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Studies on the nature of thiamine pyrophosphate binding and dependency on divalent cations of transketolase from human erythrocytes
0020-7 I I X/88 $3.00 + 0.00 Copyright C 1988 Pergamon Press plc
~nr. J. ~iochem. Vol. 20, No. I I, pp. 1255-1259, 1988 Printed in Great Britain. All rights reserved
STUDIES ON THE NATURE OF THIAMINE PYROPHOSPHATE BINDING AND DEPENDENCY ON DIVALENT CATIONS OF TRANSKETOLASE FROM HUMAN ERYTHROCYTES EUN HEE JUNG,’ TOHORU TAKEUCHI,’ KOHSUKE NISHINO’ and YOSHINORI ITOKAWA’ ‘Department of Hygiene, Faculty of Medicine, Kyoto University and *Department of Home Economics. Dohshisya Women’s College, Kyoto 606, Japan (Received 29 February
1988)
Abstract--l.
The binding kinetics for [j5S]thiamine pyrophosphate to transketolase and the dependency of transketolase on divalent cations for activity were investigated. 2. With Scatchard analysis, dissociation constant (&) and n value were calculated to be 0.2 x IO ’ M and 0.66 respectively. 3. The activity of the reconstituted enzyme increased in the order of Co*+ < Mn’+ < Ca’+ < Mg’+. The native
transketolase
contained
Mg 2+ in its molecular
lution and reconstitution of homogeneous ketolase purified from human erythrocytes.
INTRODUCTION
Transketolase (EC 2.2.1.1) appears to have multiple functions in intermediary metabolism. In the oxidative and reductive pentose phosphate cycles, it participates in the breakdown and synthesis of carbohydrates. Equally important is its role in nucleic acid metabolism as a generator of ribose S-phosphate from sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate. There are many studies on the properties of transketolase from yeast (De La Haba et al., 1985; Kochetov and Philippov, 1970; Kremer et al., 1980; Egan and Sable, 1981): transketolase releases thiamine pyrophosphate (TPP) and divalent cations,
such as Mg’+, on alkaline treatment and the resolved enzyme can be reconstituted by incubation with TPP and a divalent cation (Heinrich et al., 1972; Saitou et al., 1974). Although the physiological metal ion is Mg2+, a number of other divalent cations are capable of activating the enzyme (Heinrich et al., 1972; Morey and Juni, 1968). By means of kinetic studies, Kochetov et al. (1975) demonstrated a positive cooperative interaction between the binding sites of TPP, in the presence of Mg*+. On the contrary, Egan and Sable (1981) concluded that there was negative cooperativity in the binding of TPP to the apoenzyme in the presence of Mg*+ or Ca*+. Compared to transketolase from yeast, that from mammalian tissues has been studied less, especially that from human tissues has not been fully characterized with respect to its catalytic and chemical properties, and very little is known about the interaction between TPP and apotransketolase. This is largely because of unavailability of homogeneous enzyme preparations. The present study describes the binding nature of TPP and the effect of divalent cations through resoAbbreviations: sodium dodecyl
TPP-thiamine sulfate.
pyrophosphate;
SDS-
structure.
MATERIALS
trans-
AND METHODS
Materials Outdated human blood was donated by Kyoto University Hospital. [F.]Thiamine hydrochloride was purchased from Amersham. o-Xylulose 5-phosphate and D-sedoheptulose 7-phosphate were from Sigma. D-Ribose S-phosphate was from Boehringer. DEAE-Sephadex A-50, Sephadex G-150 and G-100, Blue Sepharose CL-6B, Mono P, Pharmalyte 8-10.5, Poly buffer 96, and PD-IO were from Pharmacia. The ultrafiltration membranes YM-30 and YMT were from
Amicon. LiChrosorb NH, (the particle size was 5 pm) was from Merck, TSK-Gel G 3000 SW for HLC was from Tow Soda, and the Bio-Rad protein assay kit was from Bio-Rad. The atomic absorption spectrophotometer was from Shimadzu, the scintillation counter was from Aloka and Biofluor scintillation solution was from NEN Research Products. All other chemicals were reagent grade materials from commercial sources. Purificarion and assay of ~ranskerolase Transketolase from human erythrocytes was purified according to Takeuchi er al. (1986). Blue Sepharose CL-6B was used to remove glyceraldehyde 3-phosphate dehydrogenase when necessary. After the chromatography on Sephadex G-150, the sample was applied to a column of
Blue Sepharose CL-6B (i7 x 150&n) equilibrated with 50mM Tris-HCl buffer (oH 8.0). For elution. a linear gradient of NaCl from 0 to 0. I5 r;?, with the flow rate of 0.9ml/min was used. The enzyme preparation thus obtained, gave only one protein band with SDS polyacrylamide gel electrophoresis, and contained 0.9 mol of TPP per 1mol of enzyme subunit. The activity of transketolase was determined by the formation rate of sedoheptulose-7-phosphate (Takeuchi et al., 1984). Preparation of apotranskerolase The method (Warnock and Prudhomme, 1982) using ammonium sulfate with acidic medium was used to resolve the enzyme with some modifications.
1255
ELJN HEE
1256
JUNG
To 800 p I of purified transketolase (440 pg/ml), I200 ~1 of ammonium sulfate solution (saturated ammonium sulfate solution/4 mM sodium acetate/l M sulfuric acid, 750: IO: 7. v/v) was added. The final concentration of ammonium sulfate was approx. 60%, and the pH of the reaction mixture was 3.5. After keeping at 0~5’C for 5 min. the mixture was centrifuged at 15,OOOg for IOmin at 4 C and the supernatant was discarded. The precipitate was resuspended in 800 ~1 of 0. I M Tris-HCI buffer (pH 7.5. at 25 C) containing I mM MgCI, and 10% (v/v) glycerol, and centrifuged again at 15,OOOg for IOmin at 4’C. The supernatant was used as apotransketolase. For characterization of the dependency on divalent cations. the buffer for resuspension did not contain MgCI,. Drtermmation
of[“SFPP
NaCl was used. The flow rate was 0.5 ml/min and a fraction volume was I ml. Mg’+ content of the fractions was deterby
flame
atomic
100
.-• 0
2 = : I-
50
\/
Lo-
0
Total Mg” content of the enzyme preparation was determined at 285.1 nm by flameless atomic absorption spectroscopy. after drying at 15O”C, ashing at 600’C and atomizing at 19OO’C. To remove nonenzyme bound Mg’+ from the enzyme preparation, we used a HLC TSK Gel G 3000 SW column (7.53 x 600 mm). For equilibration and elution, 50 mM Tris-HCI buffer (pH 7.0 containing 0.3 M
285.2 nm
ai.
