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[123] Preparation of deaminated adenine derivatives
[123]
DEAMINATED ADENINE DERIVATIVES
873
to the unknown. Because of the sensitivity of the method the extracts can usually be diluted to an extent that no inhibitory effects are observed. Other Applications. A variety of other enzymes, coenzymes, and substrates can be assayed by this method. These include myokinase, hexokinase, ATPase, apyrase, creatine-adenylic transphosphorylase, ADP, AMP, phosphocreatine, CoA, and D P N pyrophosphatase. These substances have been tested by the above procedures. It should be possible to measure quantitatively other compounds which directly or indirectly show a stoichiometrie relationship to ATP.
[123] Preparation of Deaminated Adenine Derivatives I. Preparation and Properties of Inosine Triphosphate (ITP)
By NATHAN 0. KAPLAN Preparation Principle. The procedure outlined below is based on the deamination of ATP by nitrous acid with isolation of the I T P as a barium salt in a manner described by Kleinzeller ~and Kaplan et al. ~The deamination may also be carried out enzymatically by means of takadiastase deaminase. ~ Reagents
ATP, 1 g. free acid or K or Na salts in 50 ml. of 2 N HAc. NaNO~, 8 g. dissolved in 30 ml. of H20 immediately before use. Procedure. The NaNO2 solution is added dropwise to the ATP solution. Immediately after addition of the nitrite, a suitable aliquot is removed, diluted to 3 ml., and read at 265 m~. The reaction mixture is then kept at room temperature, and aliquots are removed every 30 minutes and read at 265 m~. When there is no further decrease in the reading at 265 m~ (2 to 3 hours), the mixture is neutralized with NaOH to phenol red. Then 5.0 ml. of 25 % barium acetate is added along with 1 vol. of ethanol. The barium salt of the I T P is then collected by centrifugation and washed with 50, 95, and 100% alcohol and finally with ether. After dryingin vacuo over P~Os, the yield of barium salt is about 1.2 g.
Properties Chemical Properties. The I T P has a maximum absorption at 249 m~, the millimolar extinction coefficient being 13.9. The properties of the A. Kleinzeller, Biochem. J. 36, 729 (1924). N. O. Kaplan, S. P. Colowick, and M. M. Ciotti, J. Biol. Chem. 194, 579 (1952).
874
COENZYMES AND R E L A T E D PHOSPHA TE COMPOUNDS
[123]
compound are v e r y similar to A T P , and acid hydrolysis liberates phosp h a t e f r o m I T P at the same rate as f r o m A T P . P u r i t y can be ascertained b y comparing the labile p h o s p h a t e in the I T P with the absorption at 249 m~. T h e b a r i u m salt analyzes 8 5 % pure. Enzymatic Properties. I T P can be distinguished f r o m A T P enzymatically. I T P reacts with y e a s t hexokinase, b u t at a slower rate t h a n with A T P . I D P does not react with muscle adenylic acid kinase, and this p r o p e r t y can be used to d e t e r m i n e whether I T P or A T P is present, as illustrated in the a c c o m p a n y i n g table. I T P can also be distinguished REACTIONS OF
ITP
AND
ATP
WITH HEXOKINASE AND MUSCLE ADENYLIC ACID KINASE
(2.5 umoles of ITP and ATP is incubated with 0.01 M MgCl~, 0.05 M NaHC02, 0.05 M glucose, 13 units of yeast hexokinase,~ and with and without 0.05 ml. of muscle adenylic acid kinase,b Time of incubation is 60 minutes, and the temperature is 37 °. After treatment with TCA, the mixtures are analyzed for labile phosphate, c The results of a typical experiment are summarized below.) Additions
None Hexokinase Hexokinase and myokinase
ATP
ITP
(~moles, 7 min., P left) 5.0 2.9 0.5
(~noles, 7 min., P left) 5.0 3.1 3.1
See Volume I [32]. b See Volume II [99]. c See Volume I I I [114]. f r o m A T P in the firefly luminescence s y s t e m which does not react with the I T P . 3
II. Preparation and Properties of Deamino-DPN Preparation ~
Principle. T h e d e a m i n a t i o n is carried out as in the p r e p a r a t i o n of I T P . T h e d e a m i n a t e d coenzyme is then precipitated with acid acetone. Reagents D P N , 1 g., 9 0 % pure, dissolved in 50 ml. of 2 N H A C . NaNO2, 8 g., dissolved in 30 ml. of H 2 0 i m m e d i a t e l y before using.
