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Synthesis of uridinediphospho-[6-3H]glucose with a cell-free extract of Phytophthora cinnamomi
ANALYTICAL
26, 412414
BIOCHEMISTRY
Synthesis
(1968)
of Uridinediphospho-
Cell-Free
Extract
M. C. WANG Department
[b3H]
of Phytophfhora AND
glucose
with
a
cinnamomil
S. BARTNICKI-GARCIA
of Plant Pathology, University Riverside, California 9%@?
Received April
of California,
15, 1968
Several pr.ocedures have been published for the enzymic synthesis of uridinediphospho- [‘“Cl glucose (l-5), but apparently there are no reports on the enzymic synthesis of tritiated UDPG. An attempt to prepare UDP- [6-3H] glucose using a partially purified yeast pyrophosphorylase, routinely employed in the synthesis of UDP- [14C]glucose, resulted in a total loss of tritium to the medium (6)) due perhaps to a spurious enzymic activity present in the pyrophosphorylase preparation. We have found that a crude pyrophosphorylase from the mycelium of the phytopathogenic fungus, Phytophthora cinnamomi, may be effectively employed for the synthesis of UDPG tritiated in the glucosyl moiety without labilization of the tritium atoms. The method involves the synthesis of UDP- [6-3H] glucose from [6JH] glucose-6-phosphate by a mixture of purified phosphoglucomutase (a-n-glucose-1,6-diphosphatc: a-n-glucose-l-phosphate phosphotransferase, EC 2.7.5.1) and a crude UDP-glucose pyrophosphorylase (UTP:cy-n-glucose-l-phosphate uridylyltransferase, EC2.7.7.9) from P. cinnamomi. The reaction was driven to completion in the direction of UDPG synthesis by the inclusion of purified inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1). METHOD
Cell-free extrack of P. cinnamomi were prepared from mycelial mats grown for 7 days in a glucose-asparagine liquid medium (7) under stationary conditions at 25°C. The mats were harvested on a coarse, sintered-glass filter and thoroughly rinsed with cold Tris-HCl buffer (0.1 M, pH 7.5). The, mycelium was frozen with liquid nitrogen, disrupted with a “Hughes” press, and resuspended in a solution containing 0.025 M EDTA, pH 7.0, and 0.025 M Tris-HCI buffer, pH 7.55, at 1°C. The supernatant ‘This
work was supported in part by a grant (AI-06205) United States Public Health Service. 412
stitutes of R&h,
from the National
In-
SPNTHESI~
0~
URIDIN~~DIPBOSPH~-[~-~H]GLUCOSE
413
after centrifugation at 110,000 g for 2 hr contained the UDPG pyrophosphorylase activity. This extract (50 ml) was dialyzed overnight at 1°C against 2 liters of 5 n-J4 Tris-HCI buffer, pH 7.55, containing 0.5 mM EDTA, pH 7.0. Pyrophosphorylase activity was determined by measuring the rate of formation of. UDPG. This was assayed spectrophotometrically (8) by oxidation with commercial (Sigma Chemical Co.) UDPG dehydrogenase (UDPglucose:NAD oxidoreductase, EC 1.1.1.22). The specific activity of the crude pyrophosphorylase ranged from 0.3 to 0.5 unit (pmoles UDPG/min/mg protein at 30°C). ]6-“H] Glucose-6-phosphate (250 &pmole) was obtained through the courtesy of Dr. E. A. Evans (Radiochemical Division of Nuclear-Chicago Corporation, Des Plaines, Illinois). UPD- [6-3H] glucose was synthesized in a reaction mixture containing: 160 mpmoles (40 PC) of [ 6-3H) glucose15 mpmoles of glucose 1,6-diphosphate (Calbiochem) , 6-phosphate; IO pmoles of cysteine, pH 7.5; 2 units of rabbit phosphoglucomutase (Calbiochem, 19.9 units/mg) ; 8 pmoles of