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Enzymic synthesis of glucose-4-tritium and glucose-3-tritium
ARCHIVES
OF
BIOCHEMISTRY
Enzymic
Synthesis
ROBERT Institute
AND
for
BIOPHYSICS
of Glucose-4-Tritium
ROGNSTAD, Xedical
372-375
109,
ROBERT
Research,
and
G. KEMP,2
Cedam-Sinai
Received
(1965)
September
Hospitals,
Glucose-3-Tritium’
XKD
JOSEPH I
Los ilnyeles,
California
15, 1961
Dihydroxyacetone phosphate and n-glyceraldehyde phosphate were labeled with tritium in position 1 by enzymic exchange and isomerization in tritiated water. The labeled trioses were condensed by means of aldolase to fructose-l ,G-diphosphat,e labeled with tritium either in position 3 or 4. Labeled glucoses were prepared from the phosphate esters. The labeled sugars were obtained with a yield of 25’7 and specific activities of 5 PC per j4mole.
In an attempt to determine the source of the reductive hydrogen in fatty acid biosynthesis in adipose tissue, we undertook the synthesis of glucose-4-trit,ium (T), which yields DPNT in the glyceraldehydc phosphate dehydrogenase reaction, and glucose3-T, which yields TPNT in the phosphogluconic dehydrogenase reaction. These syntheses were based on the findings of Rose and Rieder (1, 2) on the sterospecific introduction of tritium on carbon-l of triose phosphates by means of t,he enzymes aldolase and triose phosphat’e isomerase in tritiated water. A synthesis of glucose-3-T based on these principles, but differing somewhat in procedure and yielding a sugar of lower specific activity, has previously been described (3). Principle. To synthesize glucosel-T, glyceraldehyde-3-phosphate-l-T (GAP-l-T) was prepared by incubating dihydroxyacetone-3-phosphate (DHAP) and aldolase in trit’iated water (Fig. IA), and then enzymically isomerizing the DHAP-1-T (5)4 to GAP-l-T in the presence of 2.5 M tris 1 Supported by a grant (AM 03682) to Joseph Katz from the U. S. Public Health Service. 2 Present address: Department of Biochemistry, University of Washington, Seattle, Washington. 3 Established Investigator, American Heart Association. 4 (8) and (n) refer to stereospecific position of tritium according to Cahn et al. (4).
buffer at pH 8.5 to shift, the equilibrium from the normal ratio of 95 : 3 DHAP-GAP to a ratio of approximately 95:.3 GAPDHAP.5 To synthesize glucose-3-T, DHAP-1-T (R)4 was prepared by incubating G-$P wit,h triose phosphate isomerase in tritiated water (Fig. 1B). GAP-l-T aud unlabeled DHAP, and DHAP-1-T (R) and unlabeled GAP, were condensed by means of aldolase to fructose1,6-diphosphate-4-T (FDP-4-T) and FDP3-T, respectively. The fructose diphosphates were hydrolyzed to fructose-6-phosphate (FBP), which was t,hen enzymically isomerized to glucose-A-phosphate (G61’), which in turn was dephosphorylated to glucose with acid phosphatase. EXPERIMENTAL Tritiated water (THO) was obtained from the New England Nuclear Corp., Boston, Massachusetts; DHAP dimethyl ketal and FDP were obtained from Sigma Chemical Co.. St. Louis, Missouri. All enzymes were obtained from Calbiochem, Los Angeles, California. Anion exchange resin (AG-1) was purchased from Bio-Rad Laboratories, Richmond, California and was converted to the acetate form. Tritium assay was carried out by the method of Jacobson (5) wit.h a Packard 5 The effect of high tris concentration and alkaline pH in reversing the normal GAP-DHAP equilibrium was found by Dr. 8. 1’. Rieder (personal communication to Dr. I. A. Rose).
372
SYNTHESIS
A. SYNTHESIS
OF GLUCOSE-4-T
T THO Aldolase
H-L
l=o
dH20P
:H20P
T,;~s;_“plsomerose
DHAP-I-
8. SYNTHESIS
OF DHAP -I -T ‘4 C
tritium
\C”’ H _ l _ OH
H I TriJsyp. ‘somerose
T-C-oH &O ;H20P
DHAP-I-
GAP of
T
GAP-I-T
T /S)
0
;H,OP
1. Incorporation
373
;H20P
DHAP
,, _ A-O,,
FIG. P-1-T.