Procedure. T h e NaNO~ is added dropwise with constant stirring to the D P N solution. B y measuring the decrease a t 265 m#, the course of the d e a m i n a t i o n can be followed. T h e reaction usually is completed within 2 hours. T o precipitate the d e a m i n a t e d compound, the mixture is made s See Vol. III [122].
[123]
DEAMINATED ADENINE DERIVATIVES
875
acid to congo red with HNO3. Four volumes of cold acetone are then added, and the mixture is left overnight at 4 ° . The resulting precipitate is quite oily and adheres to the walls of the flask. The supernatant from the precipitate can generally be decanted; the precipitate is dissolved in H20 and reprecipitated with 4 vol. of cold acetone after acidification with HN03. This precipitate can be washed with acetone and ether and then dried. From 1 g. of DPN, 800 mg. of deamino-DPN is obtained. No nitrite can be detected in the preparation. Deamino-DPN can also be prepared by using the takadiastase deaminase. 4
Properties
Chemical Properties. Deamino-DPN has an absorption maximum at 248 m~. The compound gives an identical reaction with cyanide as does DPN (see Vol. III [128]). From the cyanide spectrum, the compound has been estimated to be approximately 88 % pure. Enzymatic Properties. The deaminated DPN reacts equally as DPN with some dehydrogenase, whereas with other enzymes it is either inac Live or reacts at a slower rate than the natural coenzyme. 5 Neurospora DPNase attacks deamino-DPN at 2% of the rate of DPN. This difference can be used as a basis for differentiating between the two pyridine nucleotides. Because it can be distinguished from DPN enzymatically, the deamino-DPN has been used in studies dealing with the mechanism of dehydrogenase action s and also in studies involving pyridine nucleotide transhydrogenase. ~,8 III. Preparation and Properties of Inosinic Acids 9 Preparation The deamination of the 2 t-, 3 t-, and 5'-adenylic acid isomers to form the corresponding inosinic acids is carried out in the same manner as is the preparation of ITP and deamino-DPN. After the reaction is complete (as indicated by no further decrease at 265 m~), phenol red indicator is added, and the solution brought to a pale pink with phenol red; 10 ml. of 25% barium acetate are added, and the nucleotides are precipitated by the addition of 95% ethanol. The precipitates, after centrifugation, are washed with 66% ethanol, 100% ethanol, and anhydrous ether. The compounds are then placed in a desiccator over phosphorous pentoxide. 4 See Vol. I I I70]. s M. E. Pullman, S. P. Colowick, and N. O. Kaplan, J. Biol. Chem. 194, 593 (1952). E. Adams, J. Biol. Chem. 217, 325 (1955). 7 N. O. Kaplan, S. P. Colowick, and E. F. Neufeld, J. Biol. Chem. 195, 107 (1952) 8 N. O. Kaplan, S. P. Colowick, and E. F. Neufeld, J. Biol. Chem. 205, 1 (1953). 9 L. Shuster and N. O. Kaplan, J. Biol. Chem. 201, 535 (1953).
876
COENZYMESAND RELATED PHOSPHATE COMPOUNDS
[19"4]
F r o m 1 g. of adenine nucleotide, an average of 1.2 g. of the barium salts of the different inosinic acids are obtained.