UTP (Calbiochem) ; 32 pmoles of MgS04; 0.5 unit of P. cinnantomi UDPG pyrophosphorylase; 20 units of inorganic pyrophosphatase (Worthington Biochemical Corp., 1000 units/l.3 mg) ; 250 pmoles of Tris-HCl buffer, pH 7.8, in a total volume of 3.5 ml. The mixture was incubated at 30°C for 2 hr and then brought to pH 5.5 with 0.01 M acetic acid; insoluble residues were removed by centrifugation. The supernatant was adsorbed on a column of Dowex 1 (formate, 1.5 x 10 cm). UDP-[6-3H]glucose was eluted with a linear gradient of ammonium formate (O+ 1 M) at pH 5.0 and a total volume of 1 liter. The flow rate was 120 ml/hr; 10 ml fractions were collected, and their radioactivity determined in 15 ml of liquid scintillator (9) with a Packard Tri-Carb spectrometer. Only two radioactive peaks were detected, corresponding to unreacted [ 6-3H] glucose-6-phosphate and to UDP- [6-3H] glucose (95% yield). The eluate containing UDP- [6-3H] glucose was lyophilized to a small volume and desalted with a charcoal column (10). The tritiated product was eluted with 50% ethanol containing 0.05 M NH,OH. The charcoal eluate was rapidly adjusted to pH 6.8 with 0.01 M acetic acid and freeze-dried. The residue was dissolved in 5 ml of 5% ethanol; a trace of charcoal was eliminated by centrifugation. The tritiated UDPG was further purified by descending chromatography on Whatman 3 MM paper (prewashed with hot 1 M acetic acid) irrigated with ethanol/O.01 &f acetic acid (7/3, v/v). About 80-90% of the initial radioactivity wns recovered as tritiated UDPG. The identity of UDP- [6-“H] glucose was established by the following criteria : (A) Paper cochromatography with UDP- [*4C]glucose in the following solvent systems: 95% ethanol/l M ammonium acetate (7/3,
414
WANG
AND
BARTNIcXI-GARCIA
v/v, pH 3.8) ; 95% ethanol/l M ammonium acetate (7/3, v/v, pH 7.5) ; or 95% ethanol/O.01 M acetic acid (7/3, v/v), (B) Enzymic dehydrogenation with bovine liver UDPG dehydrogenase (Sigma Chemical Co., 200 units/mg), in the presence of NAD’ at 30°C for 3 hr (ll), released about 91% tritium to the medium. The rest of the radioactivity was found in [3H]NADH (1.4%) and UDP- [ 3H] glucuronic acid (7.6%). (C) UDP-[6-3H]glucose was an effective glucosyl donor for the biosynthesis of alkali-insoluble cell wall glucan of P. cinnamomi (12). SUMMARY
UDP- [6-3H] glucose (80-900/O yield) was enzymically synthesized from [ 6JH] glucose-6-phosphate utilizing commercially purified phosphoglucomutase, inorganic pyprophosphatase, and a crude UDPG pyrophosphorylase preparation from Phytophthora cinnamomi. REFERENCES 1. TRUCCO, E. R., Nature 174, 1103 (1954). 2. GLASER, L., J. Bid. Chem. 232,627 (1958). 3. ANDERSON, E. P., MAXWELL, E. S., AND BURTON, R. M., J. Am. Chem. Sot. 81, 6514 (1959). 4. WRIGHT, A., AND ROBBINS, P. W., Biochem. Biophys. Acta 104, 594 (1965). 5. FITZGERALD, D. K., AND EBNER, K. E., Anal. Biochem. 15, 150 (1966). 6. EVANS, E. A., personal communication. 7. BARTNICKI-GARCIA, S., J. Gen. Microbial. 42, 57 (1966). 8. STEELMAN, V. S., AND EBNER, K. E., Biochem. Biophys. Acta 128, 92 (1966). 9. BRAY, G. A., Anal. Biochem. 1,279 (1960). 10. LIN, T.-Y., AND HASSID, W. Z., J. Biol. Chem. 241,3283 (1966). 11. STROMINGER, J. L., MAXWELL, E. S., AXELROD, J., AND KALCKAR, H. M., J. Biol. Chem. 224, 79 (1957). 12. WANG, M. C., AND BARTNICKI-GARCIA, S., Biochem. Biophys. Research Commun. 24, 832 (1966).
