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A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range
ANALYTICAL
BIOCHEMISTRY
83,52-56
(1977)
A Rapid Quantitative Method for Measuring UTP, UDP, and UMP in the Picomole Rangel CHI P. CHEUNG Department
of Biochemistry, Philadelphia,
AND ROBERT
J. SUHADOLNIK
Temple University School Pennsylvania 19140
of Medicine,
Received January 17, 1977; accepted June 20, 1977 A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [Wlglucose lphosphate catalyzed by uridine 5’-diphosphoglucose pyrophosphorylase yielding UDP-[Wlglucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 M Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.
A direct method for the determination of uracil nucleotides is desirable for a study of the changes of these components in biological systems. Accurate UTP analysis is also required to determine the specific activity of RNA precursor pools during measurement of the rates of RNA synthesis and turnover. Enzymatic assay procedures have been described for the determination of uracil nucleotides (2-4). The first method is determined spectrophotometrically, and the sensitivity is limited to nanomole amounts (2). The second one is based on the dilution method with [3H]UTP and the separation of reaction products on a onedimensional PEI-cellulose thin-layer chromatogram (3). RNA polymerase is used in the method described in Ref. (4). However, the conditions required to determine UDP or UMP by RNA polymerase are not established. METHODS
Uracil nucleotides, glucose l-phosphate, Trizma Base, and enzymes were obtained from Sigma Chemical Co. [14C]Glucose l-phosphate was purchased from New England Nuclear Corp. Uridine 5’-diphosphoglucose pyrophosphorylase (yeast), inorganic pyrophosphatase (yeast), and nu* This research was supported by Grant No. PCM 75-01615 A01 from the National Science Foundation and Grant No. AI 12066 from the National Institutes of Health. 52 Copyright All rights
Q 1977 by Academic press, Inc. of reproduction in any form reserved.
ISSN 0003-2697
URACIL
NUCLEOTIDE
53
ASSAY
/ / ,
50 pm&s
FIG.
of
100 UTP
pmoles
510 of
UDP
1. Analysis of UTP. The assay is that described in Methods.
cleoside monophosphate kinase (beef liver) were dissolved in 20% glycerol, 20 mM KCl, 2 mM Tris-HCl (pH 7.6), 1 mM MgCl, and were maintained at 1°C while nucleoside monophosphate kinase was stored at -20°C. Trichloroacetic acid extracts (5%) of germinated wheat embryos were prepared as previously described (5). Assays UTP. A reaction mixture, in a volume of 100 ~1, containing 7 mM MgC&, 125 mM KCl, 88 mM Tris-HCl (pH 7.6), 0.1 pmol of CDP, 0.1 &i of [14C]glucose l-phosphate (specific activity, 222 mCi/mmol), 0.25 unit of uridine 5’-diphosphoglucose pyrophosphorylase, 1.25 units of inorganic pyrophosphatase, and between 25 and 100 pmol of UTP is incubated for 30 min at 37°C. The reaction is terminated by the addition of 1 ml of Trizma Base, and 0.2 ml of 2% charcoal (Merck) well suspended in water are added. The mixture is kept in ice, vortexed three times during a lo-min period, diluted with 5 ml of cold water, again kept in ice for 5 min, and finally filtered through glass fiber filters (Whatman GF/C). The filters are washed four times with 5 ml of cold water, dried, and counted in 10 ml of toluenebased scintillation fluid as previously described (1). UDP. The reaction mixture is identical to that described for the UTP assay, except for the omission of CDP and the addition of ATP (0.01 pmol) and 0.72 unit of nucleoside diphosphate kinase (bovine liver). The amount of UDP is calculated by subtracting the UTP content as determined in the UTP assay. UMP. The reaction mixture is that described for UDP with the addition
54
CHEUNG
AND SUHADOLNIK
,nl%
of UMP
100
FIG. 2. Analysis of UMP. The assay is that described in Methods.
