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[5] Enzymatic synthesis of thiamin triphosphate
24
THIAMIN:
PHOSPHATES AND ANALOGS
r51
Since the procedure was developed to determine the forms of thiamin that occur in synaptic plasma membranes,9 the applicability of the procedure was appraised after preparing subcellular fractions of rat brain as previously described. The results of this assay are shown in Fig. 2. After making the fluorescence corrections as described above, the final concentrations of thiamin and its phosphate esters are shown in the table. Using the procedure as described, only 40 mg of rat brain is required for a complete assay of all forms of the vitamin.
[Sl Enzymatic
Synthesis
By JACK
of Thiamin
R. COOPER and KOHSUKE
Triphosphate NISHINO
Although the role of thiamin pyrophosphate (ThPP) as a coenzyme in intermediary metabolism is well documented, the function of thiamin triphosphate (ThTP) is still unclear. In nervous tissue, ThTP has been implicated in Leigh’s disease (subacute necrotizing encephalomyelopathy) and considerable evidence, albeit circumstantial, suggests a role of this ester in conduction and transmission.’ Until recently, the isolation and characterization of the enzyme system catalyzing the synthesis of ThTP have met with little success. This difficulty can be understood in light of this complex enzyme system as described below. Assay Method Principle. The substrate for this phosphoryltransferase (EC 2.7.4.15, thiamin-diphosphate kinase) is not free ThPP but ThPP that is bound to a protein. This substrate is prepared by injecting rats with [35S]thiamin and subsequently isolating the [35S]ThPP-protein from liver by ion-exchange and gel chromatography. After incubation of the labeled substrate with the enzyme and cofactor, [“S]ThTP is isolated by column chromatography and the radioactivity determined.2 Reagents
Tris-HCl (pH 7.4), 0.5 M ATP, 0.1 M ’ J. R. Cooper and J. H. Pincus, Neurochen. Res. 4, 223 (1979). z K. Nishino, Y. Itokawa, N. Nishino, K. Piros, and J. R. Cooper, J. Biol. Chem. 258, 11871 (1983).
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
151
ENZYMATIC
SYNTHESIS
OF THIAMIN
TRIPHOSPHATE
25
MgC12, 0.1 M Glucose, 0.1 M [25S]ThPP-protein substrate (equivalent to 4.0-6.0 pM [35S]thiamin pyrophosphate) Enzyme preparation Trichloroacetic acid (TCA), 20% Procedure. In a final volume of 100 ~1 are added, 10 ~1 of Tris-HCl (pH 7.4), 5 ~1 ATP, 5 ~1 MgC12, 10 ~1 glucose, 50 ~1 radioactive substrate, and 20 ~1 enzyme preparation. The reaction is initiated by the addition of the enzyme after the mixture was preincubated for 2 min at 37”. Following a 10 min incubation, 10 ~1 of TCA is added to terminate the reaction, followed by centrifugation. The supernatant is removed, extracted three times with 1.5 ml water-saturated ether to remove the TCA, and the residual ether in the supernatant removed by evaporation at 37”for 5 min. Approximately 70-80 ~1 of the supernatant is then applied to an AG-50X8 (H+) column (0.7 x 2.0 cm) and the ThTP eluted with 1 ml of water. The effluent, containing 90% of the radioactive ThTP that is synthesized, is collected in a scintillation vial and the radioactivity determined after the addition of scintillant. The amount of ThTP formed is calculated from the radioactivity and the previously calculated specific activity of the labeled substrate. Controls are carried out through the procedure both using zero time and with the omission of ATP from the incubation medium. Preparation of [35SIThPP Substrate After 6 weeks on a thiamin-deficient diet, Sprague-Dawley male rats are injected ip with 100 &i of [35S]thiamin-HCl (specific activity 40-200 mCi/mmol) dissolved in 0.3 ml of physiological saline twice at a 24-hr interval. Twenty-four hours after the second injection, rats are sacrificed, and the livers removed and homogenized with 4 volumes of 0.25 M sucrose containing 30 mM Tris-HCl (pH 8.6) and 0.1 mM EDTA. After centrifugation at 40,000 g for 20 min at O-4”, the supernatant is removed and centrifuged at 105,000 g for 90 min. The resultant supernatant is dialyzed overnight against 30 mM Tris-HCl buffer (pH 8.6) at O-4” and then applied to a DEAE-Sephadex A-25 column (6 x 40 cm) equilibrated with 30 mM Tris-HCl (pH 8.6). Elution with the same buffer yields the unabsorbed labeled substrate. These fractions are pooled, the pH adjusted to 7.4, and the pool is concentrated under a nitrogen atmosphere using an Amicon PM10 apparatus. The concentrate is then applied to a Sephadex G-100 column (4.2 x 150 cm) equilibrated with the same buffer. Radioactive fractions (Fig. 1) are collected and concentrated similarly to that described above. This fraction can be used as substrate for most
26
THIAMIN:
35
40
PHOSPHATES
45 Fraction
AND
50
[51
ANALOGS
55
60
65
Number
FIG. 1. Elution pattern of substrate protein on Sephadex G-100 column chromatography. The concentrated et&tent containing substrate activity from DEAE-Sephadex A-25 chromatography was applied to a Sephadex G-100 column preequilibrated with 50 mM potassium phosphate buffer (pH 7.4), and eluted with the same buffer. Solid line, protein concentration; (0) total thiamin content of substrate protein.
experiments although it does contain a small amount of phosphoryltransferase. To block this activity, 1.OM potassium phosphate buffer (pH 8.0), and 0.1 M iodoacetamide are added to the Sephadex G-100 eluant in a final concentration of 0.1 and 20 mM, respectively. The eluant is incubated at 37” for 30 min, centrifuged to remove the resultant precipitate, and then dialyzed against 20 mM potassium phosphate buffer (pH 7.4) overnight at O-4”. The dialyzed sample is then concentrated with the Amicon PM 10 membrane. Purification
of ThPP-ATP
Phosphoryl Transferase
Step 1. Acetone Powder Preparation. Fresh bovine brain cortex, after the removal of meninges, is homogenized with 9 volumes of ice-cold 0.32 M sucrose containing 20 mM potassium phosphate buffer (pH 7.4), and 40 PM disodium EDTA in a Waring blender. After centrifugation at 1000 g for 10 min, the supernatant is removed and centrifuged at 10,800g for 20 min. The supematant is removed and the pellet is resuspended in 50 mM potassium phosphate buffer (pH 7.4), and recentrifuged for 20 min at 10,000 g. The resultant pellet (Pz) is resuspended in a small volume of the phosphate buffer and mixed with 9 volumes of acetone (-20”), and the
151
ENZYMATIC
SYNTHESIS
OF THIAMIN
27
TRIPHOSPHATE
/ I /
I
El E2 0.5
I' I 0.1
I
c-2 -2
/ I
/
I
I I 1 I 35
\
I
/I\
I
I
I
/
I
g
.07
I
/I I
I
40
45 Fraction
I
I
I
I
50
55
60
65
Number
FIG. 2. Elution pattern of cellulose DE-52 column chromatography. The enzyme prepa-
ration was applied to a DE-52 column and was eluted with a liner gradient of equal volumes of 20 mM potassium phosphate buffer (pH 7.6), containing 20% glycerol and 1.O mM DTT, and 20 mM potassium phosphate buffer (pH 7.6), containing 20% glycerol, 1.0 m&f DTT, and 1.0 A4 NaCl. Solid line, protein concentration; dotted line, NaCl concentration; (0) enzyme activity.