26, 412414
BIOCHEMISTRY
Synthesis
(1968)
of Uridinediphospho-
Cell-Free
Extract
M. C. WANG Department
[b3H]
of Phytophfhora AND
glucose
with
a
cinnamomil
S. BARTNICKI-GARCIA
of Plant Pathology, University Riverside, California 9%@?
Received April
of California,
15, 1968
Several pr.ocedures have been published for the enzymic synthesis of uridinediphospho- [‘“Cl glucose (l-5), but apparently there are no reports on the enzymic synthesis of tritiated UDPG. An attempt to prepare UDP- [6-3H] glucose using a partially purified yeast pyrophosphorylase, routinely employed in the synthesis of UDP- [14C]glucose, resulted in a total loss of tritium to the medium (6)) due perhaps to a spurious enzymic activity present in the pyrophosphorylase preparation. We have found that a crude pyrophosphorylase from the mycelium of the phytopathogenic fungus, Phytophthora cinnamomi, may be effectively employed for the synthesis of UDPG tritiated in the glucosyl moiety without labilization of the tritium atoms. The method involves the synthesis of UDP- [6-3H] glucose from [6JH] glucose-6-phosphate by a mixture of purified phosphoglucomutase (a-n-glucose-1,6-diphosphatc: a-n-glucose-l-phosphate phosphotransferase, EC 2.7.5.1) and a crude UDP-glucose pyrophosphorylase (UTP:cy-n-glucose-l-phosphate uridylyltransferase, EC2.7.7.9) from P. cinnamomi. The reaction was driven to completion in the direction of UDPG synthesis by the inclusion of purified inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1). METHOD
Cell-free extrack of P. cinnamomi were prepared from mycelial mats grown for 7 days in a glucose-asparagine liquid medium (7) under stationary conditions at 25°C. The mats were harvested on a coarse, sintered-glass filter and thoroughly rinsed with cold Tris-HCl buffer (0.1 M, pH 7.5). The, mycelium was frozen with liquid nitrogen, disrupted with a “Hughes” press, and resuspended in a solution containing 0.025 M EDTA, pH 7.0, and 0.025 M Tris-HCI buffer, pH 7.55, at 1°C. The supernatant ‘This
work was supported in part by a grant (AI-06205) United States Public Health Service. 412
stitutes of R&h,
from the National
In-
SPNTHESI~
0~
URIDIN~~DIPBOSPH~-[~-~H]GLUCOSE
413
after centrifugation at 110,000 g for 2 hr contained the UDPG pyrophosphorylase activity. This extract (50 ml) was dialyzed overnight at 1°C against 2 liters of 5 n-J4 Tris-HCI buffer, pH 7.55, containing 0.5 mM EDTA, pH 7.0. Pyrophosphorylase activity was determined by measuring the rate of formation of. UDPG. This was assayed spectrophotometrically (8) by oxidation with commercial (Sigma Chemical Co.) UDPG dehydrogenase (UDPglucose:NAD oxidoreductase, EC 1.1.1.22). The specific activity of the crude pyrophosphorylase ranged from 0.3 to 0.5 unit (pmoles UDPG/min/mg protein at 30°C). ]6-“H] Glucose-6-phosphate (250 &pmole) was obtained through the courtesy of Dr. E. A. Evans (Radiochemical Division of Nuclear-Chicago Corporation, Des Plaines, Illinois). UPD- [6-3H] glucose was synthesized in a reaction mixture containing: 160 mpmoles (40 PC) of [ 6-3H) glucose15 mpmoles of glucose 1,6-diphosphate (Calbiochem) , 6-phosphate; IO pmoles of cysteine, pH 7.5; 2 units of rabbit phosphoglucomutase (Calbiochem, 19.9 units/mg) ; 8 pmoles of UTP (Calbiochem) ; 32 pmoles of MgS04; 0.5 unit of P. cinnantomi UDPG pyrophosphorylase; 