100
binding
[“S]TPP was prepared according to Nishino er al. (1983). The specific activity of [?j]TPP was 127.3 cpm/pmol. 100 ~1 of the reaction mixture containing 3Apg of apotransketolase and various concentrations of [‘5S]TPP in 50 mM Tris-HCI buffer (pH 7.5, at 25’C) was incubated at 37 C. After 30 min, 400 ~1 of the same ice-cold buffer was added to stop the reaction. The mixture was applied to YMT membrane and centrifuged at IOOOg for IOmin at 4 C. The membrane was washed 3 times with 500~1 of 50 mM Tris-HCI buffer containing 0. I mM non-radioactive TPP. to remove nonbound [?i]TPP. Finally. 500 ~rl of 2% trichloroacetic acid solution was added to the membrane and centrifuged to eliminate enzyme-bound [“SITPP. IO ml of Biofluor scintillation mixture were added to the effluent and the radioactivity was determined with scintillation counter.
mined at troscopy.
et
absorption
spec-
TPP was identified and determined by the method of Ishii et al. (1979) using a LiChrosorb NH, column (4 x 150 mm). Protein was determined with a Bio-Rad protein assay kit using bovine serum albumin as standard. Polyacrylamide gel electrophoresis was performed in the presence of SDS on 10% polyacrylamide slab gels according to the procedure of Laemmi (I 970).
RESULTS
While transketolase from yeast could be resolved into TPP and apotransketolase in alkaline medium (De La Haba et al., 1955; Heinrich et al., 1972), that from human erythrocytes could be resolved in acidic medium. TPP was dissociated from the enzyme at pH
l\d-
4
c
5
^
A
6
PH Fig. 1. Effect of pH on dissociation of TPP from transketolase. The enzyme preparations previously adjusted to pH 3.5-6.0 with 50 mM sodium acetate buffer were centrlfuged using MPS-I Micropartition system. The enzyme protein was extracted from the membrane with 50mM Tris-HCI buffer (pH 8.0) and was used to determine the catalytic activity. The effluent that traversed the membrane was collected and used to determine the content of TPP. The activity content are given as percentages of those obtained in the assay of the native enzyme.
below 4.0, although the activity was not restored by incubation with TPP. (Fig. 1) However another method using acidic medium (pH 3.5) with ammonium sulfate was successful in restoring the activity by addition of TPP and Mg*+. Restoration of the activity by addition of TPP and Mg*+ was 5&60% and the specific activity was the same as that of the native enzyme. The resolved enzyme preparation could be stored at -9O’C for at least 4 days without significant loss of activity. [3sS]TPP binding to the transketolase was specific. (Table 1) A large amount of nonradioactive TPP which was added to the reaction mixture containitig 0.5 PM [35S]TPP inhibited the binding of radioactive TPP competitively. The optimum binding between TPP and apotransketolase was observed at pH 7.5. (Fig. 2) The reconstitution of the resolved enzyme was much affected with incubation time and temperature. The optimum binding between TPP and apotransketolase was observed at 37’C with an incubation time of 45 min. (Fig. 3) However in case of an incubation for 60min the binding seemed to decrease, but the activity decreased slightly. The decrease in binding suggested that there may have been some degeneration of the enzyme. Therefore we chose an incubation time of 30 min in our experiments. We found that the features of TPP binding to enzyme were the same as those of catalytic activity of enzyme. (Fig. 4) The binding was characterized by a single, noninteracting binding site with a dissociation constant
Table I. SpAicily of [‘?S]TPP binding to apotransketolase: each mixture containing 5.1 p’g of apoenzyme and 0.5 PM [‘SS]TPP was incubated at 37 C for 10 min with or without 5 mM nonradioactive TPP. The following procedure was the same as described in the text
cpm [“SITPP
with nonradioactwe
without nonradioactive
0.5 uM
19.5
1510.4
Thiamine pyrophosphate
1257
binding
I::jA
1:: i
.-c” 0
.E m
c loGO-
-25
I 1
d 0
I 2
I 3
I 4
.g t a
I 5
TPP+M)
Fig. 4. [“S]TPP binding to apotransketolase and catalytic activitv of the reconstituted enzvme as a function of [‘%]TPP concent;ation.
PH
Fig. 2. Effect of pH on [%]TPP binding to apotransketolase from human erythrocytes. Apoenzyme (5.1 pg) and [%]TPP in 100~1 of 50mM sodium acetate @H 4.W.O) or 50 mM Tris-HCl buffer (pH 7.G9.0) of the indicated pH value were incubated at 37°C for IO min. The procedure followed is described in the text.
of 0.2 x IOebM. (Fig. 5) Because we found that the molar ratio of TPP and transketolase (monomer) was 0.9:1, we calculated the dissociation constant under the assumption of n = 1, however in fact the plot showed n = 0.66.
Time (mini
Fig. 3. Time dependence of [%]TPP binding to apotransketolase and catalytic activity of the reconstituted enzyme. Apoenzyme (4.0 fig) and 0.5 PM of [%]TPP in 100 ~1 of 50mM Tris-HC1 buffer (pH 7.5, at 25°C) were incubated at 37°C for the indicated time. The procedure followed is described in the text. For determination of the catalytic activity, 3.9 pg of apoenzyme was incubated with 0.5 FM [3sS]TPP. Each mixture was transferred to the MPS-I Micropartition system and centrifuged at IOOOgfor 10min at 4°C. After washing (the buffer did not contain TPP) to remove the nonbound [%]TPP, the enzyme was extracted from the membrane with 200~1 of 50mM Tris-HC1 buffer (PH 8.0). The solution containing the extracted enzyme was used to determine the catalytic activity.