Properties T h e inosinic acids can be characterized by their absorption maximum. The 2'- and 3'-isomers can be distinguished from the 5'-isomer b y their acid lability. 1° T h r o u g h the use of the specific 3'-nucleotidase 11 the 3'-isomer can be identified. The inosinic acids in the free acid form are somewhat more insoluble t h a n the adenine mononucleotides. 1~ 10See Vol. III [114]. 11See Vol. II [86]. ~ Uridine nucleotides can be prepared from cytosine nueleotides with nitrous acid in the same manner as the hypoxanthine nucleotides are obtained from the adenine derivatives (see footnote #9).
[124] I s o l a t i o n o f D i p h o s p h o p y r i d i n e N u c l e o t i d e a n d
Triphosphopyridine Nucleotide I. Diphosphopyridine Nucleotide ( D P N , Cozymase, Coenzyme I)
By ARTHUR KORNBERG Principle. D P N is extracted from yeast with hot water. After removal of other substances from the extract b y basic lead acetate, D P N is precipitated as a silver salt. This is decomposed with hydrogen sulfide, and the D P N is obtained as the free acid b y precipitation with acetone. F u r t h e r purification is achieved by anion exchange chromatography. Method of Preparation. The preparation of D P N from a n u m b e r of source materials b y various methods has been reviewed. 1 T h e present method ~ is a modification of the procedure of Williamson and Green. 3 Ion exchange chromatography of D P N has been employed independently by others. 4
Purification Procedure Starting Material. Starch-free bakers' yeast is obtained from an Anheuser-Buseh yeast plant. I t is shipped in bulk (not compressed) in an insulated carton containing dry ice and is used 2 days after shipping. I G. A. LePage, B~ochem.Preparations 1, 28 (1949). 2 A. Kornberg and W. E. Pricer, Jr., Biochem. Preparations 3, 20 (1953). 3 S. Williamson and D. E. Green, J. Biol. Chem. 155, 345 (1940). 4j. B. Neilands and/~./~keson, J. Biol. Chem. 188, 307 (1951).
DEAMINATED ADENINE DERIVATIVES
873
to the unknown. Because of the sensitivity of the method the extracts can usually be diluted to an extent that no inhibitory effects are observed. Other Applications. A variety of other enzymes, coenzymes, and substrates can be assayed by this method. These include myokinase, hexokinase, ATPase, apyrase, creatine-adenylic transphosphorylase, ADP, AMP, phosphocreatine, CoA, and D P N pyrophosphatase. These substances have been tested by the above procedures. It should be possible to measure quantitatively other compounds which directly or indirectly show a stoichiometrie relationship to ATP.
[123] Preparation of Deaminated Adenine Derivatives I. Preparation and Properties of Inosine Triphosphate (ITP)
By NATHAN 0. KAPLAN Preparation Principle. The procedure outlined below is based on the deamination of ATP by nitrous acid with isolation of the I T P as a barium salt in a manner described by Kleinzeller ~and Kaplan et al. ~The deamination may also be carried out enzymatically by means of takadiastase deaminase. ~ Reagents
ATP, 1 g. free acid or K or Na salts in 50 ml. of 2 N HAc. NaNO~, 8 g. dissolved in 30 ml. of H20 immediately before use. Procedure. The NaNO2 solution is added dropwise to the ATP solution. Immediately after addition of the nitrite, a suitable aliquot is removed, diluted to 3 ml., and read at 265 m~. The reaction mixture is then kept at room temperature, and aliquots are removed every 30 minutes and read at 265 m~. When there is no further decrease in the reading at 265 m~ (2 to 3 hours), the mixture is neutralized with NaOH to phenol red. Then 5.0 ml. of 25 % barium acetate is added along with 1 vol. of ethanol. The barium salt of the I T P is then collected by centrifugation and washed with 50, 95, and 100% alcohol and finally with ether. After dryingin vacuo over P~Os, the yield of barium salt is about 1.2 g.
