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Bioscience Reports 3, 141-151 (1983) Printed in Great Britain 141 13C nuclear magnetic r e s o n a n c e studies of anaerobic glycolysis in Trypanosoma b r u c e i spp. N. E. MACKENZIE' 82 3ames Edwin HALL%, I. W. FLYNNw and A. I. SCOTT*82 *Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland; %Department of Parasitology and Laboratory Practices, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27514, U.S.A.; and w of Biochemistry, University of Edinburgh, Edinburgh EH8 9XD, Scotland (Received 24 January 1983) A n a e r o b i c glycolysis in Trypanosoma brucei spp. has been studied by 13C NMR at 50 and 75.5 MHz. The uptake of [U-13C]glucose by cell suspensions of T. b. b r u c e i was monitored by time-course spectroscopy, and while no a n o m e r i c s p e c i f i c i t y was found, t h e . e n d products of glycolysis were confirmed as glycerol and pyruvate together with alanine and dihydroxypropionate. The i n t e r m e d i a c y of L-glycerol-3-phosphate was also ascertained. The incorporation of C-I of [t-13C]glucose and of C-6 of [6-13C]glucose into glycerol and pyruvate in T. b. gambiense was quantified by measurement of t h e l o n g i t u d i n a l r e l a x a t i o n t i m e s of the s p e c i e s involved. An incorporation to the extent of 66% of each substrate into equimolar amounts of glycerol and p y r u v a t e i n d i c a t e that Keq for the triosephosphateisomerase-mediated reaction approaches unity. The use of high-resolution 13C nuclear magnetic resonance (NMR) in the noninvasive monitoring of metabolism in vivo in both microo r g a n i s m s (1-3) and intact organs (4,5) is now well established. These studies have typically involved the observation of chemical shift c h a n g e s r e s u l t i n g from biochemical transformations of singly l~Cenriched substrates. There still exists, however, the problem of the rigorous assignment of carbon-13 nuclear-magnetic-resonance signals in vivo and while u n a m b i g u o u s i d e n t i f i c a t i o n can only be made by i s o l a t i o n of e n r i c h e d m e t a b o l i t e s this is not always possible or practical. Comparison of extracts with authentic samples of suspected compounds combined with variation of chemical shift with pH are most commonly employed for the identification of unknown species. 82 Present address: Center for Biological NMR, Department of Chemistry, Texas A&M University, College Station, Texas 77843, U.S.A. 01983 The Biochemical Society 142 MACKENZIE ET AL. The use of multiply or uniformly laC-enriched substrates facilitates the rigorous assignment of metabolites, especially transient specie% and analysis of the more complex spectra obtained gives information of cycling and pathway convergence (6) which may be obscured in the case of singly 13C-enriched substrates. In many studies involving singly enriched substrates much of the d a t a e x t r a c t a b l e from the 13C NMR s p e c t r a o b t a i n e d from a time-course has been used to draw conclusions about the kinetics of t h e biological s y s t e m . While the d a t a base is sound, certain constraints of the technique should be fully realized before embarking on c o m p l e x k i n e t i c arguments. In particular the 13C-NMR signal intensities obtained from spectra (even when normalized against an i n e r t i n t e r n a l s t a n d a r d ) c a n n o t be used as a direct method of quantification of metabolite concentration. Signal intensities are also dependent on the longitudinal relaxation time (TI), which differs from one carbon nucleus to another in a molecule; the extent of coupling to adjacent and remote