suspension stirred for 10 min at O-4”. After filtration on a Biichner funnel through Whatman 3 MM filter paper, the acetone powder is washed with cold ether and air dried. The acetone powder is stored at -20” in a vacuum desiccator. Step 2. Extraction. The acetone powder is extracted with 30 volumes of 20 mM potassium phosphate buffer (pH 7.6), containing 1.0 mM dithiothreitol (DTT) via homogenization. After centrifugation at 105,000 g for 90 min, the supematant is removed, the pellet reextracted and centrifuged as above, and the two supematants combined. Glycerol to a final concentration of 20% is then added to stabilize the enzyme. Step 3. DE-52 Column Chromatography. The enzyme solution is applied to a DE-52 column (6.5 x 30 cm) that has been preequilibrated with 20 mM potassium phosphate buffer (pH 7.6), containing 20% glycerol and 1.O mM DTT. The column is washed with the same buffer and the enzyme eluted with a linear gradient of equal volumes (1500 ml) of the equilibrating buffer and the buffer containing 1.O A4 NaCl. From this procedure, as shown in Fig. 2, two peaks of enzyme activity were collected. Each
28
THIAMIN:
40
PHOSPHATES
AND
50
55
45 Fraction
151
ANALOGS
60
Number
FIG. 3. Elution pattern of Sephacryl S-200 column chromatography. Sephacryl S-200 column (4.4 X 90 cm) was employed. Ten to 15 ml of the enzyme preparation of Step 3 (fractions El and E2) was applied separately and eluted with 50 mM potassium phosphate buffer (pH 7.2), containing 20% glycerol and 1.O mM DTT. The elution speed was 4.0 cm/hr and about 12 l-ml fractions were collected. Solid line, protein; (0) enzyme activity.
enzyme fraction was individually concentrated under nitrogen by ultrafiltration using an Amicon PM10 filter membrane. Step 4. Sephacryl S-200 Column Chromatography. The two enzyme fractions (El and E2) from Step 3 are separately applied to a column of Sephacryl S-200 (4.4 x 90 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7.2), containing 20% glycerol and 1 mA4 DTT and eluted with the same buffer. As shown in Fig. 3, the El fraction gives two active fractions, referred to as El-l and El-2, and the E2 fraction yields one peak of activity, referred to as E2. Step 5. Chromatofocusing Chromatography. The active fractions from Step 4 were separately applied to a chromatofocusing column (1 x 37 cm) that was preequilibrated with 0.025 M histidine-HCI buffer (pH 6.2), containing 20% glycerol and 1.0 mM DTT. The eluant was polybuffer 74 HCl, pH 4.0 (0.0075 mmol/pH unit), containing 20% glycerol and 1.0 mM DTT. Active fractions were eluted at pH 5.1 t 0.02. In order to eliminate polybuffer, which inactivates enzyme activity, the eluted frac-
151
ENZYMATIC
SYNTHESIS
OF THIAMIN
TRIPHOSPHATE
29
tions were immediately applied to a Sephadex G-75 column (3.2 X 10 cm) preequilibrated with 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, and elution carried out with the same buffer. To concentrate and retain enzyme activity, the eluants from the Sephadex chromatography were applied to an organomercurial agarose column (Affi-Gel 501, 0.7 x 4.0 cm) which was preequilibrated with 50 mM potassium phosphate buffer (pH 7.4), containing 20% glycerol and eluted with 3 ml of 50 mM potassium phosphate buffer (pH 7.4), containing 20% glycerol and 20 mM DTT. Properties Purity and Physicochemical Properties. The three active fractions (El-l, El-2, and E2) on disc gel electrophoresis showed a single protein band with the same Rf: the molecular weight of the enzyme was calculated to be 103,000. The pH optimum was 7.5, with Tris-HCl exhibiting slightly higher activity than phosphate buffer. Dependencies of the Reaction. The reaction has an absolute dependence on ATP, Mg2+, the cofactor (presumably glucose), and ThPP that is protein bound. When the bound substrate is replaced by free ThPP, no synthesis of ThTP is observed. Inhibitions. p-Chloromercuribenzoate at 5 x 10e4M inhibits activity by 79.5% and N-ethylmaleimide (1 X 10s2 M) inhibits by 64%. Miscellaneous. In addition to the substrate for the reaction being protein bound, ThTP is also synthesized bound to a protein from which it is released for assay on deproteinization. Both the protein-bound substrate and the enzyme are found in both brain and liver. We chose to prepare the substrate protein from liver because of the yield; we prepared the enzyme from brain since earlier work3 had indicated that patients with Leigh’s disease (subacute necrotizing encephalomyelopathy) excrete material which inhibits the synthesis of ThTP using a crude brain preparation but not the liver preparation.