20 units of inorganic pyrophosphatase (Worthington Biochemical Corp., 1000 units/l.3 mg) ; 250 pmoles of Tris-HCl buffer, pH 7.8, in a total volume of 3.5 ml. The mixture was incubated at 30°C for 2 hr and then brought to pH 5.5 with 0.01 M acetic acid; insoluble residues were removed by centrifugation. The supernatant was adsorbed on a column of Dowex 1 (formate, 1.5 x 10 cm). UDP-[6-3H]glucose was eluted with a linear gradient of ammonium formate (O+ 1 M) at pH 5.0 and a total volume of 1 liter. The flow rate was 120 ml/hr; 10 ml fractions were collected, and their radioactivity determined in 15 ml of liquid scintillator (9) with a Packard Tri-Carb spectrometer. Only two radioactive peaks were detected, corresponding to unreacted [ 6-3H] glucose-6-phosphate and to UDP- [6-3H] glucose (95% yield). The eluate containing UDP- [6-3H] glucose was lyophilized to a small volume and desalted with a charcoal column (10). The tritiated product was eluted with 50% ethanol containing 0.05 M NH,OH. The charcoal eluate was rapidly adjusted to pH 6.8 with 0.01 M acetic acid and freeze-dried. The residue was dissolved in 5 ml of 5% ethanol; a trace of charcoal was eliminated by centrifugation. The tritiated UDPG was further purified by descending chromatography on Whatman 3 MM paper (prewashed with hot 1 M acetic acid) irrigated with ethanol/O.01 &f acetic acid (7/3, v/v). About 80-90% of the initial radioactivity wns recovered as tritiated UDPG. The identity of UDP- [6-“H] glucose was established by the following criteria : (A) Paper cochromatography with UDP- [*4C]glucose in the following solvent systems: 95% ethanol/l M ammonium acetate (7/3,
414
WANG
AND
BARTNIcXI-GARCIA
v/v, pH 3.8) ; 95% ethanol/l M ammonium acetate (7/3, v/v, pH 7.5) ; or 95% ethanol/O.01 M acetic acid (7/3, v/v), (B) Enzymic dehydrogenation with bovine liver UDPG dehydrogenase (Sigma Chemical Co., 200 units/mg), in the presence of NAD’ at 30°C for 3 hr (ll), released about 91% tritium to the medium. The rest of the radioactivity was found in [3H]NADH (1.4%) and UDP- [ 3H] glucuronic acid (7.6%). (C) UDP-[6-3H]glucose was an effective glucosyl donor for the biosynthesis of alkali-insoluble cell wall glucan of P. cinnamomi (12). SUMMARY
UDP- [6-3H] glucose (80-900/O yield) was enzymically synthesized from [ 6JH] glucose-6-phosphate utilizing commercially purified phosphoglucomutase, inorganic pyprophosphatase, and a crude UDPG pyrophosphorylase preparation from Phytophthora cinnamomi. REFERENCES 1. TRUCCO, E. R., Nature 174, 1103 (1954). 2. GLASER, L., J. Bid. Chem. 232,627 (1958). 3. ANDERSON, E. P., MAXWELL, E. S., AND BURTON, R. M., J. Am. Chem. Sot. 81, 6514 (1959). 4. WRIGHT, A., AND ROBBINS, P. W., Biochem. Biophys. Acta 104, 594 (1965). 5. FITZGERALD, D. K., AND EBNER, K. E., Anal. Biochem. 15, 150 (1966). 6. EVANS, E. A., personal communication. 7. BARTNICKI-GARCIA, S., J. Gen. Microbial. 42, 57 (1966). 8. STEELMAN, V. S., AND EBNER, K. E., Biochem. Biophys. Acta 128, 92 (1966). 9. BRAY, G. A., Anal. Biochem. 1,279 (1960). 10. LIN, T.-Y., AND HASSID, W. Z., J. Biol. Chem. 241,3283 (1966). 11. STROMINGER, J. L., MAXWELL, E. S., AXELROD, J., AND KALCKAR, H. M., J. Biol. Chem. 224, 79 (1957). 12. WANG, M. C., AND BARTNICKI-GARCIA, S., Biochem. Biophys. Research Commun. 24, 832 (1966).