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[10] d -Ribulose 5-phosphate
46
PREPARATION
OF SUBSTRATES
[I0]
pentulose ester on a scale ten times that described by the authors with excellent yields. The hydrazone is readily obtained by adding an excess of hydrazine before precipitating the barium salt with ethanol.
[ 10] D - R i b u l o s e 5 - P h o s p h a t e By SANDROPON~MOLI
Preparation Principle. v-Ribulose 5-phosphate is prepared by the TPN-coupled enzymatic oxidation of D-glueonate 6-phosphate followed by treatment with charcoal and precipitation of the phosphorylated product as barium or lithium salt? Reagents Glycylglycine buffer, 0.3 M pH 7.6 v-Gluconate 6-phosphate (Na salt), 0.2 M Sodium pyruvate, 0.3 M TPN, 0.1 M Crystalline lactic dehydrogenase (from rabbit muscle, purchased from Boehringer and SShne, Germany) Crystalline D-gluconate 6-phosphate dehydrogenase (specific activity 190 units per milligram of protein). For the preparation of the enzyme, see this volume [26]. Both enzymes must be centrifuged before use and dissolved in water to avoid the introduction of excessive amounts of ammonium sulfate. Procedure. The reaction mixture (8.0 ml) contains: 3.7 ml of water, 1.2 ml of D-gluconate 6-phosphate, 1.0 ml of glycylglycine buffer, 1.6 ml of sodium pyruvate, 0.2 ml of TPN, 0.24 ml (1.2 mg) of lactic dehydrogenase, and 0.06 ml (0.6 mg) of D-gluconate 6-phosphate dehydrogenase. The reaction is started by the addition of the D-gluconate 6-phosphate dehydrogenase. The contents are mixed and incubated at 30 °. Aliquots are assayed at intervals for D-gluconate 6-phosphate. After 45 minutes when less than 1% remains, the reaction mixture is rapidly chilled in an ice bath and treated with 0.78 ml of 45% trichloroaeetic acid and 100 mg of acid-washed charcoal, 2 shaken, and left 10 minutes in ice. The suspension is filtered through a sintered glass filter, and the residue is washed with 2.5 ml of water. Preparation of the Barium Salt. To the combined filtrate and wash1S. Pontremoli and G. Mangiarotti, J. Biol. Chem. 237, 643 (1962). 2For the preparation of the charcoal, see this volume [26].
[10]
DoRIBULOSE 5-PHOSPHATE
47
ings (11 ml) is added 1 ml of 1 M barium acetate. After 20 minutes in ice the turbid suspension is centrifuged, the precipitate is washed with 1 ml of water, and the supernatant solution and washing are combined (12.2 ml). This solution is treated with 3.6 ml of saturated Ba(OH),, adjusted to pH 6.6 with saturated KOH, and precipitated with 4 volumes of cold absolute ethanol. After 30 minutes the precipitate is collected by centrifugation, washed with 80% ethanol, and dried in vacuo. The yield of dried barium salt is 104.5 mg (first barium salt precipitate). An aliquot of the barium salt (55 mg) is dissolved in 5.0 ml of 0.1 M CH3COOH, the solution is adjusted to pH 6.6 with 0.01 ml of saturated KOH and 0.5 ml of saturated Ba(OH)2 and is precipitated with 4 volumes of cold absolute ethanol. After 45 minutes the precipitate is collected by centrifugation, washed with 80% ethanol, and dried in vavu~. The yield of dried barium salt is 44 mg (second barium salt precipitate). Preparation o] the Lithium Salt. To obtain the lithium salt, 49.5 mg of the first barium salt precipitate is dissolved in 4 ml of 0.1M CH3COOH, an aliquot (0.5 ml) is removed for analysis, and the remaining solution is passed through a Dowex 50 H + column to remove Ba+÷. The effluent and washings (4.1 ml) are combined and brought to pH 6.3 with 4.5 ml of 0.1 N LiOH. The lithium salt is precipitated at 0 ° with 100 ml of 10% methanol in acetone. After 30 minutes the precipitate is collected by centrifugation, washed with 10 ml of the acetone-methanol mixture, and dried in vacuo. The yield of the dried lithium salt is 19 mg. Properties and Purity of the Product. The barium and lithium salts of the product, prepared as described, have been characterized as D-ribulose 5-phosphate by chemical and enzymatic analyses. The properties of the phosphorylated sugar are summarized in the table. On the basis of CHEMICAL AND ENZYMATIC PROPERTIES OF D-RIBULOSE a-PHosPHATE LITHIUM AND BARIUM SALTS Ba salt, 1st ppt.