2.5 M Tris
-OH
d=o
H
-3-T
OF GAP-l-T
H H-L-OH
ASD
in
T(R)
glyceraldehyde-3.P,l-T
scintillation count’er. Assay for triose phosphates and for FDP was performed by the method of Biicher and Hohorst (6). Hexose phosphates were determined by the method of Hohorst (7). Glucose assay was carried out by the method of Slein (8). Tritiation was performed in a 30-ml roundbottom flask connected by a Y-tube to a trap so that unused THO could be recovered by lyophilization. Special caut.ion should be exercised in the first steps because of the very high activity of the THO used. Synthesis oj” glucose-4-l’. DHAP was prepared from the ketal according to the manufacturers instructions. A solution containing 800 ~~moles of DHAP, 50 rmoles EDTA, and 50 rmoles GSH was chilled, adjusted to pH 7 with dilute NaOH, and lyophilized. It, n-as incubated for 2 hours with 5 ml of THO that, contained approximately 3 curies and 200 units (0.5 ml) of aldolase. The solution was lyophilized and the THO was recovered in a trap for use in later experiments. The enzyme was inactivated by adding 2 ml of 1096 TCA to the dry residue. The solution was passed through a 15-ml AG-1 (acetate) column which was then washed extensively with water to remove any residual THO and readily exchangeable tritium. The column was eluted with 0.1 LY HCl, and the DHAP-T fractions were reduced in volume on a rotary vacuum evaporator. Assay (6) at this point showed 510 pmoles of DHAP whose specific activity was the same as the tritiated water used in the incubation. Four Jf t,ris base was added to the DHAP-1-T solution until the pH was 8.5, and then s&i-
and dihydroxyacetone-
cient 4 IV tris buffer (pH 8.5) was added to bring the tris concentration to 2.5 M. Twenty-four hundred units of triose phosphate isomerase were added and the mixture was incubated for 30 minutes. Assay (6) showed 15 pmoles DHAP and 450 rmoles GAP. The solution was chilled and passed through a 20-ml Dowex 50 (H+) column to acidify the solution and inactivate the enzyme. To avoid reversing the triose phosphate equilibrium, this step was carried out in the cold room. The eluate was reduced in volume to about 10 ml on a rotary vacuum evaporator and then adjusted to pH 7 with dilute NaOH. Seven hundred pmoles of unlabeled DHAP and 100 units of aldolase were added. After a l-hour incubation, assay (6) showed 310 pmoles DHAP, 18 pmoles GAP, and 400 rmoles FDP. The mixture was heated to inactivate the enzyme and then passed through a small Dowex 50 (H+) column and on to a 20-ml AG-1 (acetate) column. (The passage through the cation column increases the efficiency of absorption by the acetate resin and thus permits the use of smaller columns.) Monophosphates were eluted with 0.08 llrl ammonium acetate (NH~Ac), pH 6.5, and FDP was subsequently eluted with0.1 h’HC1. The FDP fractions were concentrated until the HCl concentration was approximately 1 i\r, and were then incubated for 1 week at 37°C. Assay (6, 7) then showed 340 pmoles F6P and 5 pmoles FDP. The pH was adjusted to 7 with NaOH, 500 units of phosphohexose isomerase was added, and the mixture was incubated for 2 hours at 25°C and then overnight at 5°C (9). Assay (7) indicated
374
BOGNSTAD,
KEMP,
that 285 pmoles G6P and 55 rmoles FGP were present. The mixture was acidified at 5°C by passing it through a Dowex 50 (H+) column, boiled several minutes to inactivate the enzyme, and placed on a 20-ml AG-1 (acetate) column. GlucoseB-phosphate and FGP were separated by a slight variation of the method of Khym and Cohn (10). Inorganic phosphate was first eluted with 0.01 M borate and 0.08 M NHqAc, pH 8.5; GOP was next eluted with 0.01 Jf borate and 0.08 M NHdAc, pH 6.5; and FGP was then eluted with 0.5 &I NH(Ac, pH 6.5. The GGP fractions were put t)hrough a 20.ml Dowex 50 (H+) column to remove NH4+, and acetic acid was removed by evaporation. The solution was put on a 5ml A(:-1 (acetate) column, and boric acid was removed by washing with water. Glucose-S-phosphate was eluted with 0.1 N HCI, and the fractions were concentrated; assay (6) showed 230 pmoles G6P. The solution was adjusted to pH 5 with acetate buffer and incubated overnight with 10 mg of potato acid phosphatase. The enzyme was precipitated by adding trichloroacetic acid to a final concentration of 5?;, and the supernatant was put through a mixed bed Amberlite MB-3 column and concentrated. Final yield was 220 @moles glucose whose specific activity was approximately 5 PC per pmole. Synthesis of y/~cose-S-T. The calcium salt of GAP was prepared by the low-temperature periodate oxidation of FDP according to the method Szewczuk et al. (11). Eight hundred rmoles of CaGAP was passed through Dowex 50 (H+) to remove Ca++. The solution was chilled, adjusted to pH 6.6 with dilute NaOH, and lyophilieed. Five ml of THO, containing approximately 3 curies, and 4800 units of triose phosphate isomerase were added, and the mixture was incubated for 2 hours. Two-tenths ml of 6X HCl was added, the mixture was lyophilized, and the residual THO was recovered. One ml of 10% TCA was added to ensure TABLE YIELDS
