oxygen or for intermediate redox systems. It is thus most appropriate for G. intestinalis whose
30
natural environment is low in oxygen tension and which is generally regarded as being microaerophilic or a facultative anaerobe. Although the arginine dihydrolase pathway occurs in a number of prokaryotes, the only eukaryotic organism in which it has been previously observed is another parasitic protozoan, Trichomonas vaginalis [4]. In a previous short communication we reported activities for the enzymes of the arginine dihydrolase pathway [3]. We now report results demonstrating the biological potential of this pathway in Giardia together with the characterisation of the basic properties of the individual enzymes of the pathway.
Materials and Methods
Organism. G. intestinalis trophozoites (Portland 1 strain) were grown in TYI-S-33 medium as described previously [2]. Materials. [14C-guanidino]arginine and [14Ccarbamoyl]citrulline were obtained from Amersham. All other chemicals were of the highest grade commercially available.
Preparation of extracts. The trophozoites were detached from the vessel wall by cooling in ice, collected by centrifugation and sonicared in 0.25 M sucrose with 5 x 20 s bursts at 80 W. The homogenate was centrifuged for 2 min (Beckman Microfuge) and the supernatant used for enzyme assays. General method jor 14C0 2 collection. Trophozoites or extracts were incubated in conical flasks sealed with rubber seals. The final total volume was 1 ml. The flasks contained an inner small glass vial seated in a larger glass vial. The inner glass vial contained 100 ~tl 1 M hyamine hydroxide in methanol. The incubation was initiated by the addition of cells or extract, the flask immediately sealed, and the reaction allowed to proceed at 37°C. At the end of the appropriate incubation time the reaction was terminated by the injection of 1 ml 1.3 M perchloric acid. Absorption of the liberated
14C0 2 was allowed to proceed for 1 hour in a shaking water bath. The inner well containing the absorbed ~4CO2 in hyamine was then removed, placed in a scintillation vial containing 10 ml scintillant (0.5% 2,5-diphenyloxazole in toluene) and the radioactivity determined in a Packard Tricarb 300 scintillation counter.
Arginine JTux. The flux through the arginine dihydrolase pathway was determined from the 14COz production from [14C-guanidino] arginine. The incubation mixture contained, in a total volume of 1 ml of phosphate buffered saline (PBS)/1.8 mM KH2PO4/5 mM KzHPO4/0.9% (w/v) NaCI pH 7.4/1 mM arginine (0.1 /iCi) and trophozoites (approx. 0.2 m~ protein which is equivalent to approx. 5 x 10°cells). The reaction was initiated by the addition of the cells, and the ~4CO2 release determined. For the determination of the effect of pH on arginine flux, the buffer used was 150 mM NaCI/5 m M KC1/10 mM Mes/10 mM Hepes for the pH range 5-8. Glucose Jlux. The flux through glucose metabolism was determined from the release of 14CO2 from [1-14C]glucose. The incubation mixture contained, in a total volume of 1 ml, PBS pH 7.4, 1 mM [1-14C]glucose (0.1/~Ci) and trophozoites (approx. 5 x 106 cells). The ~4CO2 released was determined as above. Arginine deiminase (EC 3.5.3.6).
The activity was assayed by the colorimetric determination of the product citrulline. The assay is essentially that described by Linstead and Cranshaw [4]. The assay mixture contained 1 mM arginine/40 mM Mes pH 7 and extract (approx. 0.1 mg protein). The incubation time was 20 min at 37°C. The reaction was terminated by the addition of 0.2 ml 6% (w/v) trichloroacetic acid and citrulline was determined by the method of Boyde and Rahmatullah [5].