L
0
0.5
1.0
1.5
r/[TPP],
2.0
2.5
3.0
3.5
x IO6
Fig. 5. Scatchard plot of [“S]TPP binding to apotransketolase from human erythrocytes. 100 ~1 of reaction mixture containing apoenzyme (3.2 pg) and various concentrations of [‘%]TPP in 50 mM Tris-HC1 buffer (pH 7.5, at 25°C) were incubated at 37°C for 30min. The procedure followed is described in the text. Kd and n values were estimated from least square methods (r = 0.96). r is the value of the ratio of enzyme-bound TPP to moles of transketolase, and [TPP], is the concentration of free TPP. The activation of transketolase was not observed in the absence of either Mg*+ or TPP. (Table 2) This means that transketolase absolutely requires Mg*+ for its catalytic activity. To clarify whether this result is specific to Mg*+ or not, various divalent cations were used instead of Mg*+. Other divalent cations also could restore the activity. (Table 3J The activity increased in the order of of the enzyme Co*+ < Mn*+ < Ca*+ < Mg*+ , whereas Zn*+ and Cu*+ could not restore any activity. Using atomic absorption spectroscopy, we found that our enzyme preparation contained Mg*+, but not Mn’+. To ascertain whether Mg*+ is bound to the enzyme molecule or not, we tried to remove nonbound Mg*+, using a HLC TSK Gel G 3000 SW column. The elution profile of transketolase (Fig. 6) shows two Table 2. Cofactor requirement in stitution of transketolase
recon-
Activity (%I Complete mixture* -TPP -Mg’+ -TPP, Mg2+
100 2 2 0
I mM EDTA. 2mM MgCI,, IOpM TPP and apoenzyme in 50mM Tris-HCI buffer
*The complete mixture contained
(pH 7.5, at 25 C). They were incubated at 37°C for 30min, and the activities were measured as described in the text.
1258
EUN HEE JUNG Table 3. Effect of divalent cauons on the reconstitution: each mixture contamed I mM EDTA. IOpM TPP, ZmM divalent cation (MgCI,, C&I, 2H,O. M&I, 4H,O. C&I, 6H,O, CuQ 2H,O. ZnCI,) and apoenzyme in 50 mM Tns-HCI buffer Divalent cations
Activity
Mg:’ Ca’
(%)
100
X9
+
Mll’+ Co?C”-”
86 42 0
Zll’
0
Mg’+ peaks: while the second peak is in agreement with the peak of MgCl, (free Mg’+), the first peak is in agreement with that of the enzyme, suggesting that Mg 2+ is bound to the enzyme molecule. DlSCUSSlON
Various methods have been used to resolve the coenzyme from transketolase. Yeast transketolase could be resolved at alkaline medium e.g. by dialysis against 0.12 M KC1 solution (pH 7.4) containing 16 mM EDTA for 48 hr (Racker er al., 1953), by dialysis against I .6 M ammonium sulfate solution (pH 7.8) for 16 hr (Datta and Racker, 196l), and by keeping in 25 mM glycylglycine buffer (pH 7.6) at O’C for 48 hr (Ozawa et al., 1971). In contrast to yeast transketolase, mammalian transketolase could be resolved in acidic medium (Tomita et al., 1979; Horecker et al., 1953; Tate and Nixon, 1987; Jeyasingham et al., 1986). We could resolve the transketolase using ammonium sulfate in acidic medium (pH 3.5) with 50-60% of recovery of the activity. The specific activity of the resolved enzyme was not changed, suggesting that the loss of the activity was due to the loss of the enzyme protein. The catalytic properties of transketolase were most active in the pH range of 7.6-8.0 (Takeuchi et al., 1986; Philippov et al., 1980; Heinrich and Wiss, 197 1:
.
60
600
li
0
^o
i
400 = ,a & 200 r” 0 600 2
~~~
2 400
E ,c
200
$ 2
0
5
10
15
20
25
30
35
Fractions
Fig. 6. TSK Gel G 3000 SW column chromatography. The enzyme solution was applied to a TSK Gel G 3000 SW column (7.53 i.d. x 600 mm) previously equilibrated with 50 mM Tris-HC1 buffer containing 0.3 M NaCl (pH 7.0) and eluted with the same buffer. The flow rate was 0.5 ml/min, and a fraction volume was I ml. O-_-i, A---a, elution profile of Mg*+ content of fractions; 200~1 of IOppm MgCI,; O-0, activity of the enzyme: n -m, protein content of fractions.
er al.
Paoletti, 1983). Since the strongest interaction between TPP and apotransketolase from yeast was observed at pH 5.0 (Heinrich ef al.. 1972), it was of interest to determine the effect of pH on TPP binding to apotransketolase from human erythrocytes. TPP was tightly bound to transketolase from human erythrocytes around pH range of 7.5-8.0, suggesting that TPP binding to the enzyme has a direct influence on the catalytic activity. Heinrich et al. (1972) proposed that the enzyme must be incubated with Mg’+ and TPP for 30 min in order to produce the maximum amount of quenching of fluorescence which occurred upon binding. And according to Tate and Nixon (1987), a period approaching to 60min is necessary before resolved preparations of transketolase are fully reconstituted with TPP at 24 C. In the present study when TPP was incubated with apoenzyme beyond 45 min. the binding decreased as the incubation time increased and the same tendency was okserved in case of catalytic activity. It may be because of denaturing of the enzyme itself due to long incubation. The results of TPP binding to transketolase were the same as that of the catalytic activity (Figs 3 and 4). AccordingIS. it can be supposed that the determination of the enzyme activity can substitute for TPP binding to the enzyme. In contrast to the results of Kochetov et d. ( 1975). we showed in the presence of Mg”, a single, noninteracting TPP binding site in Scatchard plot, which is in accord with the results of Heinrich rt ul. (1977) demonstrating no cooperativity in the binding of the TPP to the apoenzyme. And in the Scatchard plot we also showed that the n value was 0.66. As described previously, the apoenzyme was incubated with TPP for 30 min, but if the incubation was done for longer time not exceeding 60 min, a higher n value might be obtained. Horecker et al. (1953) reported that while spinach transketolase required Mg?+ for full activation. no such requirement was observed with liver transketolase. Warnock and Prudhomme (1982) suggested that Mg-‘+ is not required by the isolated human erythrocyte apoenzyme. II was found that although the addition of Mg” alone did not restore any activity. the addition of TPP was effective in relatively high concentration in the absence of divalent cations (Datta and Racker. 1961; Heinrich rt al.. 1973). This is in agreement with the findings of Kochetov and Philippov (1970). Without metal ions, they observed 60-70% of the activity that was found in the presence of both metal ions and coenzyme. Heinrich (‘I (I/. (1972) suggested that it was difficult to study the effect of divalent cations as determined by atomic absorption spectroscopy. Morey and Juni (1968) also proposed that the restoration of the activity of the resolved TPP dependent enzyme in the presence of TPP alone, might indicate that the resolved preparation or some of the reagents contained traces of divalent cations. We also found a lot of metal contamination in the experimental environment, that is. in chemicals, glass tools, and even in Eppendorf tubes. Therefore we used a buffer containing EDTA in resolution and reconstitution to study the effect of divalent cations. The metal contamination could be blocked by EDTA. It was suggested that the EDT4
Thiamine
pyrophosphate
should compete with TPP for the available Mg2+ as soon as any Mg’+ is dissociated from the holoenzyme (Egan and Sable, 1981). because there was a little effect. if at all, when EDTA was added to the enzyme that already reconstituted (Saitou et al., 1974; Egan and Sable. 1981). It was found that Mg’+ as well as TPP is absolutely necessary for the catalytic activity of transketolase from human erythrocytes. (Table 2) Instead of Mg2+, the addition of Cu” or Zn” could not restore any which is in accordance with the results of Heinrich et al. (1972). Our results showed that Mg?+ is the most effective divalent cation in the catalytic activity of transketolase from human erythrocytes. We found that the enzyme preparation contained Mg:’ , but not Mn’+. However exogenous Mn?+ could replace the Mg’ + for the catalytic activity. The function of Mg?+ is little known yet but can be supposed to be some modification of the enzyme structure in complex formation with TPP (Egan and activity,
Sable,
1981).