Properties Chemical Properties. The I T P has a maximum absorption at 249 m~, the millimolar extinction coefficient being 13.9. The properties of the A. Kleinzeller, Biochem. J. 36, 729 (1924). N. O. Kaplan, S. P. Colowick, and M. M. Ciotti, J. Biol. Chem. 194, 579 (1952).
874
COENZYMES AND R E L A T E D PHOSPHA TE COMPOUNDS
[123]
compound are v e r y similar to A T P , and acid hydrolysis liberates phosp h a t e f r o m I T P at the same rate as f r o m A T P . P u r i t y can be ascertained b y comparing the labile p h o s p h a t e in the I T P with the absorption at 249 m~. T h e b a r i u m salt analyzes 8 5 % pure. Enzymatic Properties. I T P can be distinguished f r o m A T P enzymatically. I T P reacts with y e a s t hexokinase, b u t at a slower rate t h a n with A T P . I D P does not react with muscle adenylic acid kinase, and this p r o p e r t y can be used to d e t e r m i n e whether I T P or A T P is present, as illustrated in the a c c o m p a n y i n g table. I T P can also be distinguished REACTIONS OF
ITP
AND
ATP
WITH HEXOKINASE AND MUSCLE ADENYLIC ACID KINASE
(2.5 umoles of ITP and ATP is incubated with 0.01 M MgCl~, 0.05 M NaHC02, 0.05 M glucose, 13 units of yeast hexokinase,~ and with and without 0.05 ml. of muscle adenylic acid kinase,b Time of incubation is 60 minutes, and the temperature is 37 °. After treatment with TCA, the mixtures are analyzed for labile phosphate, c The results of a typical experiment are summarized below.) Additions
None Hexokinase Hexokinase and myokinase
ATP
ITP
(~moles, 7 min., P left) 5.0 2.9 0.5
(~noles, 7 min., P left) 5.0 3.1 3.1
See Volume I [32]. b See Volume II [99]. c See Volume I I I [114]. f r o m A T P in the firefly luminescence s y s t e m which does not react with the I T P . 3
II. Preparation and Properties of Deamino-DPN Preparation ~
Principle. T h e d e a m i n a t i o n is carried out as in the p r e p a r a t i o n of I T P . T h e d e a m i n a t e d coenzyme is then precipitated with acid acetone. Reagents D P N , 1 g., 9 0 % pure, dissolved in 50 ml. of 2 N H A C . NaNO2, 8 g., dissolved in 30 ml. of H 2 0 i m m e d i a t e l y before using.
Procedure. T h e NaNO~ is added dropwise with constant stirring to the D P N solution. B y measuring the decrease a t 265 m#, the course of the d e a m i n a t i o n can be followed. T h e reaction usually is completed within 2 hours. T o precipitate the d e a m i n a t e d compound, the mixture is made s See Vol. III [122].