IX THE
SYNTHESIS
AND
KATZ
inactivation of the enzyme. (At this stage there is a small amount of GAP-2-T present which will subsequently give rise to tritium in the 5 position of glucose. The GAP-2-T may be destroyed by Br2 oxidation at pH 8 or with glyceraldehyde phosphate dehydrogenase in the presence of arsenate. Alternatively, a large excess of unlabeled GAP in the aldolase condensation will reduce the activity in the 5 position to a low level.) The solution containing the tritiated trioses was put on a 25-ml AC;-1 (acetate) column, and the column was washed with water until the eluates contained little activity. The column was eluted with 0.05 N HCl, and the trioseB fractions were concentrated on a rotary vacuum evaporator. Fifteen hundred pmoles of unlabeled G4P, 50 pmoles of GSH, and 50 pmoles of EDTA were added and the pH was adjusted to 7 with dilute NaOH. Two hundred units of aldolase were added. After a 2-hour incubation, 500 pmoles FDP were formed as indicated by assay (6). The remaining procedure was the same as for glucose-4-T. The final yield in this experiment was 200 pmoles of glucose-3-T whose specific activity was approximately 5 PC per jmole. Table I gives the percentage yields in the two experiments described above for glucose-4-T and glucose-3-T. Final yields in these experiments were higher than in other experiments m-here selective precipitation methods were tried in lieu of column separations. DISCUSSION
Enzymic methods of synthesis involving the use of tritiated water have in common certain advantages and limitations (12). Their chief merit resides in the complete sterospecificit,y of enzymic mechanisms. All these methods have the limitaI
OF GI,u~osE-~-T
AND
GLUCOSE-~-~?
Glucose-4-T step
C0IllpiXlnd
o/oYield
Initial Tritiation
DHAP IjHAP
Isomerization Condensation Dephosphorylation Isomerization Separation Dephosphorylation
GAP FDP F6P G6P GGP Glucose
56 50 42 36 29 27
the amount
of initial
a In both experiments
Compound
y0 Yield
Initial Tritiation and Isomerization
G-41’ DHAP
100 75
Condensation Dephosphorylation Isomerization Separation Dephosphorylation
FDP F6P G6P GGP Glucose
62 50 40 32 25
step
100
64
triose
phosphate
was 800 pmoles.
SYNTHESIS
OF GLUCOSE-4-T
tion that. the specific activity of the replaceable hydrogens of the product cannot exceed that of the hydrogen of the water. However, t’he THO can be largely recovered since only a fraction of a per cent is incorporated into the product and used repeatedly in further experiments. It is likely that our procedure can be improved by using higher concentrations of triose phosphates (0.5-1.0 M) in THO. A large excess of enzyme or prolonged incubation t,imes would then be required to ensure equilibration with THO because of prodiscrimination.6 It nounced isotope should also be possible to increase specific activities by the use of tritiated water of higher isotope concentration. An advantage of the method is t’he intermediate production of specifically labeled hexose phosphates that may be of value in the study of individual enzymes and cell free systems. Kahn and Kohn (13) have recent’ly described an alternative procedure for the chemical synthesis of glucose-4-T. Di-Oisopropylidene-4-keto-n-glucose dimethyl acet*al was prepared and reduced with lithium aluminum t,ritide to yield a mixture of glucose-4-T and galactose-4-T. Through the use of glucose-3-T and glucose-4-T it has been possible to estimate quantit,at,ively the role of DPNH and TPNH in reductive biosynthesis in adipose tissue (14). It was shown that the incorporation of tritium into fatty acids was approximately twice as large from glucose-3-T as from glucose-l-T, and that with glucose3-T (but not with glucose-l-T) as substrate, there was a correlation between TPNH generation and CO2 production in the pentose cycle. This work indicates the marked advantage of glucose-3-T over glucose-l-T in following the fate of TPNH pJ in biological reduction. 6 The discrimination against tritium isomerization of G3P to DHAP is about (R. G. Kemp, unpublished observation).