Although
besides
Mg’+
and
Mn?‘,
Ca”
has been considered as a probable divalent cation in transketolase (Kochetov and Philippov, 1970; Heinrich rt al., 1972; Takeuchi et al., 1986) we could not determine Ca’+ content in the enzyme because of small amounts of the enzyme preparation. The mechanism by which divalent cations act as potential cofactors for transketolase would seem to require more detailed investigation. Ackno~r~ledgemenr-We wish to thank Mr Hisaaki Kabata for his technical advice on determination of Mg2+.
REFERENCES
Datta A. G. and Racker E. (1961) Mechanism of action of transketolase. 1. Properties of the crystalline yeast enzyme. J. hiol. Chem. 236, 617-623. De La Haba G., Leder I. G. and Racker E. (1955) Crystalline transketolase from baker’s yeast: isolation and properties. J. biol. Chem. 214, 409426. Egan R. M. and Sable H. Z. (1981) Transketolase kinetics: the slow reconstitution of the holoenzyme is due to rate-limiting dimerization of the subunits. J. biol. Chem. 256, 48774833. Heinrich P. C. and Wiss 0. (1971) Transketolase from human erythrocytes purification and properties. Heh. chim. Acra 54, 2658-2668. Heinrich P. C., Steffen H., Janser P. and Wiss 0. (1972) Studies on the reconstitution of apotransketolase with thiamine pyrophosphate and analogs of the coenzyme. Eur. J. Biochem. 30, 553-541. Horecker B. L.. Smyrniotis P. Z. and Klenow H. (1953) The formation of sedoheptulose phosphate from pentose phosphate. J. biol. Chem. 205, 661-682. Ishii K.. Sarai K.. Sanemori H. and Kawasaki T. (1979) Analysis of thiamine and its phosphate esters by high-
binding
1259
performance liquid chromatography. Analyt. Biochem. W, 191~195. Jeyasingham M. D., Pratt 0. E., Thomson A. D. and Shaw G. K. (1986) Reduced stability of rat brain transketolase after conversion to the apo form. J. Neurochem. 47, 278-28 I. Kochetov G. A. and Philippov P. P. (1970) The function of calcium-cofactors of transketolase from baker’s yeast. FEBS Ler~. 6, 49-5 I. Kochetov G. A., Tikhomirova N. K. and Philippov P. P. (1975) The bindina of thiamine ovroohosohate with transketolase in equilibrium condi&ns: B&hem. hiopi7ys. Rex. Commun. 63, 924-930. Kremer A. B.. Egan R. M. and Sable H. Z. (1980) The active site of transketolase: two argine residues are essential for activity. J. biol. Chem. 255, 2405-2410. Laemmi U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Narurr. Lond. 221, 68&685. Morey A. V. and Juni E. (1968) Studies on the nature of the binding of thiamine pyrophosphate to enzymes. J. hiol. Chem. 243, 3009-3019. Nishino K., Itokawa Y.. Nishino N., Piros K. and Cooper J. R. (1983) Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase. J. biol. Chem. 258, 11871-l 1878. Ozawa T., Saitou S. and Ishikawa M. (1971) The coenzyme activities of 2’-substituted thiamine derivatives. II. The activities of 2’-northiamine and 2’-ethylthiamine as coenzyme for apotransketolase. Vitamins.44, 303-307. Paoletti F. (1983) Purification and oronerties of transketolase from fresh rat liver. Archs Bioch’em. Eiophys. 222, 489496. Philippov P. P., Shestakov I. K., Tikhomirova N. K. and Kochetov G. A. (1980)Characterization and properties of pig liver transketolase. Biochim. biophys. Acla 613, 359-369. Racker E., De La Haba G. and Leder I. G. (1953) Thiamine pyrophosphate, a coenzyme of transketolase. J. Am. Chem. Sot. 75, 1010-1011. Saitou S.. Ozawa T. and Tomita 1. (1974) The purification and some properties of brewer’s yeast transketolase. FEBS Left. 40, 114118. Takeuchi T., Nishino K. and Itokawa Y. (1984) Improved determination of transketolase activity in erythrocytes. Clin. Chem. 30, 658-661. Takeuchi T., Nishino K. and Itokawa Y. (1986) Purification and characterization of, and preparation of antibody to, transketolase from human red blood cells. Biochim. biophys. Acta 872, 2432. Tate J. R. and Nixon P. F. (1987) Measurement of Michaelis constant for human erythrocyte transketolase and thiamine diphosphate. Analyt. Biochem. 160, 78-87. Tomita I., Saitou S. and Ishikawa M. (1979) Purification and properties of transketolase from pig liver. I. An attempt to resolve the enzyme into apoenzyme and cofactors. J. Nutr. Sci. Vitamin. 25. 175-184. Warnock L. G. and Prudhomme CR. (1982) The isolation and preliminary characterization of apotransketolase from human erythrocytes. biochem. Biophys. Res. Commun. 106, 719. 723.