Ornithine transcarbamoylase ( EC 2.1.3.3) assayed in the direction of citrulline utilisation. The activity was determined by the release of
31
14CO2from L-[~4C-carbamoyl]citrulline in the presence of added ADP and Pi. The incubation mixture contained 40 mM Mes, pH 7/10 mM MgC12/1 mM ADP/10 mM Pi/5 mM citrulline (0.1 #Ci) and extract (approx. 0.1 mg protein). Fhe mixture was incubated at 37°C for 10 min, and the ~4CO2 released determined as described above. Ornithine transcarbamoylase ( EC 2.1.3.3) assayed in the direction of ornithine utilisation. The activity was determined from the formation of citrulline from carbamoyl phosphate and ornithine. The reaction mixture contained l0 mM carbamoyl phosphate/10 mM ornithine/100 mM Mes pH 7 and extract (approx. 0.02 mg protein) in a total volume of 1 ml. After incubation for 5 min at 37°C, the reaction was terminated by the addition of 0.2 ml 10% (w/v) trichloroacetic acid, centrifuged, and the citrulline in the supernatant determined colorimetrically [5].
Carbamate kinase (EC 2.7.2.2). The activity was determined spectrophotometrically at 30°C by coupling to hexokinase and NADdependent glucose 6-phosphate dehydrogenase. Preliminary experiments established that the optimal ADP concentration for the giardial enzyme was 0.2 mM. The reaction mixture contained 1 mM carbamoyl phosphate/10 mM glucose/30 mM MgC12/1 mM NAD/0.2 mM ADP/1 I.U. NAD-dependent glucose 6-phosphate dehydrogenase/10 I.U. hexokinase/20 mM Tris pH 8.3 and extract in a total volume of 3 ml. The reaction was determined from the change in absorption at 340 nm. Effect of inhibitors on arginine flux. The cells were incubated in ! ml PBS, containing 1 mM arginine (0.25/~Ci) and inhibitor. The inhibitors used were 5 mM sodium azide, 1 mM sodium cyanide, 3 ~M nigericin, 0.1 #M valinomycin. The cells were incubated for 30 min at 37°C, and 14CO2 was determined as before. Protein.
For whole cells the method of Lowry et al. [6] was used, and for extracts the method of Bradford [7].
Kinetic analysis.
Data were fitted to the Michaelis-Menten equation using translations into BASIC of the F O R T R A N programs HYPER and HYPERL described by Cleland [8].
Results
Flux through the arginine dihydrolase pathway. When intact trophozoites of G. intestinal& were incubated in PBS with 2.5 mM [14Cguanidino]arginine, 14CO2 was rapidly produced. The rate of production was constant for at least 2 h. In order to confirm that PBS was indeed a suitable alternative to the nondefined and complex growth medium of Diamond's TYI-S-33, trophozoites were similarly incubated in Diamond's TYI-S-33 medium (which contains approximately 2.5 mM arginine) and 50 mM glucose to which trace [14C-guanidino]arginine was added. 14CO2 production in the complex medium was about 20% less than that in PBS, clearly indicating that PBS was an appropriate but simple defined medium in which to measure flux rates over periods of up to 2 h. On the basis of these observations, PBS was used in all subsequent studies unless indicated otherwise. The saturability of the complete pathway was assessed by incubating intact trophozoites with varied arginine concentrations (range 0.25 mM) and the release of 14COz determined. The system was saturable with respect to arginine, and exhibited apparent hyperbolic kinetics with half maximal velocity at an arginine concentration of 0.15 + 0.01 mM and an effective Vmax of approximately 40 nmol min-1 (rag protein)-1 (Fig. 1). Metabolism of arginine via the dihydrolase pathway generates citrulline. To determine whether intact trophozoites can also utilise citrulline, the trophozoites were incubated with 1 mM [14C-carbamoyl]citrulline in the absence of arginine. Citrulline was not significantly metabolised to 14CO2 by intact trophozoites; the rate was 0.9 nmol min-1 (mg protein)-1, less than 3% of the rate with arginine under the same conditions. Furthermore, 1 mM
32
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1/[Argininel (raM) -1 Fig. 1. Double reciprocal plot for the effect of arginine concentration on the liberation of J4CO2 from [tacguanidino]arginine by intact G. intestinalis trophozoites. The trophozoites were incubated in PBS at 37°C containing varied concentrations of arginine, and the liberated ~4CO2 determined as described in Materials and Methods.
unlabelled citrulline had no effect on the conversion of [~4C-guanidino]arginine to 14CO2 The most likely reason for the failure of citrulline to be metabolised to CO2 was that the giardial membrane is impermeable to citrulline.