in the 12-fold
AND
-3-T
375
ACKNOWLEDGMENT The authors wish to acknowledge the most valuable advice of Dr. I. A. Rose, who suggested these methods of synthesis. Note added in proof. An alternate procedure for the synthesis of glucose-3-T has recently been described by Gabrill and Ashwell [Federation Proc. 23, 380 (1964)l. They oxidized sucrose enzymatically to X-ketosucrose, and reduced that with NaBTy. From the reduced disaccharide glucose-3-T in a 50/, yield was obtained. REFERFNCES ii
I
1. ROSE, I. A., AND RIEDER, S. I-., J. Riol. Chem. 231, 315 (1958). 2. RIEDER, S. V., AND ROSE, I. A., J. Biol. Chem. 234, 1007 (1959). 3. KEMP, R. G., Doctoral dissertation, Yale University (1964). 4. CAHN, R. S., INGOLD, C. K., AS-D P’HELOG, V. Ezperientia 12, 81 (1956). 5. JACOBSON, H. J., GUPTA, G. N., FERNANDEZ, C., HENNIX, S., AND JENSEN, E. V., Arch. Biochem. Biophys. 86, 89 (1960). fi. BUCHER, T., AND HOHORST, M., in “Methods of Enzymatic Analysis” (M. Bergmeyer, ed.), p. 246. Academic Press, New York (1963). 7. HOHORST, M., in “Methods of Enzymatic Analysis” (M. Bergmeyer, ed.), p. 134. Academic Press, New York (1963). 8. SLEIX, M., in “Methods of Enzymatic Analysis” (M. Bergmeyer, ed.), p. 117. Academic Press, New York (1963). 9. KAHAFA, S. E., LOWRY, 0. H., SCHULZ, D. W., PASSONXEAU, J. V. AND CRAWFORD, E. J., J. Biol. Chem. 236, 2178 (1960). 10. KHYM, J. Y., AND COHN, W. E., .I. Anh. C’hem. sot. 75, 1153 (1953). II. S~EWCZUK, A., WOLNY, E., WOLNY, M., AND l’., ilcta Biochim. Polon 8, BARANOWSKI, 201 (1961). 12. GijNTHER, T., AND WENZEL, M., HoppeSeylers 8. Physiol. Chem. 333, 286 (1963). 13. KOHN, B., AND KOHN, P., J. Org. Chem. 23, 1037 (1963). 14. KATZ, J., RO~NSTAD, IL., AND KEMP, R. C., J. Biol. Chem. In press.
OF
BIOCHEMISTRY
Enzymic
Synthesis
ROBERT Institute
AND
for
BIOPHYSICS
of Glucose-4-Tritium
ROGNSTAD, Xedical
372-375
109,
ROBERT
Research,
and
G. KEMP,2
Cedam-Sinai
Received
(1965)
September
Hospitals,
Glucose-3-Tritium’
XKD
JOSEPH I
Los ilnyeles,
California
15, 1961
Dihydroxyacetone phosphate and n-glyceraldehyde phosphate were labeled with tritium in position 1 by enzymic exchange and isomerization in tritiated water. The labeled trioses were condensed by means of aldolase to fructose-l ,G-diphosphat,e labeled with tritium either in position 3 or 4. Labeled glucoses were prepared from the phosphate esters. The labeled sugars were obtained with a yield of 25’7 and specific activities of 5 PC per j4mole.