[123]
DEAMINATED ADENINE DERIVATIVES
875
acid to congo red with HNO3. Four volumes of cold acetone are then added, and the mixture is left overnight at 4 ° . The resulting precipitate is quite oily and adheres to the walls of the flask. The supernatant from the precipitate can generally be decanted; the precipitate is dissolved in H20 and reprecipitated with 4 vol. of cold acetone after acidification with HN03. This precipitate can be washed with acetone and ether and then dried. From 1 g. of DPN, 800 mg. of deamino-DPN is obtained. No nitrite can be detected in the preparation. Deamino-DPN can also be prepared by using the takadiastase deaminase. 4
Properties
Chemical Properties. Deamino-DPN has an absorption maximum at 248 m~. The compound gives an identical reaction with cyanide as does DPN (see Vol. III [128]). From the cyanide spectrum, the compound has been estimated to be approximately 88 % pure. Enzymatic Properties. The deaminated DPN reacts equally as DPN with some dehydrogenase, whereas with other enzymes it is either inac Live or reacts at a slower rate than the natural coenzyme. 5 Neurospora DPNase attacks deamino-DPN at 2% of the rate of DPN. This difference can be used as a basis for differentiating between the two pyridine nucleotides. Because it can be distinguished from DPN enzymatically, the deamino-DPN has been used in studies dealing with the mechanism of dehydrogenase action s and also in studies involving pyridine nucleotide transhydrogenase. ~,8 III. Preparation and Properties of Inosinic Acids 9 Preparation The deamination of the 2 t-, 3 t-, and 5'-adenylic acid isomers to form the corresponding inosinic acids is carried out in the same manner as is the preparation of ITP and deamino-DPN. After the reaction is complete (as indicated by no further decrease at 265 m~), phenol red indicator is added, and the solution brought to a pale pink with phenol red; 10 ml. of 25% barium acetate are added, and the nucleotides are precipitated by the addition of 95% ethanol. The precipitates, after centrifugation, are washed with 66% ethanol, 100% ethanol, and anhydrous ether. The compounds are then placed in a desiccator over phosphorous pentoxide. 4 See Vol. I I I70]. s M. E. Pullman, S. P. Colowick, and N. O. Kaplan, J. Biol. Chem. 194, 593 (1952). E. Adams, J. Biol. Chem. 217, 325 (1955). 7 N. O. Kaplan, S. P. Colowick, and E. F. Neufeld, J. Biol. Chem. 195, 107 (1952) 8 N. O. Kaplan, S. P. Colowick, and E. F. Neufeld, J. Biol. Chem. 205, 1 (1953). 9 L. Shuster and N. O. Kaplan, J. Biol. Chem. 201, 535 (1953).
876
COENZYMESAND RELATED PHOSPHATE COMPOUNDS
[19"4]
F r o m 1 g. of adenine nucleotide, an average of 1.2 g. of the barium salts of the different inosinic acids are obtained.
Properties T h e inosinic acids can be characterized by their absorption maximum. The 2'- and 3'-isomers can be distinguished from the 5'-isomer b y their acid lability. 1° T h r o u g h the use of the specific 3'-nucleotidase 11 the 3'-isomer can be identified. The inosinic acids in the free acid form are somewhat more insoluble t h a n the adenine mononucleotides. 1~ 10See Vol. III [114]. 11See Vol. II [86]. ~ Uridine nucleotides can be prepared from cytosine nueleotides with nitrous acid in the same manner as the hypoxanthine nucleotides are obtained from the adenine derivatives (see footnote #9).
[124] I s o l a t i o n o f D i p h o s p h o p y r i d i n e N u c l e o t i d e a n d
Triphosphopyridine Nucleotide I. Diphosphopyridine Nucleotide ( D P N , Cozymase, Coenzyme I)
By ARTHUR KORNBERG Principle. D P N is extracted from yeast with hot water. After removal of other substances from the extract b y basic lead acetate, D P N is precipitated as a silver salt. This is decomposed with hydrogen sulfide, and the D P N is obtained as the free acid b y precipitation with acetone. F u r t h e r purification is achieved by anion exchange chromatography. Method of Preparation. The preparation of D P N from a n u m b e r of source materials b y various methods has been reviewed. 1 T h e present method ~ is a modification of the procedure of Williamson and Green. 3 Ion exchange chromatography of D P N has been employed independently by others. 4
Purification Procedure Starting Material. Starch-free bakers' yeast is obtained from an Anheuser-Buseh yeast plant. I t is shipped in bulk (not compressed) in an insulated carton containing dry ice and is used 2 days after shipping. I G. A. LePage, B~ochem.Preparations 1, 28 (1949). 2 A. Kornberg and W. E. Pricer, Jr., Biochem. Preparations 3, 20 (1953). 3 S. Williamson and D. E. Green, J. Biol. Chem. 155, 345 (1940). 4j. B. Neilands and/~./~keson, J. Biol. Chem. 188, 307 (1951).