Ba salt, 2nd ppt.
0rcinol test,=870 m~ (total micromoles)
212
86.2
Cysteine carbazole, b 540 m~ (total micromoles)
211
85.5
Test
P~ (micromoles) 0 Bromine oxidationc No v-Ribose5-phosphate~formed at equilibrium (%) Not done
0 No 70
Li salt
64 62 0 No 71
= F. Dickens and D. H. WiUiamson, B~chem. J. 64, 657 (1956). b G. Ashwell and J. Hiekman, J. Biol. Chem. $26, 65 (1957). c At 28 °, pH 6.0 in 6 minutes. d Incubated with n-ribose 5-phosphate isomerase prepared according to J. Hurwitz, A. Weissbach, B. L. Horecker, and P. Z. Smyrniotis, J. Biol. Chem. 218, 769 (1956).
48
PREPARATION OF SUBSTRATES
[11]
these data the product appears to be D-ribulose 5-phosphate in almost pure form. The presence of D-ribose 5-phosphate is excluded since the product is not oxidized by bromine in a period of time sufficient to oxidize 70% of the aldopentose. By this procedure quantities of D-ribose 5-phosphate approaching 10% of the total would be readily detected. The presence of D-xylulose 5-phosphate is excluded by the equilibrium ratio obtained with D-ribose 5-phosphate isomerase. The D-ribulose 5phosphate preparation reacts with T P N H + H* in the presence of CO2 and D-gluconate 6-phosphate dehydrogenase to produce D-gluconate 6-phosphate and TPN. The yield based on D-gluconate 6-phosphate was 80%. The product is stable if stored at 2 ° in a vacuum desiccator. C o m m e n t s . Previous methods for the enzymatic preparation of D-ribulose 5-phosphate by enzymatic oxidation of D-gluconate 6-phosphate, ~ isomerization of n-ribose 5-phosphate 4 or phosphorylation of D-ribulose, 5 required separation of the phosphorylated sugar by ion exchange chromatography since other phosphate esters or inorganic phosphate were present in the reaction mixture. The yield was low, and the product was contaminated by other phosphate esters. With the present method, pure ribulose 5-phosphate may be obtained as the lithium or the barium salt with an overall yield of 80%. SB. L. Horecker and P. Z. Smyrniotis, J. Biol. Chem. 193, 383 (1951). P. Srere, J. R. Cooper, M. Tabachnick, and E. Racker, Arch. Biochem. Biophys.
74, 295 (1958). F. J. Simpson and W. A. Wood, J. Biol. Chem. 230, 473 (1958).
[ 11] L-Ribulose 5-Phosphate
By R. L. ANDERSON The enzymatic procedure described here is based on the use of L-ribulokinase from an L-ribulose 5-phosphate 4-epimeraseless mutant of Escherichia coli. 1 The use of this mutant obviates the need to purify L-ribulokinase to obtain a product free from D-xylulose 5-phosphate and D-ribulose 5-phosphate. Crystalline L-ribulokinase has been prepared, 2 however, and could be used if desired. ~-Ribulose 5-phosphate has also E. Englesberg, R. L. Anderson, R. Weinberg, N. Lee, P. Hoffee, G. Huttenhauer, and H. Boyer, J, Bacteriol. 84, 137 (1962). See this volume [80].