carbons; and the contribution from the nuclear Overhauser e f f e c t . Since these aspects have not been emphasized in previous detailed analyses of spectra derived from metabolic processes (2,7), we now report on IaC-NMR measurements of the anaerobic metabolism of [U-laC]glucose by Trypanosoma b r u c e i b r u c e i and of [l-13C]glucose and [6-13C]glucose by Trypanosoma brucei gambiense which illustrate the above principles and, at the same tim% clarify certain aspects of the glycolytic pathway examined in a previous study of trypanosomes (8). M a t e r i a l s and M e t h o d s Tr ypanosomes Both T. b. gambiense and T. b. brucei were used in these experiments. All handling procedures, including isolation and incubat i o n p r o c e d u r e s , for the monomorphic strain (TxTAT-I) of T. b. g a m b i e n s e have been described (8). The monomorphic strain (TREU 55) of T. b. b r u c e i used in these experiments was maintained as stabilates at -70~ in 10% glycerol in Buffer B containing 50 mM glucose. Adult Wistar rats from a breeding colony at the University of Edinburgh were inoculated intraperitoneally with--I06 trypanosomes. B l o o d s t r e a m f o r m s were harvested by cardiac puncture of ethera n a e s t h e t i z e d rats at peak parasitemia approximately 72 h after inoculation. Blood was collected into ice-cold 0.15% heparin in Buffer B~ pH 8.0, containing 50 mM glucose. The blood was centrifuged at 1200 g for 10 min in an MSE 18 model centrifuge, the parasite layer removed and washed once, in the above buffer, by centrifugation. The trypanosomes were separated from remaining blood cells by column chromatography on DEAE-cellulose (9) and washed once in cold Buffer A or Buffer B, pH 7.4. The trypanosomes were resuspended to 5 x 108 cells/ml in Buffer A or B and kept on ice until incubation. Incubat ion Incubations were performed either in Buffer A or Buffer B to which 13C-enriched glucose was added. Immediately after incubation t3C-NMR STUDIES OF GLYCOLYSIS IN T. BRUCEI 1~3 an aliquot of trypanosome suspension was washed once by centrifugation with 4 vol. of ice-cold Buffer A or Buffer B, then resuspended to 5 x 108 cells/ml in ice-cold Buffer A or Buffer B. Time-course experiments involved the addition of [U-13C]glucose to 15 ml of a cell suspension in Buffer A or Buffer B to give the final c o n c e n t r a t i o n s d e s c r i b e d in e a c h f i g u r e . The suspensions were incubation at 35~ with shaking, under anaerobic (N 2) conditions. Cell viability was monitored throughout by microscopic-examination. Aliquots (2.5 ml) were taken at intervals (Table l) and immediately frozen in an ethanol-dry ice mixture. These were allowed to thaw at t~~ and were subjected to f r e e z e / t h a w cycles until cell lysis occurred. 13C-NMR spectra acquisition was performed on 2 ml of each aliquot without further t r e a t m e n t after the addition of deuterium oxide (10% v/v) as an internal lock. The remaining 0.5 ml of aliquot was used for enzymatic assay. Acid-lysed samples were prepared by addition of 6 M perchloric acid, pelleting the precipitated protein by centrifugation at 1200 g for l0 min, and r e a d j u s t i n g the supernatant to pH 7.4 with 0.33 M potassium hydroxide. Assays The NMR s p e c t r a were recorded on either a Bruker WM 300 wide-bore spectrometer at 75.5 MHz or a Varian XL-200 spectrometer at 50.5 MHz for 13C nuclei. All spectra were measured as indicated in f i g u r e l e g e n d s , and ISC chemical shifts are given relative to tetramethylsilane at 0 ppm. Glycerol was assayed by a coupled enzymatic assay using glycerok i n a s e , p y r u v a t e kinase, and lactic dehydrogenase (10,11). Since pyruvate was present in all samples, the addition of glycerol kinase to the reaction mixture was delayed until a stable absorbance at 340 nm was obtained (12). Pyruvate was measured by the standard method (13) of sample incubation with NADH in the presence of lactate dehydrogenase and measurement of the 'end-point' drop in absorbance a t 340 nm b r o u g h t about by oxidation of the NADH. Pyruvate c o n c e n t r a t i o n s may then be calculated using the molar extincting coefficient of 6.22 x 103 for NADH. Similarly, glucose was assayed by addition of hexokinase. Materials Buffer A was the standard Roswell Park Memorial Institute (RPMI) m e d i u m ( f o r m u l a t i o n 1640) p r e p a r e d by mixing the individual chemicals (Sigma) according to the formulation but omitting glucose. Buffer B was prepared by addition of sodium chloride (130 raM) and potassium chloride (5 mM) to a 20 mM phosphate buffer at pH 7.4. [ U - 1 3 C ] g l u c o s e (7996 atom enrichment) was purchased from KOR Isotopes (Cambridge, MA). [l-I3C]glucose, [6-I3C]glucose (90% atom enrichment), and deuterium oxide were purchased from Merck and Co. (St. Louis, MO). The glycerol and pyruvate assay materials were purchased from Sigma. 144 MACKENZIE ET AL. Table i. Anaerobic incubation 5 ~mol ml -I of [U-13C]glucose by 5 x 10 8 cells ml -I of T. b. brucei in Buffer A (see 'Materials and Methods') Time (min) 1 3 5 Ii 22 Concentrations (~oI ml -I) of Motility Pyruvate G!ycerg~ Glucose 0.66 1.23 1.60 2.74 4.81 0.67 1.50 2.01 3.75 5.43 4.41 2.18 0.74 0.71 0.68 very low R e s u l t s and D i s c u s s i o n The bloodstream parasites T. b. brucei and T. b. gambiense are the causative agents of nagana in cattle and sleeping sickness in man, respectively. The now generally accepted mechanism of glucose catabolism in the bloodstream form of these eukaryotes is the Embden-Meyerhof scheme, first proposed by Grant and Fulton (1$). Under anaerobic conditions however, when equimolar quantities of pyruvate and glycerol are produced (12,14), reservations have been expressed as to the validity of this pathway (15,16). Aerobically, pyruvate is considered to be the exclusive end-product of glycolysis (1%17) as it cannot be further metabolized, due to the absence of both a functional tricarboxylic acid cycle (18,19) and the enzyme lactate dehydrogenase (20) and is finally excreted in the host blood. The transport of glucose, the sole energy source of bloodstream trypanosomes, across the cell membrane has been determined as the rate-limiting step in its catabolism (21). Subsequent glycolysis is extremely rapid and the steady-state concentrations of intermediates so low as to v i t i a t e the monitoring of glycolysis by time-course spectroscopy using [ I - 1 3 C ] - a n d [6-t3C]glucose (8). This was c o n f i r m e d by incubation of [U-laC]glucose with cell suspensions of T. b . b z u c e i . Although time-course spectroscopy failed to detect any glycolytic intermediate, the gross features of anaerobic metabolism could be observed. The aJ~ equilibrium ratio of the I a C - N M R signals of C - I of glucose is 36:64 (22) and in these e x p e r i m e n t s does not change within experimental error throughout glucose utilization. This indicates that there is no increase in the r a t e of a n o m e r i z a t i o n on the cell surface and that no anomeric s p e c i f i c i t y exists in T . b . brucei, in contrast to other systems (1,2,23). Nine glycolytic enzymes and glycerol kinase are sequestered within an organelle in T. b. b r u c e i and other trypanosome species. The existence of this glycosome has been invoked to explain the anaerobic pathway