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[14] Thiamine phosphate pyrophosphorylase
[14]
THIAMINE
PHOSPHATE
PYROPHOSPHORYLASE
69
The fluorescent bands are visualized with UV light, and according to this strip the other strips are cut into pieces corresponding to the band to be measured. The thiamine compounds are eluted from the pieces of paper with 3 ml of 50% ethanol (v/v) for 45 rain. After removal of the paper, fluorometric determination is carried out as described in this volume [ 10]. Comments With this method thiamine phosphate esters can be separated from mixtures containing 0.37 M perchloric acid and 70 m M glycylglycine, sodium acetate, potassium phosphate, or sucrose. When thiamine compounds are determined from biological material, the proteins of the sample are first precipitated with perchloric acid. The sample is neutralized with K2COs and then lyophilized in order to concentrate the contents of thiamine. Blanks are determined using benzenesulfonylchloride to prevent the oxidation of thiamine to thiochrome as described in this volume [10].
[14]
Thiamine
Phosphate
Pyrophosphorylase
By TAKASHI KAWASAKI Mg2+
HMP-PP~ + Th-P .
" TMP + PP~
The systematic name for thiamine pyrophosphorylase is 2-methyl4-amino-5-hydroxymethylpyrimidinepyrophosphate:4-methyl-5-(2'-phosphoethyl)-thiazole 2-methyl-4-aminopyrimidine-5-methenyltransferase (EC 2.5.1.3). Assay M e t h o d
Principle. T M P formed is h y d r o l y z e d to thiamine by Taka-Diastase, oxidized to thiochrome with alkaline cyanogen bromide, and then determined fluorometrically. 2 Reagents. The reagents for e n z y m e activity assay are: Abbreviations used: HMP (thiamine pyrimidine), 2-methyl-4-amino-5-hydroxymethylpyrimidine; Th (thiamine thiazole), 4-methyl-5-hydroxyethylthiazole; HMP-P, HMP phosphate; HMP-PP, HMP pyrophosphate; Th-P, Th phosphate; TMP, thiamine monophosphate. 2 A. Fujita, Y. Nose, S. Kozuka, T. Tashiro, K. Ueda, and S. Sakamoto,J. Biol. Chem. 196, 289 (1952).
METHODS IN ENZYMOLOGY, VOL. 62
Copyright t~ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181962-0
70
THIAMINE: PHOSPHATES A N D ANALOGS
[14]
Tris.HCl buffer, 0.5 M, pH 7.5 HMP-PP, 05 mM Th-P, 0.5 mM MgC12, 0.1 M Enzyme Those for TMP determination are: Sodium acetate buffer, 1 M, pH 4.5 Taka-Diastase solution, 0.02%, in 0.1 M sodium acetate buffer, pH 4.5 Cyanogen bromide, 0.3 M NaOH, 1 M Procedure. The reaction mixture contains 0.5 ml each of Tris.HCl buffer, HMP-PP, and Th-P, 0.3 ml of MgCI,, and enzyme in a total volume of 4 ml. The enzyme is diluted by 50 mM Tris.HC1 buffer, pH 7.5, containing 5 mM 2-mercaptoethanol when necessary. The mixture is preincubated for 5 min at 37°; the reaction is started by the addition of enzyme and allowed to incubate for 5 min. The reaction is stopped by the addition of 0.5 ml of acetate buffer and heated for 15 min at 85°. The mixture is then incubated for 30 min at 45 ° after adding 0.5 ml of TakaDiastase and centrifuged for 10 min at 3000 rpm to remove denatured proteins. An aliquot of the supernatant is brought to 3.5 ml with water, and 0.5 ml of cyanogen bromide freshly prepared before use is added and mixed, followed by the addition of 1.0 ml of NaOH. Fluorescence intensity of the solution is determined (excitation, 365 mn; emission, 430 nm) with thiochrome solution as the standard at an appropriate concentration. A blank mixture composed of Tris.HCl buffer and enzyme in the same total volume is treated in the same manner. The blank value is subtracted from that of the sample determined. Definition of Specific Activity. Specific activity is defined as nanomoles of TMP formed per milligram of protein per minute. Purification Procedure A partial purification of this enzyme has been described?