~nr. J. ~iochem. Vol. 20, No. I I, pp. 1255-1259, 1988 Printed in Great Britain. All rights reserved
STUDIES ON THE NATURE OF THIAMINE PYROPHOSPHATE BINDING AND DEPENDENCY ON DIVALENT CATIONS OF TRANSKETOLASE FROM HUMAN ERYTHROCYTES EUN HEE JUNG,’ TOHORU TAKEUCHI,’ KOHSUKE NISHINO’ and YOSHINORI ITOKAWA’ ‘Department of Hygiene, Faculty of Medicine, Kyoto University and *Department of Home Economics. Dohshisya Women’s College, Kyoto 606, Japan (Received 29 February
1988)
Abstract--l.
The binding kinetics for [j5S]thiamine pyrophosphate to transketolase and the dependency of transketolase on divalent cations for activity were investigated. 2. With Scatchard analysis, dissociation constant (&) and n value were calculated to be 0.2 x IO ’ M and 0.66 respectively. 3. The activity of the reconstituted enzyme increased in the order of Co*+ < Mn’+ < Ca’+ < Mg’+. The native
transketolase
contained
Mg 2+ in its molecular
lution and reconstitution of homogeneous ketolase purified from human erythrocytes.
INTRODUCTION
Transketolase (EC 2.2.1.1) appears to have multiple functions in intermediary metabolism. In the oxidative and reductive pentose phosphate cycles, it participates in the breakdown and synthesis of carbohydrates. Equally important is its role in nucleic acid metabolism as a generator of ribose S-phosphate from sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate. There are many studies on the properties of transketolase from yeast (De La Haba et al., 1985; Kochetov and Philippov, 1970; Kremer et al., 1980; Egan and Sable, 1981): transketolase releases thiamine pyrophosphate (TPP) and divalent cations,
such as Mg’+, on alkaline treatment and the resolved enzyme can be reconstituted by incubation with TPP and a divalent cation (Heinrich et al., 1972; Saitou et al., 1974). Although the physiological metal ion is Mg2+, a number of other divalent cations are capable of activating the enzyme (Heinrich et al., 1972; Morey and Juni, 1968). By means of kinetic studies, Kochetov et al. (1975) demonstrated a positive cooperative interaction between the binding sites of TPP, in the presence of Mg*+. On the contrary, Egan and Sable (1981) concluded that there was negative cooperativity in the binding of TPP to the apoenzyme in the presence of Mg*+ or Ca*+. Compared to transketolase from yeast, that from mammalian tissues has been studied less, especially that from human tissues has not been fully characterized with respect to its catalytic and chemical properties, and very little is known about the interaction between TPP and apotransketolase. This is largely because of unavailability of homogeneous enzyme preparations. The present study describes the binding nature of TPP and the effect of divalent cations through resoAbbreviations: sodium dodecyl
TPP-thiamine sulfate.
pyrophosphate;
SDS-
structure.
MATERIALS
trans-
AND METHODS
Materials Outdated human blood was donated by Kyoto University Hospital. [F.]Thiamine hydrochloride was purchased from Amersham. o-Xylulose 5-phosphate and D-sedoheptulose 7-phosphate were from Sigma. D-Ribose S-phosphate was from Boehringer. DEAE-Sephadex A-50, Sephadex G-150 and G-100, Blue Sepharose CL-6B, Mono P, Pharmalyte 8-10.5, Poly buffer 96, and PD-IO were from Pharmacia. The ultrafiltration membranes YM-30 and YMT were from
Amicon. LiChrosorb NH, (the particle size was 5 pm) was from Merck, TSK-Gel G 3000 SW for HLC was from Tow Soda, and the Bio-Rad protein assay kit was from Bio-Rad. The atomic absorption spectrophotometer was from Shimadzu, the scintillation counter was from Aloka and Biofluor scintillation solution was from NEN Research Products. All other chemicals were reagent grade materials from commercial sources. Purificarion and assay of ~ranskerolase Transketolase from human erythrocytes was purified according to Takeuchi er al. (1986). Blue Sepharose CL-6B was used to remove glyceraldehyde 3-phosphate dehydrogenase when necessary. After the chromatography on Sephadex G-150, the sample was applied to a column of
Blue Sepharose CL-6B (i7 x 150&n) equilibrated with 50mM Tris-HCl buffer (oH 8.0). For elution. a linear gradient of NaCl from 0 to 0. I5 r;?, with the flow rate of 0.9ml/min was used. The enzyme preparation thus obtained, gave only one protein band with SDS polyacrylamide gel electrophoresis, and contained 0.9 mol of TPP per 1mol of enzyme subunit. The activity of transketolase was determined by the formation rate of sedoheptulose-7-phosphate (Takeuchi et al., 1984). Preparation of apotranskerolase The method (Warnock and Prudhomme, 1982) using ammonium sulfate with acidic medium was used to resolve the enzyme with some modifications.
1255
ELJN HEE
1256
JUNG
To 800 p I of purified transketolase (440 pg/ml), I200 ~1 of ammonium sulfate solution (saturated ammonium sulfate solution/4 mM sodium acetate/l M sulfuric acid, 750: IO: 7. v/v) was added. The final concentration of ammonium sulfate was approx. 60%, and the pH of the reaction mixture was 3.5. After keeping at 0~5’C for 5 min. the mixture was centrifuged at 15,OOOg for IOmin at 4 C and the supernatant was discarded. The precipitate was resuspended in 800 ~1 of 0. I M Tris-HCI buffer (pH 7.5. at 25 C) containing I mM MgCI, and 10% (v/v) glycerol, and centrifuged again at 15,OOOg for IOmin at 4’C. The supernatant was used as apotransketolase. For characterization of the dependency on divalent cations. the buffer for resuspension did not contain MgCI,. Drtermmation
of[“SFPP
NaCl was used. The flow rate was 0.5 ml/min and a fraction volume was I ml. Mg’+ content of the fractions was deterby
flame
atomic
100
.-• 0
2 = : I-
50
\/
Lo-
0
Total Mg” content of the enzyme preparation was determined at 285.1 nm by flameless atomic absorption spectroscopy. after drying at 15O”C, ashing at 600’C and atomizing at 19OO’C. To remove nonenzyme bound Mg’+ from the enzyme preparation, we used a HLC TSK Gel G 3000 SW column (7.53 x 600 mm). For equilibration and elution, 50 mM Tris-HCI buffer (pH 7.0 containing 0.3 M
285.2 nm
ai.