In an attempt to determine the source of the reductive hydrogen in fatty acid biosynthesis in adipose tissue, we undertook the synthesis of glucose-4-trit,ium (T), which yields DPNT in the glyceraldehydc phosphate dehydrogenase reaction, and glucose3-T, which yields TPNT in the phosphogluconic dehydrogenase reaction. These syntheses were based on the findings of Rose and Rieder (1, 2) on the sterospecific introduction of tritium on carbon-l of triose phosphates by means of t,he enzymes aldolase and triose phosphat’e isomerase in tritiated water. A synthesis of glucose-3-T based on these principles, but differing somewhat in procedure and yielding a sugar of lower specific activity, has previously been described (3). Principle. To synthesize glucosel-T, glyceraldehyde-3-phosphate-l-T (GAP-l-T) was prepared by incubating dihydroxyacetone-3-phosphate (DHAP) and aldolase in trit’iated water (Fig. IA), and then enzymically isomerizing the DHAP-1-T (5)4 to GAP-l-T in the presence of 2.5 M tris 1 Supported by a grant (AM 03682) to Joseph Katz from the U. S. Public Health Service. 2 Present address: Department of Biochemistry, University of Washington, Seattle, Washington. 3 Established Investigator, American Heart Association. 4 (8) and (n) refer to stereospecific position of tritium according to Cahn et al. (4).
buffer at pH 8.5 to shift, the equilibrium from the normal ratio of 95 : 3 DHAP-GAP to a ratio of approximately 95:.3 GAPDHAP.5 To synthesize glucose-3-T, DHAP-1-T (R)4 was prepared by incubating G-$P wit,h triose phosphate isomerase in tritiated water (Fig. 1B). GAP-l-T aud unlabeled DHAP, and DHAP-1-T (R) and unlabeled GAP, were condensed by means of aldolase to fructose1,6-diphosphate-4-T (FDP-4-T) and FDP3-T, respectively. The fructose diphosphates were hydrolyzed to fructose-6-phosphate (FBP), which was t,hen enzymically isomerized to glucose-A-phosphate (G61’), which in turn was dephosphorylated to glucose with acid phosphatase. EXPERIMENTAL Tritiated water (THO) was obtained from the New England Nuclear Corp., Boston, Massachusetts; DHAP dimethyl ketal and FDP were obtained from Sigma Chemical Co.. St. Louis, Missouri. All enzymes were obtained from Calbiochem, Los Angeles, California. Anion exchange resin (AG-1) was purchased from Bio-Rad Laboratories, Richmond, California and was converted to the acetate form. Tritium assay was carried out by the method of Jacobson (5) wit.h a Packard 5 The effect of high tris concentration and alkaline pH in reversing the normal GAP-DHAP equilibrium was found by Dr. 8. 1’. Rieder (personal communication to Dr. I. A. Rose).
372
SYNTHESIS
A. SYNTHESIS
OF GLUCOSE-4-T
T THO Aldolase
H-L
l=o
dH20P
:H20P
T,;~s;_“plsomerose
DHAP-I-
8. SYNTHESIS
OF DHAP -I -T ‘4 C
tritium
\C”’ H _ l _ OH
H I TriJsyp. ‘somerose
T-C-oH &O ;H20P
DHAP-I-
GAP of
T
GAP-I-T
T /S)
0
;H,OP
1. Incorporation
373
;H20P
DHAP
,, _ A-O,,
FIG. P-1-T.
2.5 M Tris
-OH
d=o
H
-3-T
OF GAP-l-T
H H-L-OH
ASD
in
T(R)
glyceraldehyde-3.P,l-T
scintillation count’er. Assay for triose phosphates and for FDP was performed by the method of Biicher and Hohorst (6). Hexose phosphates were determined by the method of Hohorst (7). Glucose assay was carried out by the method of Slein (8). Tritiation was performed in a 30-ml roundbottom flask connected by a Y-tube to a trap so that unused THO could be recovered by lyophilization. Special caut.ion should be exercised in the first steps because of the very high activity of the THO used. Synthesis oj” glucose-4-l’. DHAP was prepared from the ketal according to the manufacturers instructions. A solution containing 800 ~~moles of DHAP, 50 rmoles EDTA, and 50 rmoles GSH was chilled, adjusted to pH 7 with dilute NaOH, and lyophilized. It, n-as incubated for 2 hours with 5 ml of THO that, contained approximately 3 curies and 200 units (0.5 ml) of aldolase. The solution was lyophilized and the THO was recovered in a trap for use in later experiments. The enzyme was inactivated by adding 2 ml of 1096 TCA to the dry residue. The solution was passed through a 15-ml AG-1 (acetate) column which was then washed extensively with water to remove any residual THO and readily exchangeable tritium. The column was eluted with 0.1 LY HCl, and the DHAP-T fractions were reduced in volume on a rotary vacuum evaporator. Assay (6) at this point showed 510 pmoles of DHAP whose specific activity was the same as the tritiated water used in the incubation. Four Jf t,ris base was added to the DHAP-1-T solution until the pH was 8.5, and then s&i-
and dihydroxyacetone-
cient 4 IV tris buffer (pH 8.5) was added to bring the tris concentration to 2.5 M. Twenty-four hundred units of triose phosphate isomerase were added and the mixture was incubated for 30 minutes. Assay (6) showed 15 pmoles DHAP and 450 rmoles GAP. The solution was chilled and passed through a 20-ml Dowex 50 (H+) column to acidify the solution and inactivate the enzyme. To avoid reversing the triose phosphate equilibrium, this step was carried out in the cold room. The eluate was reduced in volume to about 10 ml on a rotary vacuum evaporator and then adjusted to pH 7 with dilute NaOH. Seven hundred pmoles of unlabeled DHAP and 100 units of aldolase were added. After a l-hour incubation, assay (6) showed 310 pmoles DHAP, 18 pmoles GAP, and 400 rmoles FDP. The mixture was heated to inactivate the enzyme and then passed through a small Dowex 50 (H+) column and on to a 20-ml AG-1 (acetate) column. (The passage through the cation column increases the efficiency of absorption by the acetate resin and thus permits the use of smaller columns.) Monophosphates were eluted with 0.08 llrl ammonium acetate (NH~Ac), pH 6.5, and FDP was subsequently eluted with0.1 h’HC1. The FDP fractions were concentrated until the HCl concentration was approximately 1 i\r, and were then incubated for 1 week at 37°C. Assay (6, 7) then showed 340 pmoles F6P and 5 pmoles FDP. The pH was adjusted to 7 with NaOH, 500 units of phosphohexose isomerase was added, and the mixture was incubated for 2 hours at 25°C and then overnight at 5°C (9). Assay (7) indicated
374
BOGNSTAD,
KEMP,
that 285 pmoles G6P and 55 rmoles FGP were present. The mixture was acidified at 5°C by passing it through a Dowex 50 (H+) column, boiled several minutes to inactivate the enzyme, and placed on a 20-ml AG-1 (acetate) column. GlucoseB-phosphate and FGP were separated by a slight variation of the method of Khym and Cohn (10). Inorganic phosphate was first eluted with 0.01 M borate and 0.08 M NHqAc, pH 8.5; GOP was next eluted with 0.01 Jf borate and 0.08 M NHdAc, pH 6.5; and FGP was then eluted with 0.5 &I NH(Ac, pH 6.5. The GGP fractions were put t)hrough a 20.ml Dowex 50 (H+) column to remove NH4+, and acetic acid was removed by evaporation. The solution was put on a 5ml A(:-1 (acetate) column, and boric acid was removed by washing with water. Glucose-S-phosphate was eluted with 0.1 N HCI, and the fractions were concentrated; assay (6) showed 230 pmoles G6P. The solution was adjusted to pH 5 with acetate buffer and incubated overnight with 10 mg of potato acid phosphatase. The enzyme was precipitated by adding trichloroacetic acid to a final concentration of 5?;, and the supernatant was put through a mixed bed Amberlite MB-3 column and concentrated. Final yield was 220 @moles glucose whose specific activity was approximately 5 PC per pmole. Synthesis of y/~cose-S-T. The calcium salt of GAP was prepared by the low-temperature periodate oxidation of FDP according to the method Szewczuk et al. (11). Eight hundred rmoles of CaGAP was passed through Dowex 50 (H+) to remove Ca++. The solution was chilled, adjusted to pH 6.6 with dilute NaOH, and lyophilieed. Five ml of THO, containing approximately 3 curies, and 4800 units of triose phosphate isomerase were added, and the mixture was incubated for 2 hours. Two-tenths ml of 6X HCl was added, the mixture was lyophilized, and the residual THO was recovered. One ml of 10% TCA was added to ensure TABLE YIELDS