Source and Cultivation of Bacterium. A thiamine regulatory mutant of Escherichia coli K12, strain PT-R1, containing a derepressed level of TMP pyrophosphorylase4 is used. Strain PT-R1 is aerobically grown at 37° in the minimal medium of Davis and Mingioli5 containing 0.2% glucose as a carbon source; at 15-17 hr after incubation, the cells are harvested by 3 y . Kayama and T. Kawasaki, Arch. Biochem. Biophys. 158, 242 0973). 4 T. Kawasaki and Y. Nose, J. Biochem. (Tokyo) 65, 417 (1969). 5 B. D. Davis and E. S. Mingioli, J. Bacteriol. 60, 17 (1950).
[14]
THIAMINE PHOSPHATE PYROPHOSPHORYLASE
71
centrifugation (12,000 g, 4 °) and washed once in 50 mM Tris.HC1 buffer, pH 7.5, 5 mM 2-mercaptoethanol, 1 mM EDTA (medium A). The washed cells obtained are approximately 10 g wet weight from 60 liters of the culture medium. Step 1. Preparation of Crude Extract. The washed cell pastes are resuspended in 100 ml of medium A, and all further steps are carried out below 4 °. The cell suspension is subjected to sonic disruption for 20 min and then centrifuged for 20 min at 15,000 g. The sediment is discarded. Step 2. Removal of Nucleic Acid. The supernatant fluid is adjusted to pH 6.0, and 2% protamine sulfate is added with vigorous stirring at a ratio of 0.1 nag to milligram of protein and then centrifuged. This treatment resuits in an improved absorption ratio of 280: 260 nm of the supernatant to 0.9-1.0. Step 3, First Ammonium Sulfate Fractionation. Solid ammonium sulfate (24.3 g/100 ml) is added to bring the solution to 40% saturation with constant stirring. The pH of the solution is maintained at 7.5 by dropwise addition of 5 M ammonium hydroxide during the addition of ammonium sulfate. The suspension is allowed to stand for 30 min before centrifugation and the precipitate is discarded. The supernatant is brought to 60% saturation by adding ammonium sulfate (13.2g/100 ml) with constant stirring and standing for another 30 min. The precipitate obtained after centrifugation is dissolved in 10 ml of medium A (ammonium sulfate I fraction). Step 4. First Sephadex Column Chromatography. The ammonium sulfate I fraction is applied to a Sephadex G-100 column (2.5 x 50 cm) previously equilibrated with medium A and elution is carried out with the same buffer system to collect 10-ml fractions at approximately 2 ml/min. The fractions containing high enzyme activity are combined (Sephadex I fraction). Step 5. Second Ammonium Sulfate Fraction. Solid ammonium sulfate (35.1 g/100 ml) is added to the Sephadex I fraction to give 55% saturation, while the solution is maintained at pH 7.5 as described above. After standing for 30 min, the precipitate is collected by centrifugation and dissolved in 20 ml of medium A (ammonium sulfate II fraction). Step 6. DEAE-Cellulose Column Chromatography. The ammonium sulfate II fraction is dialyzed for 3 hr against 5 liters of medium A and applied to a DEAE-cellulose column (2.5 x 50 cm) previously equilibrated with medium A. Elution is carried out with 300 ml of medium A and then with a linear gradient of NaC1 consisting of 200 ml of medium A in the mixing flask and an equal volume of medium A containing 0.4 M NaCI in the reservoir flask. Fractions of 5 ml are collected. High enzyme activity is obtained by elution at 0.2-0.25 M NaCI, and these fractions are pooled
72
THIAMINE:
PHOSPHATES
AND
[14]
ANALOGS
PURIFICATION OF Escherichia coli T M P PYROPHOSPHORYLASEa
Fraction
Total protein (mg)
Crude extract~ Ammonium sulfate P Sephadex Ib Ammonium sulfate IF DEAE-cellulosec Sephadex IF
16,900 7740 1920 475 90 5.2
Total Specific activity activity Purification Yield (nmol/min) (nmol/mg/min) (fold) (%) 6250 4650 2550 1920 966 338
0.37 0.60 1.3 4.0 11 65
1..0 1.6 3.6 11 29 176
100 74.5 40.8 30.7 15.4 5.4
a From Y. Kayama and T. Kawasaki, Arch. Biochem. Biophys. 158, 242 (1973). Protein was determined by the biuret method [A. G. Gornall, C. S. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1941)]. Protein was determined by the method of O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941). and concentrated to a p p r o x i m a t e l y 3 ml in a collodion bag ( D E A E fraction). Step 7. Second Sephadex Column Chromatography. The D E A E fraction is applied to a S e p h a d e x G-100 column (2.5 x 50 cm) previously equilibrated with m e d i u m A and eluted with the same medium. The fractions of 5 ml containing high e n z y m e activity are pooled and concentrated to a p p r o x i m a t e l y 1 ml in a collodion bag (Sephadex II fraction). The purification p r o c e d u r e is s u m m a r i z e d in the table. Properties
Substrate Specificity. The e n z y m e is specific for both o f the substrates, H M P - P P and Th-P. H M P , H M P - P , and Th are not replaced as substrates. Kinetic Properties. When a s s a y e d at p H 8.5 and 40 ° in the p r e s e n c e of 6 m M MgC12, the a p p a r e n t Km values for H M P - P P and Th-P are calculated to be 8.5 × l0 -7 M and 4.0 × l0 -7 M, respectively. Metal Requirement. The purified e n z y m e requires the p r e s e n c e of divalent cation for its maximal activity. Mg 2+ is m o s t effective at 6 m M and Mn 2+ at 6 m M is equally effective. Co 2+, Ca 2+, and Zn 2+ are only 20% as effective as Mg 2+. T h e a p p a r e n t Km value for Mg 2+ is calculated to be 6.3 × l0 -s M under the standard a s s a y conditions. Effect of High-Energy Phosphate Compounds. High-energy phosphate c o m p o u n d s at 4 m M are all inhibitory in the p r e s e n c e o f 6 m M MgC12. Percentage inhibitions by these c o m p o u n d s of the activity are: ATP, 64.7; ADP, 42.4; G T P , 76.7, U T P , 71.6; CTP, 58.7; acetyl phos-
[15]
S Y N T H E S I S OF P Y R I M I D I N E 14C-LABELED T H I A M I N E
73
phate, 82.8; phosphoenol pyruvate, 31.1; and phosphocreatine, 63.8. Acetyl phosphate is the most effective inhibitor and causes 50% inhibition at 0.5 mM. Practically no inhibition of the activity is found with AMP, pyridine nucleotides, and sugar phosphates. Kinetics indicate that the inhibition caused by ATP and acetyl phosphate is uncompetitive for either HMP-PP or Th-P. A partially purified TMP pyrophosphorylase of yeast is also inhibited by high-energy phosphate compounds. 6 Molecular Weight. The molecular weight of the enzyme measured by the method of Andrews 7 is approximately 17,000. Other Properties. The purified preparation retains full catalytic and regulatory properties for at least several months when stored at - 20°. Optimal pH for activity is 8.5 with 50 mM Tris.HC1 buffer, and optimal temperature is 40 °. The enzyme is completely inactivated when incubated for 5 min at 45 ° without addition of both substrates. The enzyme is inhibited by inorganic pyrophosphate and 50% inhibition results at 50/.tM concentration. 6 T. Kawasaki and K. Esaki, Biochem. Biophys. Res. Commun. 40, I468 (1970). 7 p. Andrews, Biochem. J. 91,222 (1964).
[15] Synthesis
of Pyrimidine
1*C-labeled Thiamine
By E. E. EDWIN Thiamine labeled in its thiazole moiety either with 14C or asS is commercially available. 1 However, it is difficult to obtain the pyrimidine-labeled compound, and no detailed accounts of its synthesis have been published. Since it is a useful compound in studies in many areas of thiamine metabolism, a procedure that has been successfully used in our laboratory is described. It has been adapted from a number of published methods to suit semimicro and radiochemical techniques. The method yields compounds labeled either on the 2-methyl substituent or the T-position of the pyrimidine ring, depending on whether [1t4C]acetonitrile or [2-~4C]acetonitrile is used as a starting material. Principle. [1-~4C]Acetonitrile or [2-~4C]acetonitrile is converted into acetamidine 2 and condensed a with ~-ethoxymethylene malononitrile. The resulting cyanomethylpyrimidine is reduced to give the aminomethyl 1 Radiochemical Centre, A m e r s h a m , England.
z A. W. Dox, in "Organic S y n t h e s e s " (A. H. Blatt, ed), Collective Vol. I. p. 5. Wiley, N e w York, 1941. a R. Greive, Hoppe-Seyler's Z. PhysioL Chem. 242, 5 (1941).
METHODS 1N ENZYMOLOGY, VOL. 62
Copyright © 1979by AcademicPress, lnc, All rightsof reproduction in any form reserved. ISBN 0-12~181962-0
THIAMINE
PHOSPHATE
PYROPHOSPHORYLASE
69
The fluorescent bands are visualized with UV light, and according to this strip the other strips are cut into pieces corresponding to the band to be measured. The thiamine compounds are eluted from the pieces of paper with 3 ml of 50% ethanol (v/v) for 45 rain. After removal of the paper, fluorometric determination is carried out as described in this volume [ 10]. Comments With this method thiamine phosphate esters can be separated from mixtures containing 0.37 M perchloric acid and 70 m M glycylglycine, sodium acetate, potassium phosphate, or sucrose. When thiamine compounds are determined from biological material, the proteins of the sample are first precipitated with perchloric acid. The sample is neutralized with K2COs and then lyophilized in order to concentrate the contents of thiamine. Blanks are determined using benzenesulfonylchloride to prevent the oxidation of thiamine to thiochrome as described in this volume [10].
[14]
Thiamine
Phosphate
Pyrophosphorylase
By TAKASHI KAWASAKI Mg2+
HMP-PP~ + Th-P .
" TMP + PP~
The systematic name for thiamine pyrophosphorylase is 2-methyl4-amino-5-hydroxymethylpyrimidinepyrophosphate:4-methyl-5-(2'-phosphoethyl)-thiazole 2-methyl-4-aminopyrimidine-5-methenyltransferase (EC 2.5.1.3). Assay M e t h o d
Principle. T M P formed is h y d r o l y z e d to thiamine by Taka-Diastase, oxidized to thiochrome with alkaline cyanogen bromide, and then determined fluorometrically. 2 Reagents. The reagents for e n z y m e activity assay are: Abbreviations used: HMP (thiamine pyrimidine), 2-methyl-4-amino-5-hydroxymethylpyrimidine; Th (thiamine thiazole), 4-methyl-5-hydroxyethylthiazole; HMP-P, HMP phosphate; HMP-PP, HMP pyrophosphate; Th-P, Th phosphate; TMP, thiamine monophosphate. 2 A. Fujita, Y. Nose, S. Kozuka, T. Tashiro, K. Ueda, and S. Sakamoto,J. Biol. Chem. 196, 289 (1952).
METHODS IN ENZYMOLOGY, VOL. 62
Copyright t~ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181962-0
70
THIAMINE: PHOSPHATES A N D ANALOGS
[14]
Tris.HCl buffer, 0.5 M, pH 7.5 HMP-PP, 05 mM Th-P, 0.5 mM MgC12, 0.1 M Enzyme Those for TMP determination are: Sodium acetate buffer, 1 M, pH 4.5 Taka-Diastase solution, 0.02%, in 0.1 M sodium acetate buffer, pH 4.5 Cyanogen bromide, 0.3 M NaOH, 1 M Procedure. The reaction mixture contains 0.5 ml each of Tris.HCl buffer, HMP-PP, and Th-P, 0.3 ml of MgCI,, and enzyme in a total volume of 4 ml. The enzyme is diluted by 50 mM Tris.HC1 buffer, pH 7.5, containing 5 mM 2-mercaptoethanol when necessary. The mixture is preincubated for 5 min at 37°; the reaction is started by the addition of enzyme and allowed to incubate for 5 min. The reaction is stopped by the addition of 0.5 ml of acetate buffer and heated for 15 min at 85°. The mixture is then incubated for 30 min at 45 ° after adding 0.5 ml of TakaDiastase and centrifuged for 10 min at 3000 rpm to remove denatured proteins. An aliquot of the supernatant is brought to 3.5 ml with water, and 0.5 ml of cyanogen bromide freshly prepared before use is added and mixed, followed by the addition of 1.0 ml of NaOH. Fluorescence intensity of the solution is determined (excitation, 365 mn; emission, 430 nm) with thiochrome solution as the standard at an appropriate concentration. A blank mixture composed of Tris.HCl buffer and enzyme in the same total volume is treated in the same manner. The blank value is subtracted from that of the sample determined. Definition of Specific Activity. Specific activity is defined as nanomoles of TMP formed per milligram of protein per minute. Purification Procedure A partial purification of this enzyme has been described?
Source and Cultivation of Bacterium. A thiamine regulatory mutant of Escherichia coli K12, strain PT-R1, containing a derepressed level of TMP pyrophosphorylase4 is used. Strain PT-R1 is aerobically grown at 37° in the minimal medium of Davis and Mingioli5 containing 0.2% glucose as a carbon source; at 15-17 hr after incubation, the cells are harvested by 3 y . Kayama and T. Kawasaki, Arch. Biochem. Biophys. 158, 242 0973). 4 T. Kawasaki and Y. Nose, J. Biochem. (Tokyo) 65, 417 (1969). 5 B. D. Davis and E. S. Mingioli, J. Bacteriol. 60, 17 (1950).
[14]
THIAMINE PHOSPHATE PYROPHOSPHORYLASE
71
centrifugation (12,000 g, 4 °) and washed once in 50 mM Tris.HC1 buffer, pH 7.5, 5 mM 2-mercaptoethanol, 1 mM EDTA (medium A). The washed cells obtained are approximately 10 g wet weight from 60 liters of the culture medium. Step 1. Preparation of Crude Extract. The washed cell pastes are resuspended in 100 ml of medium A, and all further steps are carried out below 4 °. The cell suspension is subjected to sonic disruption for 20 min and then centrifuged for 20 min at 15,000 g. The sediment is discarded. Step 2. Removal of Nucleic Acid. The supernatant fluid is adjusted to pH 6.0, and 2% protamine sulfate is added with vigorous stirring at a ratio of 0.1 nag to milligram of protein and then centrifuged. This treatment resuits in an improved absorption ratio of 280: 260 nm of the supernatant to 0.9-1.0. Step 3, First Ammonium Sulfate Fractionation. Solid ammonium sulfate (24.3 g/100 ml) is added to bring the solution to 40% saturation with constant stirring. The pH of the solution is maintained at 7.5 by dropwise addition of 5 M ammonium hydroxide during the addition of ammonium sulfate. The suspension is allowed to stand for 30 min before centrifugation and the precipitate is discarded. The supernatant is brought to 60% saturation by adding ammonium sulfate (13.2g/100 ml) with constant stirring and standing for another 30 min. The precipitate obtained after centrifugation is dissolved in 10 ml of medium A (ammonium sulfate I fraction). Step 4. First Sephadex Column Chromatography. The ammonium sulfate I fraction is applied to a Sephadex G-100 column (2.5 x 50 cm) previously equilibrated with medium A and elution is carried out with the same buffer system to collect 10-ml fractions at approximately 2 ml/min. The fractions containing high enzyme activity are combined (Sephadex I fraction). Step 5. Second Ammonium Sulfate Fraction. Solid ammonium sulfate (35.1 g/100 ml) is added to the Sephadex I fraction to give 55% saturation, while the solution is maintained at pH 7.5 as described above. After standing for 30 min, the precipitate is collected by centrifugation and dissolved in 20 ml of medium A (ammonium sulfate II fraction). Step 6. DEAE-Cellulose Column Chromatography. The ammonium sulfate II fraction is dialyzed for 3 hr against 5 liters of medium A and applied to a DEAE-cellulose column (2.5 x 50 cm) previously equilibrated with medium A. Elution is carried out with 300 ml of medium A and then with a linear gradient of NaC1 consisting of 200 ml of medium A in the mixing flask and an equal volume of medium A containing 0.4 M NaCI in the reservoir flask. Fractions of 5 ml are collected. High enzyme activity is obtained by elution at 0.2-0.25 M NaCI, and these fractions are pooled
72
THIAMINE:
PHOSPHATES
AND
[14]
ANALOGS
PURIFICATION OF Escherichia coli T M P PYROPHOSPHORYLASEa
Fraction
Total protein (mg)
Crude extract~ Ammonium sulfate P Sephadex Ib Ammonium sulfate IF DEAE-cellulosec Sephadex IF
16,900 7740 1920 475 90 5.2
Total Specific activity activity Purification Yield (nmol/min) (nmol/mg/min) (fold) (%) 6250 4650 2550 1920 966 338
0.37 0.60 1.3 4.0 11 65
1..0 1.6 3.6 11 29 176
100 74.5 40.8 30.7 15.4 5.4
a From Y. Kayama and T. Kawasaki, Arch. Biochem. Biophys. 158, 242 (1973). Protein was determined by the biuret method [A. G. Gornall, C. S. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1941)]. Protein was determined by the method of O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941). and concentrated to a p p r o x i m a t e l y 3 ml in a collodion bag ( D E A E fraction). Step 7. Second Sephadex Column Chromatography. The D E A E fraction is applied to a S e p h a d e x G-100 column (2.5 x 50 cm) previously equilibrated with m e d i u m A and eluted with the same medium. The fractions of 5 ml containing high e n z y m e activity are pooled and concentrated to a p p r o x i m a t e l y 1 ml in a collodion bag (Sephadex II fraction). The purification p r o c e d u r e is s u m m a r i z e d in the table. Properties
Substrate Specificity. The e n z y m e is specific for both o f the substrates, H M P - P P and Th-P. H M P , H M P - P , and Th are not replaced as substrates. Kinetic Properties. When a s s a y e d at p H 8.5 and 40 ° in the p r e s e n c e of 6 m M MgC12, the a p p a r e n t Km values for H M P - P P and Th-P are calculated to be 8.5 × l0 -7 M and 4.0 × l0 -7 M, respectively. Metal Requirement. The purified e n z y m e requires the p r e s e n c e of divalent cation for its maximal activity. Mg 2+ is m o s t effective at 6 m M and Mn 2+ at 6 m M is equally effective. Co 2+, Ca 2+, and Zn 2+ are only 20% as effective as Mg 2+. T h e a p p a r e n t Km value for Mg 2+ is calculated to be 6.3 × l0 -s M under the standard a s s a y conditions. Effect of High-Energy Phosphate Compounds. High-energy phosphate c o m p o u n d s at 4 m M are all inhibitory in the p r e s e n c e o f 6 m M MgC12. Percentage inhibitions by these c o m p o u n d s of the activity are: ATP, 64.7; ADP, 42.4; G T P , 76.7, U T P , 71.6; CTP, 58.7; acetyl phos-
[15]
S Y N T H E S I S OF P Y R I M I D I N E 14C-LABELED T H I A M I N E
73
phate, 82.8; phosphoenol pyruvate, 31.1; and phosphocreatine, 63.8. Acetyl phosphate is the most effective inhibitor and causes 50% inhibition at 0.5 mM. Practically no inhibition of the activity is found with AMP, pyridine nucleotides, and sugar phosphates. Kinetics indicate that the inhibition caused by ATP and acetyl phosphate is uncompetitive for either HMP-PP or Th-P. A partially purified TMP pyrophosphorylase of yeast is also inhibited by high-energy phosphate compounds. 6 Molecular Weight. The molecular weight of the enzyme measured by the method of Andrews 7 is approximately 17,000. Other Properties. The purified preparation retains full catalytic and regulatory properties for at least several months when stored at - 20°. Optimal pH for activity is 8.5 with 50 mM Tris.HC1 buffer, and optimal temperature is 40 °. The enzyme is completely inactivated when incubated for 5 min at 45 ° without addition of both substrates. The enzyme is inhibited by inorganic pyrophosphate and 50% inhibition results at 50/.tM concentration. 6 T. Kawasaki and K. Esaki, Biochem. Biophys. Res. Commun. 40, I468 (1970). 7 p. Andrews, Biochem. J. 91,222 (1964).
[15] Synthesis
of Pyrimidine
1*C-labeled Thiamine
By E. E. EDWIN Thiamine labeled in its thiazole moiety either with 14C or asS is commercially available. 1 However, it is difficult to obtain the pyrimidine-labeled compound, and no detailed accounts of its synthesis have been published. Since it is a useful compound in studies in many areas of thiamine metabolism, a procedure that has been successfully used in our laboratory is described. It has been adapted from a number of published methods to suit semimicro and radiochemical techniques. The method yields compounds labeled either on the 2-methyl substituent or the T-position of the pyrimidine ring, depending on whether [1t4C]acetonitrile or [2-~4C]acetonitrile is used as a starting material. Principle. [1-~4C]Acetonitrile or [2-~4C]acetonitrile is converted into acetamidine 2 and condensed a with ~-ethoxymethylene malononitrile. The resulting cyanomethylpyrimidine is reduced to give the aminomethyl 1 Radiochemical Centre, A m e r s h a m , England.
z A. W. Dox, in "Organic S y n t h e s e s " (A. H. Blatt, ed), Collective Vol. I. p. 5. Wiley, N e w York, 1941. a R. Greive, Hoppe-Seyler's Z. PhysioL Chem. 242, 5 (1941).
METHODS 1N ENZYMOLOGY, VOL. 62
Copyright © 1979by AcademicPress, lnc, All rightsof reproduction in any form reserved. ISBN 0-12~181962-0