converting glucose to equimolar pyruvate and glycerol while giving a net yield of ATP (24). Hammond and Bowman (25) have shown that glycerol formation proceeds within the glyeosomal compartment of the trypanosome cell via reversal of the glycerol kinase reaction resulting in ATP synthesis by L-glycerol-3-phosphate(G-3-P)-dependent phosphorylation of ADP. This t h e r m o d y n a m i c a l l y unfavorable reaction may proceed due to I3C-NMR STUDIES OF GLYCOLYSIS IN T. BRUCEI 1#5 efficient compartmentation of the trypanosome cell leading to high local concentrations of G-3-P. This factor coupled with glucose phosphorytation from G-3-P-dependent ATP formation would allow this reaction to proceed in the direction of ATP generation (26). The flux of [U-13C]glucose was conveniently monitored by freezing aliquots of anaerobic incubation mixtures for assay at regular intervals throughout the experiments, until microscopic examination revealed that the ceils were no longer viable (Table I ) . This enabled t3C-NMR data to be accumulated for much longer time blocks. The glycolytic reaction profile in Buffer A obtained from the t3C-NMR spectra (Fig. I A , B ) shows t h a t the only i n t e r m e d i a t e of glyco]ysis detected anaerobically is G-3-P which is identilied by virtue of the 13C NMR signal of C-3 (d, 668.I ppm; 3C2_C3 = 33 Hz). This is the first d i r e c t (i.e., n o n - e n z y m a t i c ) evidence for the existence of this intraglycosomal metabolite in T. b. b r u c e 1 and confirms the dramatic increase in the concentration of G-3-P found in cells undergoing anaerobic glycosysis and in cells treated with salicilylhydroxamic acid (SHAM), which mimics anaerobiosis (27). The end-products of anaerobic glycolysis were shown to be glycerol (Ct, 3 d, 663.~ 13Ct(3)_C2 = z~2 Hz; C 2 t, 673.4 13Cz_C1(3 ) = 42 Hz) and pyruvate (C I dd, 6170.9 t3C1_C2 = 62 Hz, 23Ct_C3 = 13 Hz; C 2 dd, 6205.8 13C2_C1 = 62 Hz, 13C2_C3 = 30 Hz; C 3 dd, 627.3 13C3_C2 = #0 Hz, 23C3_C1 = 13 Hz) (Fig. 2). The significant amount of alanine formed anaerobically in Buffer A is due to transamination of pyruvate (Table 1) enhanced by the amino-group donors present in this enriched buffer. These, however, must also be synthesized endogenously since alanine (C I d, 6176.9 13C1_C9 = 60 Hz; C9 dd, 651.7 13C2_C! = 60 Hz, 13C2_C3 35 Hz; C 3 d 617.# ~3C3_Cz = 35"Hz) is produced in incubations in Buffer B in which no exogenous amino group donors are present (Fig. 2). Alanine was found to be a consistent end-product of glycolysis and although ignored in the past must be considered in any future carbon-balance studies undertaken in this system. In Fig. 1B the apparently greater rate of alanine production compared with that of p y r u v a t e simply r e f l e c t s the longer T t for C-3 of pyruvate compared with T I for C-3 of alanine. The remaining 13C-NMR signals in Fig. 2 are due to 2,2-dihydroxypropionate (C 1 d, 6179.8 3C1_C2 = 60 Hz; C2 dd, 695.2 13C2C3 = t~# Hz, t J c 2 _ c t : - 6 0 Hz; C~ d, 626.# tJC3_C2 = ## Hz), arising from hydration of pyruvate (28-32), a common reaction of a-oxoacids in aqueous media (33). Despite the production of nearly equimolar quantities of pyruvate and g l y c e r o l , as determined by enzymatic assay, there is a Iarge discrepancy in the 13C-NMR resonance intensities of these metabolites. This is most obvious on comparison of the resonances assigned to C1,3 of glycerol and C-3 of pyruvate (Fig. 2), an e f l e c t previously reported (8) in the metabolism of [1-13C] - and [6-13C]glucose in suspensions of T. b . g a m b i e n s e . It was found in the latter work that the label in [ l - 1 3 C ] g l u c o s e was predominantly