of 0.03 unit of nucleoside monophosphate kinase to the initial reaction mixture. The level of UMP is calculated by subtracting the amounts of UDP and UTP as determined in the UDP assay. RESULTS AND DISCUSSION
The reaction catalyzed by uridine 5’-diphosphoglucose pyrophosphorylase is used to determine the content of UTP. The key step in this analysis is the selective adsorption of the reaction product UDP-[14C]glucose onto activated charcoal in the presence of 0.8 M Trizma Base. Figure 1 shows a standard curve for UTP, indicating a sensitivity of about 5 pmol. The assay is specific since the enzyme, uridine 5’-diphosphoglucose pyrophosphorylase, reacts only with uracil nucleotides 6). The commercially available pyrophosphorylase enzyme is contaminated with nucleoside diphosphate kinase and thus converts UDP to UTP in a biological extract which has endogenous ATP. In the described procedure this difficulty is solved by the addition of excess CDP (0.1 pmol). This suppresses the phosphorylation of UDP by removing endogenous ATP. UDP is assayed by its conversion to UTP in an incubation with excess ATP and nucleoside diphosphate kinase. A basic requirement of the assay is that UDP be completely converted to UTP. Since nucleoside diphosphate kinase is not specific for UDP, other nucleoside diphosphates can be converted to their corresponding nucleoside 5’-triphosphates. However, this does not affect the specificity of the assay for UDP since only UTP is active in the reaction with glucose l-phosphate.
URACIL NUCLEOTIDE TABLE SPECIFICITY
OF THE
URACIL
55
ASSAY
I NUCLEOTIDE
ASSAY”
14Cadsorbed onto charcoal (cpm) Nucleotide (100 pmol each) UTP UDP UMP dUTP ATP + UDP CTP GTP
UTP assay
UDP assay
UMP assay
497 13,750
1,020 16,030 16,000 1,060
1,360 16,150 16,540 17,970
1,330 803 510 512
a The assays are as described in Methods.
The assay for UMP is shown in Fig. 2. The enzyme, nucleoside monophosphate kinase, used in these determinations is unstable and must be stored at -20°C in 20% glycerol buffer. Table 1 demonstrates the specificity of the uracil nucleotide analyses. UTP is the only substrate active in the reaction catalyzed by UDPG-pyrophosphorylase. In the UDP assay, UDP is converted quantitatively to UTP, and UMP does not interfere. Similarly, in the UMP assay, quantitative conversion to UTP is obtained. The application of the procedure to the analysis of trichloroacetic acid extracts of germinated wheat embryos in shown in Table 2. When crude TABLE ANALYSIS
Crude extract a (PI)
2
OF URACIL NUCLEOTIDES OF CRUDE EXTRACT EMBRYOS AFTER 40 MIN OF GERMINATIONS
Nucleotide added (25 pmol)
-
-
2 2 2 2 -
UTP UTP UDP UDP UMP UMP
UTP assay cm 497 1960 5230 3630
Recovery
FROM
WHEAT
UDP assay cm
Recovery
1020 2350
UMP assay cm
Recovery
1360 3420
(104%) 5970 4730
(96%) 7430 5620
(94%)
a The assays are as described in Methods. * Wheat embryos, 125 mg, germinated for 40 min were extracted with 4.5 ml of 5% cold TCA. After ether extraction and neutralization with 1 M Trizma Base, an aliquot was assayed as shown.
56
CHEUNG
AND SUHADOLNIK
biological extracts are measured for the content of uracil nucleotide, an internal standard is always added. This precaution avoids any change in the specific activity of [14C]glucose l-phosphate due to different concentrations of endogenous glucose l-phosphate present in extracts. The present paper describes a condition in which such a precaution could be avoided. The amount of [14C]glucose l-phosphate used in the assay is in large excess, as compared with the endogenous level of the crude extract from isolated wheat embryos. ACKNOWLEDGMENT The authors wish to thank Dr. Abraham Marcus for his valuable assistance and advice in the preparation of this manuscript.
REFERENCES 1. Cheung, C. P., and Marcus, A. (1975)AnaZ. Biochem. 69, 131-139. 2. Kalckar, H. M., and Anderson, E. P. (1957)Zn Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, p. 975, Academic Press, New York. 3. Wiegeos, U., Kalaproth, K., and Hilz, H. (1974) FEES Letr. 47, 307-313. 4. Maxson, R. E., Jr., and Wu, R. S. (1976) Eur. J. Biochem. 62, 551-554. 5. Cheung, C. P., and Marcus, A. (1976) FEBS Left. 70, 141-144. 6. