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[38] Assay of thiamine pyrophosphokinase (ATP: Thiamine pyrophosphotransferase, EC 2.7.6.2) using14C- or35S-labeled thiamine
[38]
ASSAY OF THIAMINE PYROPIIOSPIIOKINASE
221
ratio of gel to protein is 2 : 1. The column is washed thoroughly with 0.01 M acetate buffer, pH 4.3, and the charge of supernatant solution from step 5 is placed on the column. The enzyme is recovered by gradient elution, starting with 0.1 M KH2PO4, pH 4.5, until the first inert protein peak disappears. Then a gradient is started, using 0.133 M sodium phosphate buffer, pH 6.7, in the reservoir and 100 ml of 0.1 M KH2PO4 in the mixer system. When the pH of the eluate reaches 5.2, the reservoir system is changed to 0.16 M Na2HPO4. The fractions between pH 5.2 and 5.8 contain most of the TPK activity. These are combined and brought to saturation with (NH4)2SO4. After centrifugation at high speed, the precipitate is redissolved in a minimum of water and may be stored at - 2 0 ° for several months without appreciable loss of activity. Properties The enzyme, as prepared, contains negligible activity of thiamine diphosphatase, adenylate kinase, or ATPase, is approximately 68% pure, and should have a specific activity of approximately 40-50 milliunits/mg with 10% recovery of original activity. Pyrithiamine is a potent inhibitor of thiamine pyrophosphokinase (Ki about 10-~), and oxythiamine is a 1000-fold weaker inhibitor (K~ = 10-4). The K~ for thiamine is of the order of 10-7 M. The enzyme is quite specific for ATP, although UTP has about 40% of the activity of ATP. AMP and ADP are inactive unless an ATP-generating system is operating.
(ATP:
[ 3 8 ] A s s a y of T h i a m i n e P y r o p h o s p h o k i n a s e T h i a m i n e P y r o p h o s p h o t r a n s f e r a s e , E C 2.7.6.2) using ]4C-or 35S-Labeled Thiamine By BRUNO DEUS
Labeled thiamine + ATP --* labeled thiamine pyrophosphate + AMP The rate of enzymatic conversion of thiamine to thiamine pyrophosphate (TPP) provides a convenient means of determining thiamine pyrophosphokinase activity. For the estimation of TPP, pyruvate decarboxylase (2oxoaeid carboxy-lyase, EC 4.1.1.1),1.2 and transketolase (D-sedoheptulose-7phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde transferase, EC H. G. K. Westenbrink, Vol. II [107], p. 636. 2R. N~veke, H. W. Goedde, and H. Holzer, Arch. Mikrobiol. 44, 93 (1962).
222
THIAMINE: PHOSPHATES AND ANALOGS
[38]
2.2.1.1), 3.4 which are completely dependent on their coenzyme, are used. A radiometric procedure, 5 which allows direct measurement of T P P , is described here in detail. Principle. The amount of labeled T1)P formed in the reaction from labeled thiamine and A T P is determined after separation from the reaction mixture by paper chromatography or by electrophoresis. Reagents Glycylglycine buffer, 0.5 M, pH 7.3 ATP, disodium salt, 0.1 M, pH 7.0 MgSO4, 0.1 M Thiamine hydrochloride, 6 0.01 M labeled; specific activity 1 to 3 )< l07 dpm per micromole All reagents are stored at - 15°. Procedure. In a final volume ef 0.5 ml the following solutions are pipetted into a 7 X 40-mm metal centrifuge tube: 0.1 ml glycylg]ycine buffer, 0.04 ml MgSO4, 0.06 ml ATP, and the enzyme preparation. The first three ingredients may bc added together. After equilibration, the reaction is started by addition of 0.01 ml ~4C- or 35S-labeled thiamine. The stoppered tubes are incubated at 37 ° for 60 minutes with vigorous shaking. After boiling for i minute, the tubes are placed into an ice bath. Precipitated protein is removed by centrifugation. A 0.1-ml aliquot is applied as a narrow band 6 cm from one end of a 3.8-cm strip of chromatography paper. 7 The chromatograms are developed in the descending manner at 20 ° for 12-15 hours with n-propanol-I M sodium acetate buffer, pH 5.0-water (7:1:2, v / v ) .s The solvent front runs about 40 cm under these conditions. Thiamine, thiamine monophosphate, and thiamine pyrophosphate give average R/ values of 0.46, 0.27, and 0.10, respectively, and are completely separated. The part of the air-dried chromatogram containing the T P P peak (usually within 12 cm from the start) is cut into 1 )< 3.8 cm strips. These are analyzed for radioactivity in l0 ml of scintillation medium. 9 The counting s A. G. ] ) a t t a and E. Racker, J. Biol. Chem. 236, 624 (1961). 4 L. R. Johnson and C. J. Gitbler, Biochim. Biophys. Acta 156, 85 (1968). 5 B. Deus, H. E. C. Blum, and H. Holzer, Anal. Biochem. 27, 492 (1969). s The Radiochemical Centre, Amersham, England, provides thiamine-(thiazole-2-14C) hydrochloride (10-30 mCi/millimole) and thiamine-35S hydrochloride (5-70 m C i /
millimole). 7 Chromatography paper No. 2043b Mgl (used without prewashing), Schleicher & Schfill, 3354 Dassel, Germany. s D. Siliprandi and N. Siliprandi, Biochim. Biophys. Acta 14, 52 (1954). ' Distilled toluene containing 0.3% 2,5-diphenyloxazole (PPO) and 0.03% p-bis-2(5-phenyloxazolyl)benzene (POPOP).
[38]
ASSAY OF THIAMINE PYROPHOSPHOKINASE
223
efficiency in the liquid scintillation spectrometer l° is 71 -4- 0.7% with a lower/upper discriminator setting of 8 0 - ~ and a gain of 1000. A control is obtained by running an incubation without the enzyme. Alternative Separation Procedure. In addition to paper chromatography, T P P may be separated from the reaction mixture by electrophoresis on paper? ,11 A 0.05-ml sample of the deproteinized incubation mixture is applied as a narrow band to the middle of a 3.8 X 40 cm strip of Whatman No. 3 M M paper which has been soaked with 0.05 M sodium acetate buffer, pH 5.5, previously. T P P is isolated by electrophoresis 12 for 4 hours at 0 °. The voltage gradient is set to l0 V per centimeter at 3.5 mA. After drying, T P P is located and radioactivity is estimated as described above. Definition of Units and Specific Activity. The amount of T P P produced enzymatically is calculated from the disintegrations per minute obtained in the T P P peak. It must be checked that the specific activities of thiamine and T P P are the same. One unit is defined as the amount of enzyme which catalyzes the formation of 1 micromole of T P P per minute under the conditions specified. Specific activity is expressed as units of enzyme per milligram of protein. The biuret method l~ is used for the determination of protein. Thiamine and T P P are measured by the thiochrome procedure. ~4 Comments on the Assay Procedure. The method described above has been applied to the purified enzyme from rat liver, ~ to rat liver crude extract, s and to rat hemolysate. 15 Depending on the activities of interfering enzymes, the optimal concentrations of A T P or MgS04 in the assay mixture may vary considerably. 15 Therefore these conditions nmst be checked for each enzyme source. Interfering activities which may occur are: T P P degrading enzymes, le A T P degrading enzymes, 15 and ADP- or AMP-forming enzymes. The reaction is strongly inhibited by high concentrations of ATP. In 10Packard Instrument Company, Inc., La Grange, Illinois, Tri-Carb Model 314 EX or 3375. H E. Sandner and B. Gassmann, Z. Ern(~hrungswiss. 8, 222 (1967). t~ Pherograph-Original-Frankfurt (Wieland and Pfleiderer), obtained from Hormuth, 69 Heidelberg, Germany. ~8G. Beisenherz, H. J. Boltze, T. Bticher, R. Czok, K. H. Garbade, E. Meyer-Arendt, and G. Pfleiderer, Z. Naturforsch. 8b, 555 (1953). ~4G. Kohlhaw, B. Deus, and H. Holzer, J. Biol. Chem. 240, 2135 (1965). 15 H. E. C. Blum and B. Deus, reported at the Herbsttagung der Gesellschaft ftir Biologische Chemie, MOnster, Germany, October 3 ~ , 1968; Physiol. Chem. 34q, 1240 (1968). ~e Thiamine pyrophosphate degrading activity is measured under the test conditions by substituting labeled T P P for labeled thiamine in the above incubation mixture) The decrease in the amount of T P P during the reaction time is determined. Labeled T P P is prepared enzymatically and purified by paper chromatography. 6
224
THIAMINE: PHOSPHATES AND ANALOGS
[38]
the presence of 1.2 X 10-2 M ATP, thiamine pyrophosphokinase from rat liver is inhibited about 70% by 3 × 10-3 M A D P or AMP, and about 90% by 1 X 10-2 M ADP. 5 The sensitivity of the method depends on the specific ~ctivity of the available thiamine. With thiamine of 1 to 3 X 107 dpm/micromole, 200 picomoles are easily detected. The coefficient of variation is less than ± 5 % . (The volume of the incubation mixture should not be diminished, as the results become less reproducible.) The author prefers separation of T P P from the assay system by paper chromatography because it is more sensitive and more convenient than electrophore~is, especially when many samples have to be assayed simultaneously. A Rapid Test for the Detection of Thiamine Pyrophosphokinase Activity For determining the presence of thiamine pyrophosphokinase in effluents from chromatography or gel filtration columns, a simplified procedure, which eliminates the time-consuming paper chromatography, is described. '7 Principle. The method is a modification of an assay devised for thymidine kinase. TMT P P is adsorbed to anion-exchange paper disks, '9 and thiamine is removed by a washing procedure. Procedure. The reaction mixture is as described above. If sufficiently high enzyme activities ~re to be expected, the incubation period may be reduced. Afte~ 15-60 minutes at 37 ° the reaction mixture is cooled to - 5 °. Samples of 20 ~l are spotted on anion-exchange paper disks. The papers must not be dried, but are immediately transferred to a funnel with a fritted disk of 22-ram diameter. At 15 seconds after the application of the aliquot, the disks are washed 5 times with 20 ml of 1 X 10 -4 M sodium acetate. The time interval between the washings is 0.5 minute. The eluent is withdrawn by suction. The papers are dried in hot air, then the radioactivity is measured in 5 ml of toluene scintillation medium. 9 Comments. More than 99% of the thiamine is removed from the papers by the washing procedure. Figure 1 demonstrates, that the percentage of T P P which is adsorbed to the anion-exchange paper is dependent on the absolute amount of T P P applied. In addition, it is dependent on the volume in which a constant amount of T P P is applied to the papers. Therefore, the results obtained by this procedure are qualitative rather than quantitative. However, provided the conditions in the assay system are standardized, ,7 B. Deus and H. E. C. Blum, in preparation. 18 T. R. Breitman, Biochirn. Biophys. Acta 67, 153 (1963). 10Whatman D E 81 (23 mm in diameter).
[38]
ASSAY OF THIAMINE PYROPHOSPHOKINASE
225
100%
50%
I
0
I
too
L
t
500 picomoles TPP
Fie,. 1. Percentage of '4C-labeled thiamine pyrophosphale remaining adsorbed to anion-exchange paper disks after the washing procedure as a function of the a m o u n t added. Crosses represent lhe resttlts obtained in the presence of 335 micromoles of unlabeled thiamine.
this method is applicable to the detection of thiamine pyrophosphokinase activity in effluents from columns and to related problems. Because of the highly friable condition of the wet paper disks, it is not advisable to wash the disks in a beaker all together.
Occurrence of the Enzyme The enzyme has been partially purified from brewers' yeast 2° and liver? °-22 It has been detected in several animal tissues u,2~ and in microorganisms. -~4-26In Escherichia coli it is located in the spheroplasts, 26 whereas it is found exclusively (more than 99%) in the soluble fraction of rat erythrocytes ~ and of rat liver cells) 7 ~0Nguyen-Van-Thoai and L. Chevillard, Bull. Soc. Chim. Biol. 31,204 (1949). 21 F. Leuthardt and H. Nielsen, Helv. Chim. Acta 35, 1196 (1952). ~2y . Mano, J. Biochem. (Tokyo) 47, 283 (1960). 2s K. Lohmann and P. Schuster, Biochem. Z. 294, 188 (1937). 24 R. Suzue, Bull. Inst. Chem. Res., Kyoto Univ. 36, 8, 13 (1958); Chem. Abstr. 52, 18565b-g (1958). ~5 A. K. Sinha and G. C. Chatterjee, Biochem. J. 104, 731 (1967). ~6I. Miyata, T. Kawasaki, and Y. Nose, Biochem. Biophys. Res. Commun. 27, 601 (1967).
ASSAY OF THIAMINE PYROPIIOSPIIOKINASE
221
ratio of gel to protein is 2 : 1. The column is washed thoroughly with 0.01 M acetate buffer, pH 4.3, and the charge of supernatant solution from step 5 is placed on the column. The enzyme is recovered by gradient elution, starting with 0.1 M KH2PO4, pH 4.5, until the first inert protein peak disappears. Then a gradient is started, using 0.133 M sodium phosphate buffer, pH 6.7, in the reservoir and 100 ml of 0.1 M KH2PO4 in the mixer system. When the pH of the eluate reaches 5.2, the reservoir system is changed to 0.16 M Na2HPO4. The fractions between pH 5.2 and 5.8 contain most of the TPK activity. These are combined and brought to saturation with (NH4)2SO4. After centrifugation at high speed, the precipitate is redissolved in a minimum of water and may be stored at - 2 0 ° for several months without appreciable loss of activity. Properties The enzyme, as prepared, contains negligible activity of thiamine diphosphatase, adenylate kinase, or ATPase, is approximately 68% pure, and should have a specific activity of approximately 40-50 milliunits/mg with 10% recovery of original activity. Pyrithiamine is a potent inhibitor of thiamine pyrophosphokinase (Ki about 10-~), and oxythiamine is a 1000-fold weaker inhibitor (K~ = 10-4). The K~ for thiamine is of the order of 10-7 M. The enzyme is quite specific for ATP, although UTP has about 40% of the activity of ATP. AMP and ADP are inactive unless an ATP-generating system is operating.
(ATP:
[ 3 8 ] A s s a y of T h i a m i n e P y r o p h o s p h o k i n a s e T h i a m i n e P y r o p h o s p h o t r a n s f e r a s e , E C 2.7.6.2) using ]4C-or 35S-Labeled Thiamine By BRUNO DEUS
Labeled thiamine + ATP --* labeled thiamine pyrophosphate + AMP The rate of enzymatic conversion of thiamine to thiamine pyrophosphate (TPP) provides a convenient means of determining thiamine pyrophosphokinase activity. For the estimation of TPP, pyruvate decarboxylase (2oxoaeid carboxy-lyase, EC 4.1.1.1),1.2 and transketolase (D-sedoheptulose-7phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde transferase, EC H. G. K. Westenbrink, Vol. II [107], p. 636. 2R. N~veke, H. W. Goedde, and H. Holzer, Arch. Mikrobiol. 44, 93 (1962).
222
THIAMINE: PHOSPHATES AND ANALOGS
[38]
2.2.1.1), 3.4 which are completely dependent on their coenzyme, are used. A radiometric procedure, 5 which allows direct measurement of T P P , is described here in detail. Principle. The amount of labeled T1)P formed in the reaction from labeled thiamine and A T P is determined after separation from the reaction mixture by paper chromatography or by electrophoresis. Reagents Glycylglycine buffer, 0.5 M, pH 7.3 ATP, disodium salt, 0.1 M, pH 7.0 MgSO4, 0.1 M Thiamine hydrochloride, 6 0.01 M labeled; specific activity 1 to 3 )< l07 dpm per micromole All reagents are stored at - 15°. Procedure. In a final volume ef 0.5 ml the following solutions are pipetted into a 7 X 40-mm metal centrifuge tube: 0.1 ml glycylg]ycine buffer, 0.04 ml MgSO4, 0.06 ml ATP, and the enzyme preparation. The first three ingredients may bc added together. After equilibration, the reaction is started by addition of 0.01 ml ~4C- or 35S-labeled thiamine. The stoppered tubes are incubated at 37 ° for 60 minutes with vigorous shaking. After boiling for i minute, the tubes are placed into an ice bath. Precipitated protein is removed by centrifugation. A 0.1-ml aliquot is applied as a narrow band 6 cm from one end of a 3.8-cm strip of chromatography paper. 7 The chromatograms are developed in the descending manner at 20 ° for 12-15 hours with n-propanol-I M sodium acetate buffer, pH 5.0-water (7:1:2, v / v ) .s The solvent front runs about 40 cm under these conditions. Thiamine, thiamine monophosphate, and thiamine pyrophosphate give average R/ values of 0.46, 0.27, and 0.10, respectively, and are completely separated. The part of the air-dried chromatogram containing the T P P peak (usually within 12 cm from the start) is cut into 1 )< 3.8 cm strips. These are analyzed for radioactivity in l0 ml of scintillation medium. 9 The counting s A. G. ] ) a t t a and E. Racker, J. Biol. Chem. 236, 624 (1961). 4 L. R. Johnson and C. J. Gitbler, Biochim. Biophys. Acta 156, 85 (1968). 5 B. Deus, H. E. C. Blum, and H. Holzer, Anal. Biochem. 27, 492 (1969). s The Radiochemical Centre, Amersham, England, provides thiamine-(thiazole-2-14C) hydrochloride (10-30 mCi/millimole) and thiamine-35S hydrochloride (5-70 m C i /
millimole). 7 Chromatography paper No. 2043b Mgl (used without prewashing), Schleicher & Schfill, 3354 Dassel, Germany. s D. Siliprandi and N. Siliprandi, Biochim. Biophys. Acta 14, 52 (1954). ' Distilled toluene containing 0.3% 2,5-diphenyloxazole (PPO) and 0.03% p-bis-2(5-phenyloxazolyl)benzene (POPOP).
[38]
ASSAY OF THIAMINE PYROPHOSPHOKINASE
223
efficiency in the liquid scintillation spectrometer l° is 71 -4- 0.7% with a lower/upper discriminator setting of 8 0 - ~ and a gain of 1000. A control is obtained by running an incubation without the enzyme. Alternative Separation Procedure. In addition to paper chromatography, T P P may be separated from the reaction mixture by electrophoresis on paper? ,11 A 0.05-ml sample of the deproteinized incubation mixture is applied as a narrow band to the middle of a 3.8 X 40 cm strip of Whatman No. 3 M M paper which has been soaked with 0.05 M sodium acetate buffer, pH 5.5, previously. T P P is isolated by electrophoresis 12 for 4 hours at 0 °. The voltage gradient is set to l0 V per centimeter at 3.5 mA. After drying, T P P is located and radioactivity is estimated as described above. Definition of Units and Specific Activity. The amount of T P P produced enzymatically is calculated from the disintegrations per minute obtained in the T P P peak. It must be checked that the specific activities of thiamine and T P P are the same. One unit is defined as the amount of enzyme which catalyzes the formation of 1 micromole of T P P per minute under the conditions specified. Specific activity is expressed as units of enzyme per milligram of protein. The biuret method l~ is used for the determination of protein. Thiamine and T P P are measured by the thiochrome procedure. ~4 Comments on the Assay Procedure. The method described above has been applied to the purified enzyme from rat liver, ~ to rat liver crude extract, s and to rat hemolysate. 15 Depending on the activities of interfering enzymes, the optimal concentrations of A T P or MgS04 in the assay mixture may vary considerably. 15 Therefore these conditions nmst be checked for each enzyme source. Interfering activities which may occur are: T P P degrading enzymes, le A T P degrading enzymes, 15 and ADP- or AMP-forming enzymes. The reaction is strongly inhibited by high concentrations of ATP. In 10Packard Instrument Company, Inc., La Grange, Illinois, Tri-Carb Model 314 EX or 3375. H E. Sandner and B. Gassmann, Z. Ern(~hrungswiss. 8, 222 (1967). t~ Pherograph-Original-Frankfurt (Wieland and Pfleiderer), obtained from Hormuth, 69 Heidelberg, Germany. ~8G. Beisenherz, H. J. Boltze, T. Bticher, R. Czok, K. H. Garbade, E. Meyer-Arendt, and G. Pfleiderer, Z. Naturforsch. 8b, 555 (1953). ~4G. Kohlhaw, B. Deus, and H. Holzer, J. Biol. Chem. 240, 2135 (1965). 15 H. E. C. Blum and B. Deus, reported at the Herbsttagung der Gesellschaft ftir Biologische Chemie, MOnster, Germany, October 3 ~ , 1968; Physiol. Chem. 34q, 1240 (1968). ~e Thiamine pyrophosphate degrading activity is measured under the test conditions by substituting labeled T P P for labeled thiamine in the above incubation mixture) The decrease in the amount of T P P during the reaction time is determined. Labeled T P P is prepared enzymatically and purified by paper chromatography. 6
224
THIAMINE: PHOSPHATES AND ANALOGS
[38]