IX THE
SYNTHESIS
AND
KATZ
inactivation of the enzyme. (At this stage there is a small amount of GAP-2-T present which will subsequently give rise to tritium in the 5 position of glucose. The GAP-2-T may be destroyed by Br2 oxidation at pH 8 or with glyceraldehyde phosphate dehydrogenase in the presence of arsenate. Alternatively, a large excess of unlabeled GAP in the aldolase condensation will reduce the activity in the 5 position to a low level.) The solution containing the tritiated trioses was put on a 25-ml AC;-1 (acetate) column, and the column was washed with water until the eluates contained little activity. The column was eluted with 0.05 N HCl, and the trioseB fractions were concentrated on a rotary vacuum evaporator. Fifteen hundred pmoles of unlabeled G4P, 50 pmoles of GSH, and 50 pmoles of EDTA were added and the pH was adjusted to 7 with dilute NaOH. Two hundred units of aldolase were added. After a 2-hour incubation, 500 pmoles FDP were formed as indicated by assay (6). The remaining procedure was the same as for glucose-4-T. The final yield in this experiment was 200 pmoles of glucose-3-T whose specific activity was approximately 5 PC per jmole. Table I gives the percentage yields in the two experiments described above for glucose-4-T and glucose-3-T. Final yields in these experiments were higher than in other experiments m-here selective precipitation methods were tried in lieu of column separations. DISCUSSION
Enzymic methods of synthesis involving the use of tritiated water have in common certain advantages and limitations (12). Their chief merit resides in the complete sterospecificit,y of enzymic mechanisms. All these methods have the limitaI
OF GI,u~osE-~-T
AND
GLUCOSE-~-~?
Glucose-4-T step
C0IllpiXlnd
o/oYield
Initial Tritiation
DHAP IjHAP
Isomerization Condensation Dephosphorylation Isomerization Separation Dephosphorylation
GAP FDP F6P G6P GGP Glucose
56 50 42 36 29 27
the amount
of initial
a In both experiments
Compound
y0 Yield
Initial Tritiation and Isomerization
G-41’ DHAP
100 75
Condensation Dephosphorylation Isomerization Separation Dephosphorylation
FDP F6P G6P GGP Glucose
62 50 40 32 25
step
100
64
triose
phosphate
was 800 pmoles.
SYNTHESIS
OF GLUCOSE-4-T
tion that. the specific activity of the replaceable hydrogens of the product cannot exceed that of the hydrogen of the water. However, t’he THO can be largely recovered since only a fraction of a per cent is incorporated into the product and used repeatedly in further experiments. It is likely that our procedure can be improved by using higher concentrations of triose phosphates (0.5-1.0 M) in THO. A large excess of enzyme or prolonged incubation t,imes would then be required to ensure equilibration with THO because of prodiscrimination.6 It nounced isotope should also be possible to increase specific activities by the use of tritiated water of higher isotope concentration. An advantage of the method is t’he intermediate production of specifically labeled hexose phosphates that may be of value in the study of individual enzymes and cell free systems. Kahn and Kohn (13) have recent’ly described an alternative procedure for the chemical synthesis of glucose-4-T. Di-Oisopropylidene-4-keto-n-glucose dimethyl acet*al was prepared and reduced with lithium aluminum t,ritide to yield a mixture of glucose-4-T and galactose-4-T. Through the use of glucose-3-T and glucose-4-T it has been possible to estimate quantit,at,ively the role of DPNH and TPNH in reductive biosynthesis in adipose tissue (14). It was shown that the incorporation of tritium into fatty acids was approximately twice as large from glucose-3-T as from glucose-l-T, and that with glucose3-T (but not with glucose-l-T) as substrate, there was a correlation between TPNH generation and CO2 production in the pentose cycle. This work indicates the marked advantage of glucose-3-T over glucose-l-T in following the fate of TPNH pJ in biological reduction. 6 The discrimination against tritium isomerization of G3P to DHAP is about (R. G. Kemp, unpublished observation).
in the 12-fold
AND
-3-T
375
ACKNOWLEDGMENT The authors wish to acknowledge the most valuable advice of Dr. I. A. Rose, who suggested these methods of synthesis. Note added in proof. An alternate procedure for the synthesis of glucose-3-T has recently been described by Gabrill and Ashwell [Federation Proc. 23, 380 (1964)l. They oxidized sucrose enzymatically to X-ketosucrose, and reduced that with NaBTy. From the reduced disaccharide glucose-3-T in a 50/, yield was obtained. REFERFNCES ii
I
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