3 J. R. Cooper and J. H. Pincus, J. Agric. Food Chem. 20,490 (1972).
THIAMIN:
PHOSPHATES AND ANALOGS
r51
Since the procedure was developed to determine the forms of thiamin that occur in synaptic plasma membranes,9 the applicability of the procedure was appraised after preparing subcellular fractions of rat brain as previously described. The results of this assay are shown in Fig. 2. After making the fluorescence corrections as described above, the final concentrations of thiamin and its phosphate esters are shown in the table. Using the procedure as described, only 40 mg of rat brain is required for a complete assay of all forms of the vitamin.
[Sl Enzymatic
Synthesis
By JACK
of Thiamin
R. COOPER and KOHSUKE
Triphosphate NISHINO
Although the role of thiamin pyrophosphate (ThPP) as a coenzyme in intermediary metabolism is well documented, the function of thiamin triphosphate (ThTP) is still unclear. In nervous tissue, ThTP has been implicated in Leigh’s disease (subacute necrotizing encephalomyelopathy) and considerable evidence, albeit circumstantial, suggests a role of this ester in conduction and transmission.’ Until recently, the isolation and characterization of the enzyme system catalyzing the synthesis of ThTP have met with little success. This difficulty can be understood in light of this complex enzyme system as described below. Assay Method Principle. The substrate for this phosphoryltransferase (EC 2.7.4.15, thiamin-diphosphate kinase) is not free ThPP but ThPP that is bound to a protein. This substrate is prepared by injecting rats with [35S]thiamin and subsequently isolating the [35S]ThPP-protein from liver by ion-exchange and gel chromatography. After incubation of the labeled substrate with the enzyme and cofactor, [“S]ThTP is isolated by column chromatography and the radioactivity determined.2 Reagents
Tris-HCl (pH 7.4), 0.5 M ATP, 0.1 M ’ J. R. Cooper and J. H. Pincus, Neurochen. Res. 4, 223 (1979). z K. Nishino, Y. Itokawa, N. Nishino, K. Piros, and J. R. Cooper, J. Biol. Chem. 258, 11871 (1983).
METHODS
IN ENZYMOLOGY,
VOL.
122
Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
151
ENZYMATIC
SYNTHESIS
OF THIAMIN
TRIPHOSPHATE
25
MgC12, 0.1 M Glucose, 0.1 M [25S]ThPP-protein substrate (equivalent to 4.0-6.0 pM [35S]thiamin pyrophosphate) Enzyme preparation Trichloroacetic acid (TCA), 20% Procedure. In a final volume of 100 ~1 are added, 10 ~1 of Tris-HCl (pH 7.4), 5 ~1 ATP, 5 ~1 MgC12, 10 ~1 glucose, 50 ~1 radioactive substrate, and 20 ~1 enzyme preparation. The reaction is initiated by the addition of the enzyme after the mixture was preincubated for 2 min at 37”. Following a 10 min incubation, 10 ~1 of TCA is added to terminate the reaction, followed by centrifugation. The supernatant is removed, extracted three times with 1.5 ml water-saturated ether to remove the TCA, and the residual ether in the supernatant removed by evaporation at 37”for 5 min. Approximately 70-80 ~1 of the supernatant is then applied to an AG-50X8 (H+) column (0.7 x 2.0 cm) and the ThTP eluted with 1 ml of water. The effluent, containing 90% of the radioactive ThTP that is synthesized, is collected in a scintillation vial and the radioactivity determined after the addition of scintillant. The amount of ThTP formed is calculated from the radioactivity and the previously calculated specific activity of the labeled substrate. Controls are carried out through the procedure both using zero time and with the omission of ATP from the incubation medium. Preparation of [35SIThPP Substrate After 6 weeks on a thiamin-deficient diet, Sprague-Dawley male rats are injected ip with 100 &i of [35S]thiamin-HCl (specific activity 40-200 mCi/mmol) dissolved in 0.3 ml of physiological saline twice at a 24-hr interval. Twenty-four hours after the second injection, rats are sacrificed, and the livers removed and homogenized with 4 volumes of 0.25 M sucrose containing 30 mM Tris-HCl (pH 8.6) and 0.1 mM EDTA. After centrifugation at 40,000 g for 20 min at O-4”, the supernatant is removed and centrifuged at 105,000 g for 90 min. The resultant supernatant is dialyzed overnight against 30 mM Tris-HCl buffer (pH 8.6) at O-4” and then applied to a DEAE-Sephadex A-25 column (6 x 40 cm) equilibrated with 30 mM Tris-HCl (pH 8.6). Elution with the same buffer yields the unabsorbed labeled substrate. These fractions are pooled, the pH adjusted to 7.4, and the pool is concentrated under a nitrogen atmosphere using an Amicon PM10 apparatus. The concentrate is then applied to a Sephadex G-100 column (4.2 x 150 cm) equilibrated with the same buffer. Radioactive fractions (Fig. 1) are collected and concentrated similarly to that described above. This fraction can be used as substrate for most
26
THIAMIN:
35
40
PHOSPHATES
45 Fraction
AND
50
[51
ANALOGS
55
60
65
Number
FIG. 1. Elution pattern of substrate protein on Sephadex G-100 column chromatography. The concentrated et&tent containing substrate activity from DEAE-Sephadex A-25 chromatography was applied to a Sephadex G-100 column preequilibrated with 50 mM potassium phosphate buffer (pH 7.4), and eluted with the same buffer. Solid line, protein concentration; (0) total thiamin content of substrate protein.
experiments although it does contain a small amount of phosphoryltransferase. To block this activity, 1.OM potassium phosphate buffer (pH 8.0), and 0.1 M iodoacetamide are added to the Sephadex G-100 eluant in a final concentration of 0.1 and 20 mM, respectively. The eluant is incubated at 37” for 30 min, centrifuged to remove the resultant precipitate, and then dialyzed against 20 mM potassium phosphate buffer (pH 7.4) overnight at O-4”. The dialyzed sample is then concentrated with the Amicon PM 10 membrane. Purification
of ThPP-ATP
Phosphoryl Transferase
Step 1. Acetone Powder Preparation. Fresh bovine brain cortex, after the removal of meninges, is homogenized with 9 volumes of ice-cold 0.32 M sucrose containing 20 mM potassium phosphate buffer (pH 7.4), and 40 PM disodium EDTA in a Waring blender. After centrifugation at 1000 g for 10 min, the supernatant is removed and centrifuged at 10,800g for 20 min. The supematant is removed and the pellet is resuspended in 50 mM potassium phosphate buffer (pH 7.4), and recentrifuged for 20 min at 10,000 g. The resultant pellet (Pz) is resuspended in a small volume of the phosphate buffer and mixed with 9 volumes of acetone (-20”), and the
151
ENZYMATIC
SYNTHESIS
OF THIAMIN
27
TRIPHOSPHATE
/ I /
I
El E2 0.5
I' I 0.1
I
c-2 -2
/ I
/
I
I I 1 I 35
\
I
/I\
I
I
I
/
I
g
.07
I
/I I
I
40
45 Fraction
I
I
I
I
50
55
60
65
Number
FIG. 2. Elution pattern of cellulose DE-52 column chromatography. The enzyme prepa-
ration was applied to a DE-52 column and was eluted with a liner gradient of equal volumes of 20 mM potassium phosphate buffer (pH 7.6), containing 20% glycerol and 1.O mM DTT, and 20 mM potassium phosphate buffer (pH 7.6), containing 20% glycerol, 1.0 m&f DTT, and 1.0 A4 NaCl. Solid line, protein concentration; dotted line, NaCl concentration; (0) enzyme activity.