However, there was no significant effect on the arginine flux (Table I). The arginine flux through the dihydrolase pathway was also independent of external pH. Trophozoites incubated with 1 mM [14C-guanidino]arginine in 150 mM NaC1/5 mM KCI/10 mM Hepes/10 mM Mes buffers over the pH range 5-8 showed no pH-dependent change in ~4CO2 production. Since arginine was clearly of potential importance in the energy economy of Giardia, particularly during the early stages of growth, and glucose is a documented energy source [1], it was pertinent to investigate metabolic interaction between arginine and glucose utilisation. When intact trophozoites were incubated with 1 mM arginine in the presence of varied concentrations of glucose (concentration range 0.1-10 mM), carbon dioxide production from arginine was unaffected, and conversely arginine did not inhibit the glucose flux (Table II).
Enzymes of the arginine dihydrolase pathwa.v. Linstead and Cranshaw [4] reported the arginine dihydrolase pathway in Trichomonas TABLE II Effect of glucose on the arginine flux and vice versa Arg conc. (raM)
Glucose conc. Arg flux rate (mM) nmol rain i (mg protein) i
compounds known to inhibit active membrane transport processes in other systems were tested to determine whether they affected arginine metabolism by intact trophozoites.
1 1 I 1 1
0 0.1 I 5 10
TABLE I
Glucose conc. Arg conc. (mM) (raM)
Inhibition of arginine uptake. A number of
Effect of potential inhibitors on the arginine flux Inhibitor
% control
Sodium azide (5 mM) Nigericin (3 ~M) Valinomycin (0.1 #M) Nigericin (3 pM) + valinomycin (0.1 /~M) Sodium cyanide (1 mM)
97 85 85 91 94
Intact trophozoites were incubated in 5 mM
1 1 1 1 1
[14C-
guanMino]arginine in the presence of potential inhibitors, and the flux determined from the liberated 14CO2 as described in Materials and Methods. The values are representative values from sets of triplicates.
0 0.1 I 5 10
28.9 32.6 27.9 23.9 24.4
+ + + + +
5.4 1.4 1.9 1.8 2.9
% Control
115 96 80 85
Glucose flux rate % Control nmol min i (rag protein) / 1.50 + 0.07 1.83 + 0.33 2.15 + 0.23 1.59 + 0.31
123 146 108
1.77 + 0.17
123
Trophozoites were incubated with 1 mM [14C-guanidino]arginine with varying concentration of glucose, and the flux determined from the liberated 14CO2 as described in Materials and Methods. Similarly, the glucose flux was determined from the liberation of 14CO2 from [1-14C]glucose in the presence of varied arginine concentrations. The values are expressed as mean _+ S.D. (n = 3).
33
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4
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1/[Carbamoylphosphate] (mM) -1
Fig. 2. Lineweaver-Burk plot for ornithine transcarbamoylase (anabolic direction). (A) Ornithine. The assay contained 10 mM carbamoyl phosphate/100 m M Mes pH 7.0/extract (approx 0.02 mg protein), and ornithine (0.1 10 mM) in a total volume of 1 ml. The mixture was incubated for 5 min at 37°C, the reaction terminated, and the citrulline determined colorimetrically as described in Materials and Methods. (B) Carbamoyl phosphate. The assay was as above, except that the ornithine concentration was maintained at 10 mM, and the carbamoyl phosphate concentration varied from 0.15 to 10 mM.