the presence of 1.2 X 10-2 M ATP, thiamine pyrophosphokinase from rat liver is inhibited about 70% by 3 × 10-3 M A D P or AMP, and about 90% by 1 X 10-2 M ADP. 5 The sensitivity of the method depends on the specific ~ctivity of the available thiamine. With thiamine of 1 to 3 X 107 dpm/micromole, 200 picomoles are easily detected. The coefficient of variation is less than ± 5 % . (The volume of the incubation mixture should not be diminished, as the results become less reproducible.) The author prefers separation of T P P from the assay system by paper chromatography because it is more sensitive and more convenient than electrophore~is, especially when many samples have to be assayed simultaneously. A Rapid Test for the Detection of Thiamine Pyrophosphokinase Activity For determining the presence of thiamine pyrophosphokinase in effluents from chromatography or gel filtration columns, a simplified procedure, which eliminates the time-consuming paper chromatography, is described. '7 Principle. The method is a modification of an assay devised for thymidine kinase. TMT P P is adsorbed to anion-exchange paper disks, '9 and thiamine is removed by a washing procedure. Procedure. The reaction mixture is as described above. If sufficiently high enzyme activities ~re to be expected, the incubation period may be reduced. Afte~ 15-60 minutes at 37 ° the reaction mixture is cooled to - 5 °. Samples of 20 ~l are spotted on anion-exchange paper disks. The papers must not be dried, but are immediately transferred to a funnel with a fritted disk of 22-ram diameter. At 15 seconds after the application of the aliquot, the disks are washed 5 times with 20 ml of 1 X 10 -4 M sodium acetate. The time interval between the washings is 0.5 minute. The eluent is withdrawn by suction. The papers are dried in hot air, then the radioactivity is measured in 5 ml of toluene scintillation medium. 9 Comments. More than 99% of the thiamine is removed from the papers by the washing procedure. Figure 1 demonstrates, that the percentage of T P P which is adsorbed to the anion-exchange paper is dependent on the absolute amount of T P P applied. In addition, it is dependent on the volume in which a constant amount of T P P is applied to the papers. Therefore, the results obtained by this procedure are qualitative rather than quantitative. However, provided the conditions in the assay system are standardized, ,7 B. Deus and H. E. C. Blum, in preparation. 18 T. R. Breitman, Biochirn. Biophys. Acta 67, 153 (1963). 10Whatman D E 81 (23 mm in diameter).
[38]
ASSAY OF THIAMINE PYROPHOSPHOKINASE
225
100%
50%
I
0
I
too
L
t
500 picomoles TPP
Fie,. 1. Percentage of '4C-labeled thiamine pyrophosphale remaining adsorbed to anion-exchange paper disks after the washing procedure as a function of the a m o u n t added. Crosses represent lhe resttlts obtained in the presence of 335 micromoles of unlabeled thiamine.
this method is applicable to the detection of thiamine pyrophosphokinase activity in effluents from columns and to related problems. Because of the highly friable condition of the wet paper disks, it is not advisable to wash the disks in a beaker all together.
Occurrence of the Enzyme The enzyme has been partially purified from brewers' yeast 2° and liver? °-22 It has been detected in several animal tissues u,2~ and in microorganisms. -~4-26In Escherichia coli it is located in the spheroplasts, 26 whereas it is found exclusively (more than 99%) in the soluble fraction of rat erythrocytes ~ and of rat liver cells) 7 ~0Nguyen-Van-Thoai and L. Chevillard, Bull. Soc. Chim. Biol. 31,204 (1949). 21 F. Leuthardt and H. Nielsen, Helv. Chim. Acta 35, 1196 (1952). ~2y . Mano, J. Biochem. (Tokyo) 47, 283 (1960). 2s K. Lohmann and P. Schuster, Biochem. Z. 294, 188 (1937). 24 R. Suzue, Bull. Inst. Chem. Res., Kyoto Univ. 36, 8, 13 (1958); Chem. Abstr. 52, 18565b-g (1958). ~5 A. K. Sinha and G. C. Chatterjee, Biochem. J. 104, 731 (1967). ~6I. Miyata, T. Kawasaki, and Y. Nose, Biochem. Biophys. Res. Commun. 27, 601 (1967).