100
binding
[“S]TPP was prepared according to Nishino er al. (1983). The specific activity of [?j]TPP was 127.3 cpm/pmol. 100 ~1 of the reaction mixture containing 3Apg of apotransketolase and various concentrations of [‘5S]TPP in 50 mM Tris-HCI buffer (pH 7.5, at 25’C) was incubated at 37 C. After 30 min, 400 ~1 of the same ice-cold buffer was added to stop the reaction. The mixture was applied to YMT membrane and centrifuged at IOOOg for IOmin at 4 C. The membrane was washed 3 times with 500~1 of 50 mM Tris-HCI buffer containing 0. I mM non-radioactive TPP. to remove nonbound [?i]TPP. Finally. 500 ~rl of 2% trichloroacetic acid solution was added to the membrane and centrifuged to eliminate enzyme-bound [“SITPP. IO ml of Biofluor scintillation mixture were added to the effluent and the radioactivity was determined with scintillation counter.
mined at troscopy.
et
absorption
spec-
TPP was identified and determined by the method of Ishii et al. (1979) using a LiChrosorb NH, column (4 x 150 mm). Protein was determined with a Bio-Rad protein assay kit using bovine serum albumin as standard. Polyacrylamide gel electrophoresis was performed in the presence of SDS on 10% polyacrylamide slab gels according to the procedure of Laemmi (I 970).
RESULTS
While transketolase from yeast could be resolved into TPP and apotransketolase in alkaline medium (De La Haba et al., 1955; Heinrich et al., 1972), that from human erythrocytes could be resolved in acidic medium. TPP was dissociated from the enzyme at pH
l\d-
4
c
5
^
A
6
PH Fig. 1. Effect of pH on dissociation of TPP from transketolase. The enzyme preparations previously adjusted to pH 3.5-6.0 with 50 mM sodium acetate buffer were centrlfuged using MPS-I Micropartition system. The enzyme protein was extracted from the membrane with 50mM Tris-HCI buffer (pH 8.0) and was used to determine the catalytic activity. The effluent that traversed the membrane was collected and used to determine the content of TPP. The activity content are given as percentages of those obtained in the assay of the native enzyme.
below 4.0, although the activity was not restored by incubation with TPP. (Fig. 1) However another method using acidic medium (pH 3.5) with ammonium sulfate was successful in restoring the activity by addition of TPP and Mg*+. Restoration of the activity by addition of TPP and Mg*+ was 5&60% and the specific activity was the same as that of the native enzyme. The resolved enzyme preparation could be stored at -9O’C for at least 4 days without significant loss of activity. [3sS]TPP binding to the transketolase was specific. (Table 1) A large amount of nonradioactive TPP which was added to the reaction mixture containitig 0.5 PM [35S]TPP inhibited the binding of radioactive TPP competitively. The optimum binding between TPP and apotransketolase was observed at pH 7.5. (Fig. 2) The reconstitution of the resolved enzyme was much affected with incubation time and temperature. The optimum binding between TPP and apotransketolase was observed at 37’C with an incubation time of 45 min. (Fig. 3) However in case of an incubation for 60min the binding seemed to decrease, but the activity decreased slightly. The decrease in binding suggested that there may have been some degeneration of the enzyme. Therefore we chose an incubation time of 30 min in our experiments. We found that the features of TPP binding to enzyme were the same as those of catalytic activity of enzyme. (Fig. 4) The binding was characterized by a single, noninteracting binding site with a dissociation constant
Table I. SpAicily of [‘?S]TPP binding to apotransketolase: each mixture containing 5.1 p’g of apoenzyme and 0.5 PM [‘SS]TPP was incubated at 37 C for 10 min with or without 5 mM nonradioactive TPP. The following procedure was the same as described in the text
cpm [“SITPP
with nonradioactwe
without nonradioactive
0.5 uM
19.5
1510.4
Thiamine pyrophosphate
1257
binding
I::jA
1:: i
.-c” 0
.E m
c loGO-
-25
I 1
d 0
I 2
I 3
I 4
.g t a
I 5
TPP+M)
Fig. 4. [“S]TPP binding to apotransketolase and catalytic activitv of the reconstituted enzvme as a function of [‘%]TPP concent;ation.
PH
Fig. 2. Effect of pH on [%]TPP binding to apotransketolase from human erythrocytes. Apoenzyme (5.1 pg) and [%]TPP in 100~1 of 50mM sodium acetate @H 4.W.O) or 50 mM Tris-HCl buffer (pH 7.G9.0) of the indicated pH value were incubated at 37°C for IO min. The procedure followed is described in the text.
of 0.2 x IOebM. (Fig. 5) Because we found that the molar ratio of TPP and transketolase (monomer) was 0.9:1, we calculated the dissociation constant under the assumption of n = 1, however in fact the plot showed n = 0.66.
Time (mini
Fig. 3. Time dependence of [%]TPP binding to apotransketolase and catalytic activity of the reconstituted enzyme. Apoenzyme (4.0 fig) and 0.5 PM of [%]TPP in 100 ~1 of 50mM Tris-HC1 buffer (pH 7.5, at 25°C) were incubated at 37°C for the indicated time. The procedure followed is described in the text. For determination of the catalytic activity, 3.9 pg of apoenzyme was incubated with 0.5 FM [3sS]TPP. Each mixture was transferred to the MPS-I Micropartition system and centrifuged at IOOOgfor 10min at 4°C. After washing (the buffer did not contain TPP) to remove the nonbound [%]TPP, the enzyme was extracted from the membrane with 200~1 of 50mM Tris-HC1 buffer (PH 8.0). The solution containing the extracted enzyme was used to determine the catalytic activity.