vaginalis and determined the activity and some properties of the individual enzymes. Their methods and the properties of the individual trichomonad enzymes were used as the initial guide to develop appropriate methods for defining the activities and characteristics of the giardial enzymes. Arginine deiminase (ADI) was determined by the formation of citrulline which was assayed colorimetrically. 1 m M arginine was routinely used for the assay, but at a pH of 7 which was optimal for the giardial enzyme. The specific activity was 270 + 23 nmol m i n - l (mg protein)-] (n = 4). Ornithine transcarbamoylase (OTC) was measured in the direction of citrulline utilisation by the release of i4co2 from [14Ccarbamoyl]citrulline. Initially, 0.1 m M citrulline at an assay pH of 6.0 was used, as per Linstead and Cranshaw [4], but this was found to be suboptimal. In addition, since the 14CO2 release is dependent upon the coupling to endogenous carbamate kinase, it was necessary to optimise the conditions to ensure an appropriate A D P concentration for carbamate kinase, and inorganic phosphate concen-
tration for the transcarbamoylase. From a matrix of Pi concentration (range 1-10 mM) and ADP concentration (range 0.01-10 mM), the optimal combination was found to be 10 m M Pi and 1 m M ADP. When these conditions were used, the OTC activity was found to be very much higher than previously reported. However, it was necessary to use a short incubation period (10 rain) as linearity was not retained at longer times, and a protein content of not greater than 0.1 mg in the assay• Under these conditions, the OTC activity was 170 + 22 nmol min-1 (mg protein)-1 (n = 3) which is approximately 80-fold higher than that reported for T. vaginalis and approximately 200-fold higher than reported previously by ourselves for Giardia under suboptimal conditions [3]. The OTC activity in the reverse direction, as determined by the production of citrulline from ornithine plus carbamoyl phosphate, was considerably greater. The activity was 2100 + 100 nmol min -1 (mg protein)-] (n -- 11). The optimal pH was 8.2. The apparent Km for ornithine was 180 + 24 #M (Fig. 2A), and that for carbamoyl phosphate was 3.8 + 0.7 m M (Fig. 2B).
34
10
2.0
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1/[ADP] (1~4)-1
0.0
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,
,
10
20
30
1/[Carbamoylphosphatc] (mM) 1
Fig. 3. Lineweaver-Burk plot for carbamate kinase. (A) ADP. The assay contained 1 mM carbamoyl phosphate/10 mM glucose/30 mM MgCI2/1 mM NAD/1 U NAD-dependent glucose 6-phosphate dehydrogenase/10 U hexokinase/20 mM Tris pH 8.3/ADP (0.01 to 1 mM) in a total volume of 2.4 ml, The initial velocity was determined spectrophotometrically from the change in absorption at 340 nm at 30°C. (B) Carbamoyl phosphate. The assay was as above, except that the carbamoyl phosphate concentration was varied from 0.05 to I mM, at a constant ADP concentration of 0.2 raM.
Carbamate kinase (CK) activity was assayed via a continuous spectrophotometric method by coupling the ATP production to hexokinase and glucose 6-phosphate dehydrogenase. It was necessary to optimise the ADP and magnesium concentrations. The most suitable combination was 0.2 mM ADP and 30 mM MgC12. Under these conditions, the measured CK activity at pH 7.5 was 2100 _+ 400 nmol m i n - ' (mg protein) i (n = 7). The apparent Km for ADP was 170 _+ 15 #M (Fig. 3A) and that for carbamoyl phosphate, 200 ___ 29 #M (Fig. 3B).