PREPARATION
OF SUBSTRATES
[I0]
pentulose ester on a scale ten times that described by the authors with excellent yields. The hydrazone is readily obtained by adding an excess of hydrazine before precipitating the barium salt with ethanol.
[ 10] D - R i b u l o s e 5 - P h o s p h a t e By SANDROPON~MOLI
Preparation Principle. v-Ribulose 5-phosphate is prepared by the TPN-coupled enzymatic oxidation of D-glueonate 6-phosphate followed by treatment with charcoal and precipitation of the phosphorylated product as barium or lithium salt? Reagents Glycylglycine buffer, 0.3 M pH 7.6 v-Gluconate 6-phosphate (Na salt), 0.2 M Sodium pyruvate, 0.3 M TPN, 0.1 M Crystalline lactic dehydrogenase (from rabbit muscle, purchased from Boehringer and SShne, Germany) Crystalline D-gluconate 6-phosphate dehydrogenase (specific activity 190 units per milligram of protein). For the preparation of the enzyme, see this volume [26]. Both enzymes must be centrifuged before use and dissolved in water to avoid the introduction of excessive amounts of ammonium sulfate. Procedure. The reaction mixture (8.0 ml) contains: 3.7 ml of water, 1.2 ml of D-gluconate 6-phosphate, 1.0 ml of glycylglycine buffer, 1.6 ml of sodium pyruvate, 0.2 ml of TPN, 0.24 ml (1.2 mg) of lactic dehydrogenase, and 0.06 ml (0.6 mg) of D-gluconate 6-phosphate dehydrogenase. The reaction is started by the addition of the D-gluconate 6-phosphate dehydrogenase. The contents are mixed and incubated at 30 °. Aliquots are assayed at intervals for D-gluconate 6-phosphate. After 45 minutes when less than 1% remains, the reaction mixture is rapidly chilled in an ice bath and treated with 0.78 ml of 45% trichloroaeetic acid and 100 mg of acid-washed charcoal, 2 shaken, and left 10 minutes in ice. The suspension is filtered through a sintered glass filter, and the residue is washed with 2.5 ml of water. Preparation of the Barium Salt. To the combined filtrate and wash1S. Pontremoli and G. Mangiarotti, J. Biol. Chem. 237, 643 (1962). 2For the preparation of the charcoal, see this volume [26].
[10]
DoRIBULOSE 5-PHOSPHATE
47
ings (11 ml) is added 1 ml of 1 M barium acetate. After 20 minutes in ice the turbid suspension is centrifuged, the precipitate is washed with 1 ml of water, and the supernatant solution and washing are combined (12.2 ml). This solution is treated with 3.6 ml of saturated Ba(OH),, adjusted to pH 6.6 with saturated KOH, and precipitated with 4 volumes of cold absolute ethanol. After 30 minutes the precipitate is collected by centrifugation, washed with 80% ethanol, and dried in vacuo. The yield of dried barium salt is 104.5 mg (first barium salt precipitate). An aliquot of the barium salt (55 mg) is dissolved in 5.0 ml of 0.1 M CH3COOH, the solution is adjusted to pH 6.6 with 0.01 ml of saturated KOH and 0.5 ml of saturated Ba(OH)2 and is precipitated with 4 volumes of cold absolute ethanol. After 45 minutes the precipitate is collected by centrifugation, washed with 80% ethanol, and dried in vavu~. The yield of dried barium salt is 44 mg (second barium salt precipitate). Preparation o] the Lithium Salt. To obtain the lithium salt, 49.5 mg of the first barium salt precipitate is dissolved in 4 ml of 0.1M CH3COOH, an aliquot (0.5 ml) is removed for analysis, and the remaining solution is passed through a Dowex 50 H + column to remove Ba+÷. The effluent and washings (4.1 ml) are combined and brought to pH 6.3 with 4.5 ml of 0.1 N LiOH. The lithium salt is precipitated at 0 ° with 100 ml of 10% methanol in acetone. After 30 minutes the precipitate is collected by centrifugation, washed with 10 ml of the acetone-methanol mixture, and dried in vacuo. The yield of the dried lithium salt is 19 mg. Properties and Purity of the Product. The barium and lithium salts of the product, prepared as described, have been characterized as D-ribulose 5-phosphate by chemical and enzymatic analyses. The properties of the phosphorylated sugar are summarized in the table. On the basis of CHEMICAL AND ENZYMATIC PROPERTIES OF D-RIBULOSE a-PHosPHATE LITHIUM AND BARIUM SALTS Ba salt, 1st ppt.
