the collection of Dr. R. Sager, Sidney Farber Center, New. York) and mutant strains 102 (nit-5, nit-6) and 104 (nit-4). (20), lacking XDH, and mutant 305 (nit-la), ...
Plant Physiol. (1991) 95, 126-1 30 0032-0889/91/95/01 26/05/$01 .00/0
Received for publication June 12, 1990 Accepted September 15, 1990
Distinction between Hypoxanthine and Xanthine Transport in Chiamydomonas reinhardtiil Rafael Perez-Vicente, Jacobo Cardenas*, and Manuel Pineda Departamento de Bioquimica y Biologia Molecular y Fisiologia, Facultad de Ciencias, Universidad de C6rdoba, Avda. San Alberto Magno s/n, 14071-C6rdoba, Spain ABSTRACT Chiamydomonas reinhardtii cells consumed hypoxanthine and xanthine by means of active systems which promoted purine intracellular accumulation against a high concentration gradient. Both uptake and accumulation were also observed in mutant strains lacking xanthine dehydrogenase activity. Xanthine and hypoxanthine uptake systems exhibited very similar Michaelis constants for transport and pH values, and both systems were induced by either hypoxanthine or xanthine. However, they differed greatly in the length of the lag phase before uptake induction, which was longer for hypoxanthine than for xanthine. Cells grown on ammonium and transferred to hypoxanthine media consumed xanthine before hypoxanthine, whereas cells transferred to xanthine media did not take up hypoxanthine until 2 hours after commencing xanthine consumption. Metabolic and photosynthetic inhibitors such as 2,4-dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, and carbonylcyanide m-chlorophenylhydrazone inhibited to a different extent the hypoxanthine and xanthine uptake. Similarly, N-ethylmaleimide abolished xanthine uptake but slightly affected that of hypoxanthine. Hypoxanthine consumption was inhibited by adenine and guanine whereas that of xanthine was inhibited only by urate. We conclude that hypoxanthine and xanthine in C. reinhardtii are taken up by different active transport systems which work independently of the intracellular enzymatic oxidation of these punnes.
MATERIALS AND METHODS Growth Conditions and Preparation of Enzyme Extracts
Chlamydomonas reinhardtii wild-type 6145c cells (from the collection of Dr. R. Sager, Sidney Farber Center, New York) and mutant strains 102 (nit-5, nit-6) and 104 (nit-4) (20), lacking XDH, and mutant 305 (nit-la), having XDH (9, 10), were grown with 10 mM ammonium until the midlogarithmic phase of growth. Then, cells were washed and transferred to media containing either xanthine or hypoxanthine as the sole nitrogen source, as previously described (18). Cells were harvested by centrifugation at 20,000g for 10 min and broken by freezing at -40°C and thawing with gentle stirring in 0.1 M Tris-HCl buffer (pH 8.5). The homogenate was centrifuged at 27,000g for 20 min, and the resulting supernatant was used, after filtration through a Sephadex G-25 column to eliminate purines, as a cell-free extract for activity determinations (15). Hypoxanthine and Xanthine Uptake
Xanthine and hypoxanthine uptake was measured by following their disappearance from the media after cell removal by centrifugation. For spectrophotometric measurements, 1 mL aliquots of culture medium were centrifuged at 13,000 rpm for 1 to 2 min in a Beckman Microfuge model 11. In radioactivity experiments, samples of 0.2 mL were centrifuged (13,000 rpm, 30-60 s) in 0.4 mL centrifuge vials containing 50 ,L of a silicone DC-550: bis(3,5,5-trimethyl hexyl)phthalate mixture (3:2, v/v). Absorbance or radioactivity was measured in the supernatant. Uptake rates are expressed as the decrease in purine concentration in the culture media per unit time. Intracellular hypoxanthine or xanthine content was determined by centrifuging 20 mL of cell culture at 27,000g for 10 min, and storing the washed sediment at -40°C. Frozen pellets were then thawed with vigorous stirring in 2 mL of 0.1 M Tris-HCl buffer (pH 8.5), and centrifuged as above. Purines were estimated in the supernatant. Cell fresh weight was calculated by subtracting the weight of centrifuge tubes cleaned and dried from the same tubes containing freshly precipitated cells. Kinetic experiments were performed by following the disappearance of xanthine or hypoxanthine from the medium, at 60 s intervals, until their complete exhaustion, and plotting the corresponding progress curve ([S] versus t). Kinetic param-
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Studies on purine metabolism and transport in green algae and mainly centered on the catabolic utilization of these compounds as the nitrogen source for growth (7, 18, 21). Recently we have characterized the urate uptake system in the unicellular green alga Chlamydomonas reinhardtii (16, 17) and found that both urate and xanthine are transported by energy-dependent systems (19). However, a further characterization of systems of purine translocation into algal cells and the number of systems involved in purine transport are questions still unresolved. In the present paper, we characterize the uptake of xanthine and hypoxanthine by C. reinhardtii cells and conclude that each purine is transported by a different system. Both systems are energy dependent and can be distinguished from intracellular XDH2 activity. are scarce
'This work was supported by CAICYT (Ministerio de Educaci6n Ciencia, Spain), grant PB86-0167-CO3-0 1. 2Abbreviations: XDH, xanthine dehydrogenase; CCCP, carbonylcyanide m-chlorophenyl-hydrazone; 2,4-DNP, 2,4-dinitrophenol; pHMB, p-hydroxymercuribenzoate; NEM, N-ethylmaleimide.