L
0
0.5
1.0
1.5
r/[TPP],
2.0
2.5
3.0
3.5
x IO6
Fig. 5. Scatchard plot of [“S]TPP binding to apotransketolase from human erythrocytes. 100 ~1 of reaction mixture containing apoenzyme (3.2 pg) and various concentrations of [‘%]TPP in 50 mM Tris-HC1 buffer (pH 7.5, at 25°C) were incubated at 37°C for 30min. The procedure followed is described in the text. Kd and n values were estimated from least square methods (r = 0.96). r is the value of the ratio of enzyme-bound TPP to moles of transketolase, and [TPP], is the concentration of free TPP. The activation of transketolase was not observed in the absence of either Mg*+ or TPP. (Table 2) This means that transketolase absolutely requires Mg*+ for its catalytic activity. To clarify whether this result is specific to Mg*+ or not, various divalent cations were used instead of Mg*+. Other divalent cations also could restore the activity. (Table 3J The activity increased in the order of of the enzyme Co*+ < Mn*+ < Ca*+ < Mg*+ , whereas Zn*+ and Cu*+ could not restore any activity. Using atomic absorption spectroscopy, we found that our enzyme preparation contained Mg*+, but not Mn’+. To ascertain whether Mg*+ is bound to the enzyme molecule or not, we tried to remove nonbound Mg*+, using a HLC TSK Gel G 3000 SW column. The elution profile of transketolase (Fig. 6) shows two Table 2. Cofactor requirement in stitution of transketolase
recon-
Activity (%I Complete mixture* -TPP -Mg’+ -TPP, Mg2+
100 2 2 0
I mM EDTA. 2mM MgCI,, IOpM TPP and apoenzyme in 50mM Tris-HCI buffer
*The complete mixture contained
(pH 7.5, at 25 C). They were incubated at 37°C for 30min, and the activities were measured as described in the text.
1258
EUN HEE JUNG Table 3. Effect of divalent cauons on the reconstitution: each mixture contamed I mM EDTA. IOpM TPP, ZmM divalent cation (MgCI,, C&I, 2H,O. M&I, 4H,O. C&I, 6H,O, CuQ 2H,O. ZnCI,) and apoenzyme in 50 mM Tns-HCI buffer Divalent cations
Activity
Mg:’ Ca’
(%)
100
X9
+
Mll’+ Co?C”-”
86 42 0
Zll’
0
Mg’+ peaks: while the second peak is in agreement with the peak of MgCl, (free Mg’+), the first peak is in agreement with that of the enzyme, suggesting that Mg 2+ is bound to the enzyme molecule. DlSCUSSlON
Various methods have been used to resolve the coenzyme from transketolase. Yeast transketolase could be resolved at alkaline medium e.g. by dialysis against 0.12 M KC1 solution (pH 7.4) containing 16 mM EDTA for 48 hr (Racker er al., 1953), by dialysis against I .6 M ammonium sulfate solution (pH 7.8) for 16 hr (Datta and Racker, 196l), and by keeping in 25 mM glycylglycine buffer (pH 7.6) at O’C for 48 hr (Ozawa et al., 1971). In contrast to yeast transketolase, mammalian transketolase could be resolved in acidic medium (Tomita et al., 1979; Horecker et al., 1953; Tate and Nixon, 1987; Jeyasingham et al., 1986). We could resolve the transketolase using ammonium sulfate in acidic medium (pH 3.5) with 50-60% of recovery of the activity. The specific activity of the resolved enzyme was not changed, suggesting that the loss of the activity was due to the loss of the enzyme protein. The catalytic properties of transketolase were most active in the pH range of 7.6-8.0 (Takeuchi et al., 1986; Philippov et al., 1980; Heinrich and Wiss, 197 1:
.
60
600
li
0
^o
i
400 = ,a & 200 r” 0 600 2
~~~
2 400
E ,c
200
$ 2
0
5
10
15
20
25
30
35
Fractions
Fig. 6. TSK Gel G 3000 SW column chromatography. The enzyme solution was applied to a TSK Gel G 3000 SW column (7.53 i.d. x 600 mm) previously equilibrated with 50 mM Tris-HC1 buffer containing 0.3 M NaCl (pH 7.0) and eluted with the same buffer. The flow rate was 0.5 ml/min, and a fraction volume was I ml. O-_-i, A---a, elution profile of Mg*+ content of fractions; 200~1 of IOppm MgCI,; O-0, activity of the enzyme: n -m, protein content of fractions.
er al.
Paoletti, 1983). Since the strongest interaction between TPP and apotransketolase from yeast was observed at pH 5.0 (Heinrich ef al.. 1972), it was of interest to determine the effect of pH on TPP binding to apotransketolase from human erythrocytes. TPP was tightly bound to transketolase from human erythrocytes around pH range of 7.5-8.0, suggesting that TPP binding to the enzyme has a direct influence on the catalytic activity. Heinrich et al. (1972) proposed that the enzyme must be incubated with Mg’+ and TPP for 30 min in order to produce the maximum amount of quenching of fluorescence which occurred upon binding. And according to Tate and Nixon (1987), a period approaching to 60min is necessary before resolved preparations of transketolase are fully reconstituted with TPP at 24 C. In the present study when TPP was incubated with apoenzyme beyond 45 min. the binding decreased as the incubation time increased and the same tendency was okserved in case of catalytic activity. It may be because of denaturing of the enzyme itself due to long incubation. The results of TPP binding to transketolase were the same as that of the catalytic activity (Figs 3 and 4). AccordingIS. it can be supposed that the determination of the enzyme activity can substitute for TPP binding to the enzyme. In contrast to the results of Kochetov et d. ( 1975). we showed in the presence of Mg”, a single, noninteracting TPP binding site in Scatchard plot, which is in accord with the results of Heinrich rt ul. (1977) demonstrating no cooperativity in the binding of the TPP to the apoenzyme. And in the Scatchard plot we also showed that the n value was 0.66. As described previously, the apoenzyme was incubated with TPP for 30 min, but if the incubation was done for longer time not exceeding 60 min, a higher n value might be obtained. Horecker et al. (1953) reported that while spinach transketolase required Mg?+ for full activation. no such requirement was observed with liver transketolase. Warnock and Prudhomme (1982) suggested that Mg-‘+ is not required by the isolated human erythrocyte apoenzyme. II was found that although the addition of Mg” alone did not restore any activity. the addition of TPP was effective in relatively high concentration in the absence of divalent cations (Datta and Racker. 1961; Heinrich rt al.. 1973). This is in agreement with the findings of Kochetov and Philippov (1970). Without metal ions, they observed 60-70% of the activity that was found in the presence of both metal ions and coenzyme. Heinrich (‘I (I/. (1972) suggested that it was difficult to study the effect of divalent cations as determined by atomic absorption spectroscopy. Morey and Juni (1968) also proposed that the restoration of the activity of the resolved TPP dependent enzyme in the presence of TPP alone, might indicate that the resolved preparation or some of the reagents contained traces of divalent cations. We also found a lot of metal contamination in the experimental environment, that is. in chemicals, glass tools, and even in Eppendorf tubes. Therefore we used a buffer containing EDTA in resolution and reconstitution to study the effect of divalent cations. The metal contamination could be blocked by EDTA. It was suggested that the EDT4
Thiamine
pyrophosphate
should compete with TPP for the available Mg2+ as soon as any Mg’+ is dissociated from the holoenzyme (Egan and Sable, 1981). because there was a little effect. if at all, when EDTA was added to the enzyme that already reconstituted (Saitou et al., 1974; Egan and Sable. 1981). It was found that Mg’+ as well as TPP is absolutely necessary for the catalytic activity of transketolase from human erythrocytes. (Table 2) Instead of Mg2+, the addition of Cu” or Zn” could not restore any which is in accordance with the results of Heinrich et al. (1972). Our results showed that Mg?+ is the most effective divalent cation in the catalytic activity of transketolase from human erythrocytes. We found that the enzyme preparation contained Mg:’ , but not Mn’+. However exogenous Mn?+ could replace the Mg’ + for the catalytic activity. The function of Mg?+ is little known yet but can be supposed to be some modification of the enzyme structure in complex formation with TPP (Egan and activity,
Sable,
1981).