Discussion
Our previous observations had indicated that when trophozoites of G. intestinalis were grown in Diamond's TYI-S-33 medium over a period of days, the arginine content of the medium was depleted within the first 2 days with the concurrent production of ornithine and ammonia, suggesting the operation of the arginine dihydrolase pathway. From these
observations only a gross estimate, at best, could be obtained as to the possible flux through the pathway, and ultimately the capacity of the pathway to generate energy. In order to quantify the potential flux a more defined system and a shorter time frame were used. In this context [~4C-guanidino]arginine is the most appropriate substrate for determining the flux through the pathway, since its metabolism via the dihydrolase pathway produces ~4CO2. In G. intestinalis this ~4CO2 is a specific determinant of the dihydrolase pathway since there is no evidence that it can be generated from the arginine guanidino group in any other way. Arginase is absent [3] and hence CO2 cannot be generated from the guanidino group by the concerted action of arginase and urease. The individual enzymes of the giardial dihydrolase pathway show a number of distinctive features, the foremost of which is their high activity. The activities of the 3 enzymes are between 10- and 250-fold higher than the activities reported for T. vaginalis. Indeed, the high flux rates of arginine in the
35 intact trophozoites are a reflection of these high enzyme activities, with the maximum rate of carbon dioxide release from arginine being approximately one-quarter of the activity of OTC, the least active of the 3 enzymes. The initial enzyme of the pathway, ADI, had a relatively high specific activity particularly vis-a-vis the overall flux of arginine through the complete pathway. It is thus unlikely to be the rate-controlling step of the pathway. The OTC activity in the direction of citrulline utilisation was considerably higher than that previously reported [3], and this is due to optimisation of the assay procedures. However, with an activity of approximately 170 nmol min-I (mg protein) - / i n the catabolic direction it is the most likely candidate as the rate controlling step in the overall pathway. In this assay the endogenous carbamate kinase is used as a coupling enzyme. However, since the carbamate kinase activity is approx. 10 times higher than that determined for the OTC under these assay conditions, the activity of carbamate kinase will not be limiting and the release of 14CO2 from [14C-carbamoyl]citrulline is thus a valid method for determining OTC activity. The activity in this direction is considerably less than that for citrulline production, the activity in this latter case being about 12-fold higher. Despite this imbalance, the flux through this step in vivo will favour the catabolic direction due to the overall irreversibility of the arginine dihydrolase pathway. The enzymatic properties of this pathway have been analysed in a number of bacteria [9]. The arginine deiminase is an essentially irreversible reaction, and the equilibrium of the final reaction, carbamate kinase, favours ATP synthesis [10]. These properties, together with the relatively high Km of 2.5 mM for carbamoyl phosphate for ornithine transcarbamoylase in the anabolic direction, all suggest a net flow from arginine through to ammonia and carbon dioxide. The final enzyme of the pathway, CK, has one of the highest activities ever recorded for a giardial enzyme, and this high activity together with the high affinities for ADP and carbamoyl phosphate may further function to maintain
net flux through ornithine transcarbamoylase in the direction of citrulline utilisation. The potential arginine flux, with its subsequent energy production via the substrate level phosphorylation at CK, is much greater than the potential glucose flux. The arginine flux rate at 1 mM arginine is approximately 30 nmol min -I (mg protein) -I, whereas the glucose flux, as measured by direct glucose U4Pctake(data not shown) or by the liberation of 02 from 1 mM [1-14C]glucose, is approximately 2 nmol min-1 (mg protein)-1. Thus at equimolar concentrations the potential flux through arginine in G. intestinalis is approximately 15-fold greater than the potential flux through glucose, which has previously been assumed to be the major energy source in Giardia. Under these circumstances the ATP yield from arginine (1 mM) is approximately 30 nmol min-1 (mg protein)-i, whereas that from 1 mM glucose (based on a metabolic flux rate of 2 nmol m i n - I (mg protein)-1 and a stoichiometry of 2 mol ATP per mol glucose) is of the order of only 4 nmol min -1 (mg protein) -1. Hence the potential energy yield from arginine is therefore some 7-8-fold greater than that from glucose. Clearly there is the capability in