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126
HYPOXANTHINE AND XANTHINE TRANSPORT IN CHLAMYDOMONAS
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bovine serum albumin as a standard. Xanthine and hypoxanthine were determined enzymatically with milk xanthine oxidase at 292 nm (12) or directly at 268 and 250 nm, respectively (E" = 9150 and 10500 for xanthine and hypoxanthine, respectively). Where indicated, either [8-'4C]hypoxanthine or [6-'4C]xanthine was used. Radioactivity was measured in a Beckman LS 3801 liquid scintillation counter. Results are mean values of at least three independent determinations.
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Figure 1. Uptake and intracellular accumulation of xanthine in wildtype and mutant cells of C. reinhardtii. Wild-type 6145c (A) and XDHmutant 104 (B) cells were grown with 10 mm ammonium until midlogarithmic phase of growth. Then they were harvested, washed and transferred to media containing xanthine as sole nitrogen source in the light. Intracellular xanthine concentration and XDH activity were measured at the indicated times.
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XDH (EC 1.1.1.204, formerly 1.2.1.37) activity was assayed spectrophotometrically at 340 nm in a reaction mixture containing, in a final volume of 1 mL, 100 ,umol Tris-HCl (pH 8.5), 0.6 ormol hypoxanthine, 1 ,mol NAD+, and a suitable amount of enzyme extract (40-80 ,ug) (14). The reaction was started by the addition of enzyme extract and was carried out at 30°C. One unit of enzyme activity is defined as the amount of enzyme which catalyzed the reduction of 1 ,mol of NAD+ per min under optimal assay conditions. Specific activity is expressed in munits/mg protein.
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Analytical Determinations Cell growth was measured turbidimetrically at 660 nm. Protein was determined according to Bradford (3), using
Figure 2. Uptake and intracellular accumulation of hypoxanthine in wild-type and mutant cells of C. reinhardtii. Conditions are as in the legend of Figure 1 except that hypoxanthine substituted for xanthine.
PEREZ-VICENTE ET AL.
128
up are represented in Figures lA and 2A. XDH increased linearly after a lag phase, reached maximum values at the moment of purine depletion from the medium, and then was maintained until intracellular purine concentration started decreasing. Both purine uptake and accumulation were also observed in mutants 104 (Figs. IB, 2B) and 102 (not shown) lacking XDH (9). Xanthine was taken up after a lag phase similar to that of wild-type cells and at a similar rate. Lag phase for hypoxanthine was longer in mutant 104 (8 h) than in wildtype strain (5 h), although uptake rates were very similar. Intracellular accumulation of purines in mutant strains 102 and 104 was higher than in wild-type cells (1.1-1.4 times for xanthine and 3-4 times for hypoxanthine). These increases may reflect the nondegradation of purines to uric acid by mutants lacking XDH. Mutant 305, which has XDH, accumulates a high concentration of xanthine whereas that of hypoxanthine was similar to that found in wild-type cells (Table I). Appearance of the uptake systems of xanthine and hypoxanthine depended on de novo protein synthesis, since purine consumption was not induced in the presence of 3.5 ,M cycloheximide, a known inhibitor of protein synthesis in eukaryotes. In Figure 3 the cross-induction of both uptake systems by either hypoxanthine or xanthine is shown. In the presence of hypoxanthine as inducer, ['4C]xanthine was taken by cells still incapable of consuming hypoxanthine, and the ability for xanthine consumption increased along the time (Fig. 3A). However, in the presence of xanthine as inducer cells actively consuming xanthine were unable to use ['4C]hypoxanthine until 3.5 h elapsed (Fig. 3B), when the lag phase for hypoxanthine uptake induction was ending and hypoxanthine utilization started (Fig. 3A). On the other hand, cells fully induced (>8 h) in media with either hypoxanthine or xanthine were able to consume both purines without any lag phase (results not shown). Km and pH values for xanthine and hypoxanthine uptake were very similar (1.1 and 1.4 ,M; and 6.8 and 6.5, respectively). These figures greatly differ from those of XDH (Km apparent for xanthine and hypoxanthine, 70 and 160 uM, respectively; optimum pH for both substrates, 8.4) (15). The effect of different metabolic inhibitors on hypoxan-
Table I. Intracellular Accumulation of Xanthine and Hypoxanthine in Wild-Type (6145c) and Mutant Cells of C. reinhardtii Cells grown phototrophically with 10 mm ammonium were washed and transferred to media containing 0.35 mm xanthine or hypoxanthine as the sole nitrogen source. Intracellular purine concentration was determined just after cells exhausted purines from the medium. Intracellular Concentration Strain Xanthine
Hypoxanthine
nmol/mg fresh wt 6145c wild 305 (XDH+) 102 (XDH-) 104 (XDH-)
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34
11 27
28
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Plant Physiol. Vol. 95, 1991
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