Although
besides
Mg’+
and
Mn?‘,
Ca”
has been considered as a probable divalent cation in transketolase (Kochetov and Philippov, 1970; Heinrich rt al., 1972; Takeuchi et al., 1986) we could not determine Ca’+ content in the enzyme because of small amounts of the enzyme preparation. The mechanism by which divalent cations act as potential cofactors for transketolase would seem to require more detailed investigation. Ackno~r~ledgemenr-We wish to thank Mr Hisaaki Kabata for his technical advice on determination of Mg2+.
REFERENCES
Datta A. G. and Racker E. (1961) Mechanism of action of transketolase. 1. Properties of the crystalline yeast enzyme. J. hiol. Chem. 236, 617-623. De La Haba G., Leder I. G. and Racker E. (1955) Crystalline transketolase from baker’s yeast: isolation and properties. J. biol. Chem. 214, 409426. Egan R. M. and Sable H. Z. (1981) Transketolase kinetics: the slow reconstitution of the holoenzyme is due to rate-limiting dimerization of the subunits. J. biol. Chem. 256, 48774833. Heinrich P. C. and Wiss 0. (1971) Transketolase from human erythrocytes purification and properties. Heh. chim. Acra 54, 2658-2668. Heinrich P. C., Steffen H., Janser P. and Wiss 0. (1972) Studies on the reconstitution of apotransketolase with thiamine pyrophosphate and analogs of the coenzyme. Eur. J. Biochem. 30, 553-541. Horecker B. L.. Smyrniotis P. Z. and Klenow H. (1953) The formation of sedoheptulose phosphate from pentose phosphate. J. biol. Chem. 205, 661-682. Ishii K.. Sarai K.. Sanemori H. and Kawasaki T. (1979) Analysis of thiamine and its phosphate esters by high-
binding
1259
performance liquid chromatography. Analyt. Biochem. W, 191~195. Jeyasingham M. D., Pratt 0. E., Thomson A. D. and Shaw G. K. (1986) Reduced stability of rat brain transketolase after conversion to the apo form. J. Neurochem. 47, 278-28 I. Kochetov G. A. and Philippov P. P. (1970) The function of calcium-cofactors of transketolase from baker’s yeast. FEBS Ler~. 6, 49-5 I. Kochetov G. A., Tikhomirova N. K. and Philippov P. P. (1975) The bindina of thiamine ovroohosohate with transketolase in equilibrium condi&ns: B&hem. hiopi7ys. Rex. Commun. 63, 924-930. Kremer A. B.. Egan R. M. and Sable H. Z. (1980) The active site of transketolase: two argine residues are essential for activity. J. biol. Chem. 255, 2405-2410. Laemmi U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Narurr. Lond. 221, 68&685. Morey A. V. and Juni E. (1968) Studies on the nature of the binding of thiamine pyrophosphate to enzymes. J. hiol. Chem. 243, 3009-3019. Nishino K., Itokawa Y.. Nishino N., Piros K. and Cooper J. R. (1983) Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase. J. biol. Chem. 258, 11871-l 1878. Ozawa T., Saitou S. and Ishikawa M. (1971) The coenzyme activities of 2’-substituted thiamine derivatives. II. The activities of 2’-northiamine and 2’-ethylthiamine as coenzyme for apotransketolase. Vitamins.44, 303-307. Paoletti F. (1983) Purification and oronerties of transketolase from fresh rat liver. Archs Bioch’em. Eiophys. 222, 489496. Philippov P. P., Shestakov I. K., Tikhomirova N. K. and Kochetov G. A. (1980)Characterization and properties of pig liver transketolase. Biochim. biophys. Acla 613, 359-369. Racker E., De La Haba G. and Leder I. G. (1953) Thiamine pyrophosphate, a coenzyme of transketolase. J. Am. Chem. Sot. 75, 1010-1011. Saitou S.. Ozawa T. and Tomita 1. (1974) The purification and some properties of brewer’s yeast transketolase. FEBS Left. 40, 114118. Takeuchi T., Nishino K. and Itokawa Y. (1984) Improved determination of transketolase activity in erythrocytes. Clin. Chem. 30, 658-661. Takeuchi T., Nishino K. and Itokawa Y. (1986) Purification and characterization of, and preparation of antibody to, transketolase from human red blood cells. Biochim. biophys. Acta 872, 2432. Tate J. R. and Nixon P. F. (1987) Measurement of Michaelis constant for human erythrocyte transketolase and thiamine diphosphate. Analyt. Biochem. 160, 78-87. Tomita I., Saitou S. and Ishikawa M. (1979) Purification and properties of transketolase from pig liver. I. An attempt to resolve the enzyme into apoenzyme and cofactors. J. Nutr. Sci. Vitamin. 25. 175-184. Warnock L. G. and Prudhomme CR. (1982) The isolation and preliminary characterization of apotransketolase from human erythrocytes. biochem. Biophys. Res. Commun. 106, 719. 723.