incorporated in CI,3 of glycerol while C-6 of [6-13C]glucose was apparently randomized between CI,3 of glycerol and C-3 of p y r u v a t e . This evidence could not be rationalized in the light of the Embden-Meyerhof pathway and the existence of an alternative mechanism was suggested. In order to reconcile this anomaly, [ l - t 3 C ] - and [6-13C]glucose were incubated w i t h T . b . g a m b i e n s e essentially according to the method of 146 MACKENZIE ET AL. , ,, 4~ IQ- 2, 0 2 4 ~ , ,0 ,2 ,4 ,~ ,, ,,~2 0 2 4 , , ,2 ,4 ,~ 20 22 TIMs Fig. i. Normalized 13C-NMR signal intensities taken from spectra (75.5 MHz) of 5 mM [U-13C]glucose (98% atom enrichment) anaerobically metabolized by 5 x 108 cells/ml of T. b. brucei in Buffer A. A 10-mm NMR tube was used and spectra were acquired at 5~ Peak intensities were taken from spectra which r e s u l t e d from 5000 (72~ e90 = 25 s) pulses accumulated over 38 min. The lines were broadened by 5.0 Hz by exponential multiplication of the F.I.D. The spectral width was 17 857 Hz of 16 K data points. (A) O ~ signal intensity of e~8 C-6 of glucose; A , signal intensity of C-1,3 of glycerol. (B) O , signal intensity of C-3 of L-glycerol -3-phosphate, A, signal intensity of C-3 of alanine; [] , signal intensity of C-3 of pyruvate. Mackenzie et al. (g) but without addition of dithiothreitol to acid lysates. The spectra obtained were consistent with those published previously (Fig. 3A,C) and the taC-NMR resonances assigned (see Fig. 2). The l o n g i t u d i n a l r e l a x a t i o n time (T t) of the various species involved were measured using the method of inversion recovery (34) (Table 2). While the T t values of ~C-1 and 8C-1 of glucose, C-3 of a l a n i n e , and C-3 of 2 , 2 - d i h y d r o x y p r o p i o n a t e were of anticipated magnitudes (35), C-3 of pyruvate was abnormally long at 10.68 + 1.27 s. I~C-NMR STUDIES OF GLYCOLYSIS IN T. BRUCEI 1~7 G%X~ /', - o.,,, \ ~ 9' It/. p GI \ Ckm~cal ~h~ft(pl~ Fig. 2. 13C-NMR (75.5 MHz) spectrum of the acid lysate of an incubation involving the anaerobic metabolism of 5 m M [ U - 1 3 C ] g l u c o s e (79% atom enrichment) by 7.5 x 109 cells (5 x 108 cells/ml) of T. b. brucei in Buffer B at pH 7.4 at 35~ A 20-mm NMR tube was used and temperature was maintained by~ e m p l o y i n g a gated decoupling ratio of 5:1. The spectrum represents i0 200 (90~ ~90 = 40 s) pulses accumulated over 6.26 h. The lines were broadened by 3.0 Bz by e x p o n e n t i a l multiplication of the F.I.D. The spectral width was 18 518 Hz over 16 K data points. Abbreviations: AI9 A29 A 3 - C-I 9 -29 and -3 of alanine 9 respectively; DI9 D29 D 3 - C-I -29 and -3 of dihydroxypropionate9 respectively; ~98G19 egBG 6 - e- and 8-anomers of C-I and C-6 of glucose 9 respectively; Gly I 3 and Gly 2 - C-I(3) and . 9 C-2 of glycerol 9 respectlveIy; PI~ P2~ P3 - C-19 -2~ and -3 of pyruvate, respectively. Table 2. Longitudinal relaxation times (T I) measured by the method of inversion recovery Carbon-13 @pecies T 1 (s) eC-i Glucose 0.57 +- 0.06 8C-I Glucose 0.81 + 0.05 C-193 Glycerol 1.34 + 0.07 C-3 Dihydroxypropionate 1.17 +_ 0.34 C-3 Alanine 2.81 + 1.49 C-3 Pyruvate 10.68 -+ 1.27 Its8 MACKENZIE A ET AL. B fpr~ ~,'GlYl,3 J~ I I I I 1 I IO0 I 1 f ~1 I 0 I00 C .... I 0 D /~,pG6 I IL]O I I I I I PPM 0 I IL]O I I I I PPM Fig. 3. (A) 13C-NMR (50.3 MHz) spectrum of an acid lysate of 5 m M [l-13C]~glucose (90% atom enrichment) anaerobically metabolized by 5 x 108 cells/ml of T. b. 9ambiense in Buffer B. A 10-r~n NMR tube was used and the spectrum was acquired at 0~ The spectrum represents 7200 (50~ ~90 = 14.2 ps) pulses accumulated over I h. The lines were broadened by 0.45 Hz by exponential