Ba salt, 2nd ppt.
0rcinol test,=870 m~ (total micromoles)
212
86.2
Cysteine carbazole, b 540 m~ (total micromoles)
211
85.5
Test
P~ (micromoles) 0 Bromine oxidationc No v-Ribose5-phosphate~formed at equilibrium (%) Not done
0 No 70
Li salt
64 62 0 No 71
= F. Dickens and D. H. WiUiamson, B~chem. J. 64, 657 (1956). b G. Ashwell and J. Hiekman, J. Biol. Chem. $26, 65 (1957). c At 28 °, pH 6.0 in 6 minutes. d Incubated with n-ribose 5-phosphate isomerase prepared according to J. Hurwitz, A. Weissbach, B. L. Horecker, and P. Z. Smyrniotis, J. Biol. Chem. 218, 769 (1956).
48
PREPARATION OF SUBSTRATES
[11]
these data the product appears to be D-ribulose 5-phosphate in almost pure form. The presence of D-ribose 5-phosphate is excluded since the product is not oxidized by bromine in a period of time sufficient to oxidize 70% of the aldopentose. By this procedure quantities of D-ribose 5-phosphate approaching 10% of the total would be readily detected. The presence of D-xylulose 5-phosphate is excluded by the equilibrium ratio obtained with D-ribose 5-phosphate isomerase. The D-ribulose 5phosphate preparation reacts with T P N H + H* in the presence of CO2 and D-gluconate 6-phosphate dehydrogenase to produce D-gluconate 6-phosphate and TPN. The yield based on D-gluconate 6-phosphate was 80%. The product is stable if stored at 2 ° in a vacuum desiccator. C o m m e n t s . Previous methods for the enzymatic preparation of D-ribulose 5-phosphate by enzymatic oxidation of D-gluconate 6-phosphate, ~ isomerization of n-ribose 5-phosphate 4 or phosphorylation of D-ribulose, 5 required separation of the phosphorylated sugar by ion exchange chromatography since other phosphate esters or inorganic phosphate were present in the reaction mixture. The yield was low, and the product was contaminated by other phosphate esters. With the present method, pure ribulose 5-phosphate may be obtained as the lithium or the barium salt with an overall yield of 80%. SB. L. Horecker and P. Z. Smyrniotis, J. Biol. Chem. 193, 383 (1951). P. Srere, J. R. Cooper, M. Tabachnick, and E. Racker, Arch. Biochem. Biophys.
74, 295 (1958). F. J. Simpson and W. A. Wood, J. Biol. Chem. 230, 473 (1958).
[ 11] L-Ribulose 5-Phosphate
By R. L. ANDERSON The enzymatic procedure described here is based on the use of L-ribulokinase from an L-ribulose 5-phosphate 4-epimeraseless mutant of Escherichia coli. 1 The use of this mutant obviates the need to purify L-ribulokinase to obtain a product free from D-xylulose 5-phosphate and D-ribulose 5-phosphate. Crystalline L-ribulokinase has been prepared, 2 however, and could be used if desired. ~-Ribulose 5-phosphate has also E. Englesberg, R. L. Anderson, R. Weinberg, N. Lee, P. Hoffee, G. Huttenhauer, and H. Boyer, J, Bacteriol. 84, 137 (1962). See this volume [80].