Giardia for using arginine as an energy source in preference to glucose, consistent with our previous observations that proliferating trophozoites rapidly deplete the arginine in the growth medium before the glucose is significantly utilised, suggesting that glucose is a minor source of energy during the rapid proliferative stage. The maximal arginine flux, approximately 40 nmol min -1 (mg protein) -I, is approximately 80-fold higher than the reported maximal flux of 0.46 nmol min - ~ (mg protein) -1 through the arginine dihydrolase pathway in T. vaginalis which is the only other protozoan parasite in which the pathway has been demonstrated. In T. vaginalis, it has been suggested that the pathway may make a significant contribution to the energy balance for the parasite in its natural habitat [4], even though the maximal flux through the pathway is relatively low and glycolysis is usually considered to be the major source of ATP in
36
T. vaginalis. Even at physiological ranges of arginine concentrations, the potential energy yield from arginine in G. intestinalis still greatly exceeds that from glucose. The arginine concentration of duodenal juice can be up to 0.2-0.4 mM (unpublished data). At 0.3 mM arginine concentration, which is the midpoint of this physiological concentration range, the measured arginine flux and hence ATP yield, was approximately 21 nmol rain ~ (mg protein) ~. This still represents an energy yield some 3-4-fold greater than that from the concentrations of glucose likely to be encountered in vivo, and it suggests that even within the natural habitat, rather than the artificial conditions of growth media, arginine is the major energy source for G. intestinalis. The release of 14CO2 from [14Cguanidino]arginine provides a quantitative determinant of the overall arginine metabolism which, in turn, incorporates the component of the initial transport and/or uptake of arginine. Although the CO2 liberation cannot provide direct information as to the mechanism of transport or uptake, it can nevertheless provide a substantial guide to the possible types of mechanisms present. On the basis of arginine fluxes, the arginine uptake appears quite specific. Whereas arginine is readily metabolised, [~4C-carbamoyl]citrulline is metabolised only very slightly to 1aco2 by intact trophozoites, the rate being less than 3% of that of arginine, suggesting that the giardial membrane is impermeable to citrulline. Ionophores and inhibitors such as valinomycin, nigericin, azide and cyanide at concentrations which severely inhibit energy-dependent transport processes in a number of protozoan parasites had no significant effect on arginine fluxes. Although these effects cannot be regarded as conclusive with respect to the possible mechanisms of arginine transport, they suggest that the transport is not energy dependent. From a phenomenological aspect, an energy dependent transport system for arginine would be unlikely; it would be
biologically wasteful, since the energy yield I¥om arginine metabolism is only one tool ATP per tool arginine utilised. Hence a transport system that does not require the direct intervention of ATP but which still confers specificity by means of a selective carrier is more likely. We are currently addressing the question of the nature of this arginine transport.
Acknowledgements This work was supported by the National Health and Medical Research Council.
References 1 Schofield, P.J., Edwards, M.R. and Kranz, P. (1991) Glucose metabolism in Giardia intestinalis. Mol. Biochem. Parasitol. 45, 39-48. 2 Edwards, M.R., Gilroy, F.V., Jimenez, B.M. and O'Sullivan, W.J. (1989) Alanine is a major end product of metabolism by Giardia lamblia: a proton nuclear magnetic resonance study. Mol. Biochem. Parasitol. 37, 19-26.
3 Schofield, P.J., Costello, M., Edwards, M.R. and O'Sullivan, W.J. (1990) The arginine dihydrolase pathway is present in Giardia intestinalis. Int. J. Parasitol. 20, 697 699. 4 Linstead, D. and Cranshaw, M.R. (1983) The pathway of arginine catabolism in the parasitic flagellate Trichomonas vaginalis. Mol. Biochem. Parasitol. 8, 241-252. 5 Boyde, T.R.C. and Rahmatullah, M. (1980) Optimisation of conditions for the colorimetric determination of citrulline, using diacetyl monoxime. Anal. Biochem. 107, 424431. 6 Lowry, O.H., Rosebrough, N.H., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 7 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248-254. 8 Cleland, W.W. (1979) Statistical analysis of enzyme kinetic data. Methods Enzymol. 63, 103--138. 9 Poolmam B., Driessen, A.J.M. and Konings, W.N. (1987) Regulation of arginine-ornithine exchange and the arginine deiminase pathway in Streptococcus lactis. J. Bacteriol. 169, 5597-5604. 10 Thauer, R.K., Jungermann, K. and Decker, K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41, 100 180.