multiplication of the F.I.D. Abbreviations same as for Fig. 2. (B) 13C-NMR (50.3 MHz) spectrum of the same sample as in (A) above acquired at 0~ with no nuclear O v e r h a u s e r enhancement. The spectrum represents I 0 I3C-NMR STUDIES OF GLYCOLYSIS IN T. BRUCEI 1#9 S p e c t r a of the acid l y s a t e of the cell suspensions were then a c c u m u l a t e d under c o n d i t i o n s of z e r o nuclear Overhauser e f f e c t (decoupler power o f f for 10 s~ on for 0.5 s during F.I.D.) and a small flip angle (29 ~ ) to minimize differential saturation e f f e c t s (36). Under these conditions, which optimize the C-3 of pyruvate signal/ noise~ integration of the t3C-NMR resonances gave a true representation of the relative intensities if the integral for the C-3 of pyruvate was corrected by +g%. The resonances in Fig. 3B were integrated, adjusted as above, and it was found that C - 1 of [ l - t 3 C ] g l u c o s e is incorporated preferentially into C-1 (= C-3) of glycerol to the extent of 2 to 1 over C-3 of pyruvate, alanine, and 2,2-dihydroxypropionate c o l l e c t i v e l y (the l a t t e r t w o m e t a b o l i t e s arising from pyruvate). I n t e g r a t i o n of the r e s o n a n c e s in Fig. 3D revealed that C-6 of [6-t3C]glucose was preferentially incorporated in the same ratio (2/1) i n t o C - 3 of p y r u v a t e , alanine, and 2 , 2 - d i h y d r o x y p r o p i o n a t e (collectively) over C-1~3 of glycerol. Triosephosphate isomerase interconverls glyceraldehyde-3-phosphate ( G a l d - 3 - P ) , d e r i v e d from C-6 of g l u c o s e , and dihydroxyacetone phosphate (DHAP), the initial site of C-I of glucose. The equilibrium constant (Keq) has been assigned the value 22 to 2# (37,3g) for DHAP/Gald-3-P in rabbit-muscle enzyme. More recently, however~ it was determined that in neutral solutions, Gald-3-P is 96.7% hydrated to the gem diol and DHAP is ##% hydrated (39,#0). Since only the unhydrated substrates are bound by the enzyme, the corrected value of his 367 (DHAP/Gald-3-P) at 25~ Ke lc~ trypanosome glycolysis an earlier study (1#) showed that under a n a e r o b i c c o n d i t i o n s C-1 of [1-t~C]glucose was preferentially incorporated into glycerol despite equimolar production of this metabolite and pyruvate as end-products. It was concluded from this result that the rates of formation of glycerol from DHAP and pyruvate from Gald-3-P were equal and greater than the rate of the triosephosphate-isomerase-mediated conversion of Gald-3-P into DHAP. We have shown in this study that C-1 of glucose is incorporated into glycerol to the extent of 66%, in agreement with earlier results (1#). However, the same incorporation (66%) of C-6 of glucose into p y r u v a t e and the e q u i m o l a r production of glycerol and pyruvate i n d i c a t e s that Ke for t h e t r i o s e p h o s p h a t e i s o m e r a s e r e a c t i o n approaches unity in ~. b r u c e i spp. 4000 (29 ~ ~90 = 14.2 ~s) pulses with an acquisition time of 0.5 s and delay time of 10 s. Lines were broadened as above by 1Hz. (C) 13C-NMR (50.3 MHz) spectrum of an acid lysate of 5 m M [6-13C] glucose (90% atom enrichment) anaerobically metabolized by 5 x 108 cells/ml of T. b. gambiense in Buffer B. Spectrum accumulation condition as for (A) above. (D) 13C-NMR (50.3 MHz) spectrum of the same sample as in (C) above accumulated under conditions as for (B) above. 150 MACKENZIE ET AL. R a t i o n a l i z a t i o n of this incongruity c a n n o t be found in the high degree of c o m p a r t m e n t a t i o n in T. b r u c e i spp. as no t r a n s m e m b r a n e processes are involved. 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