suspension stirred for 10 min at O-4”. After filtration on a Biichner funnel through Whatman 3 MM filter paper, the acetone powder is washed with cold ether and air dried. The acetone powder is stored at -20” in a vacuum desiccator. Step 2. Extraction. The acetone powder is extracted with 30 volumes of 20 mM potassium phosphate buffer (pH 7.6), containing 1.0 mM dithiothreitol (DTT) via homogenization. After centrifugation at 105,000 g for 90 min, the supematant is removed, the pellet reextracted and centrifuged as above, and the two supematants combined. Glycerol to a final concentration of 20% is then added to stabilize the enzyme. Step 3. DE-52 Column Chromatography. The enzyme solution is applied to a DE-52 column (6.5 x 30 cm) that has been preequilibrated with 20 mM potassium phosphate buffer (pH 7.6), containing 20% glycerol and 1.O mM DTT. The column is washed with the same buffer and the enzyme eluted with a linear gradient of equal volumes (1500 ml) of the equilibrating buffer and the buffer containing 1.O A4 NaCl. From this procedure, as shown in Fig. 2, two peaks of enzyme activity were collected. Each
28
THIAMIN:
40
PHOSPHATES
AND
50
55
45 Fraction
151
ANALOGS
60
Number
FIG. 3. Elution pattern of Sephacryl S-200 column chromatography. Sephacryl S-200 column (4.4 X 90 cm) was employed. Ten to 15 ml of the enzyme preparation of Step 3 (fractions El and E2) was applied separately and eluted with 50 mM potassium phosphate buffer (pH 7.2), containing 20% glycerol and 1.O mM DTT. The elution speed was 4.0 cm/hr and about 12 l-ml fractions were collected. Solid line, protein; (0) enzyme activity.
enzyme fraction was individually concentrated under nitrogen by ultrafiltration using an Amicon PM10 filter membrane. Step 4. Sephacryl S-200 Column Chromatography. The two enzyme fractions (El and E2) from Step 3 are separately applied to a column of Sephacryl S-200 (4.4 x 90 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7.2), containing 20% glycerol and 1 mA4 DTT and eluted with the same buffer. As shown in Fig. 3, the El fraction gives two active fractions, referred to as El-l and El-2, and the E2 fraction yields one peak of activity, referred to as E2. Step 5. Chromatofocusing Chromatography. The active fractions from Step 4 were separately applied to a chromatofocusing column (1 x 37 cm) that was preequilibrated with 0.025 M histidine-HCI buffer (pH 6.2), containing 20% glycerol and 1.0 mM DTT. The eluant was polybuffer 74 HCl, pH 4.0 (0.0075 mmol/pH unit), containing 20% glycerol and 1.0 mM DTT. Active fractions were eluted at pH 5.1 t 0.02. In order to eliminate polybuffer, which inactivates enzyme activity, the eluted frac-
151
ENZYMATIC
SYNTHESIS
OF THIAMIN
TRIPHOSPHATE
29
tions were immediately applied to a Sephadex G-75 column (3.2 X 10 cm) preequilibrated with 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, and elution carried out with the same buffer. To concentrate and retain enzyme activity, the eluants from the Sephadex chromatography were applied to an organomercurial agarose column (Affi-Gel 501, 0.7 x 4.0 cm) which was preequilibrated with 50 mM potassium phosphate buffer (pH 7.4), containing 20% glycerol and eluted with 3 ml of 50 mM potassium phosphate buffer (pH 7.4), containing 20% glycerol and 20 mM DTT. Properties Purity and Physicochemical Properties. The three active fractions (El-l, El-2, and E2) on disc gel electrophoresis showed a single protein band with the same Rf: the molecular weight of the enzyme was calculated to be 103,000. The pH optimum was 7.5, with Tris-HCl exhibiting slightly higher activity than phosphate buffer. Dependencies of the Reaction. The reaction has an absolute dependence on ATP, Mg2+, the cofactor (presumably glucose), and ThPP that is protein bound. When the bound substrate is replaced by free ThPP, no synthesis of ThTP is observed. Inhibitions. p-Chloromercuribenzoate at 5 x 10e4M inhibits activity by 79.5% and N-ethylmaleimide (1 X 10s2 M) inhibits by 64%. Miscellaneous. In addition to the substrate for the reaction being protein bound, ThTP is also synthesized bound to a protein from which it is released for assay on deproteinization. Both the protein-bound substrate and the enzyme are found in both brain and liver. We chose to prepare the substrate protein from liver because of the yield; we prepared the enzyme from brain since earlier work3 had indicated that patients with Leigh’s disease (subacute necrotizing encephalomyelopathy) excrete material which inhibits the synthesis of ThTP using a crude brain preparation but not the liver preparation.
3 J. R. Cooper and J. H. Pincus, J. Agric. Food Chem. 20,490 (1972).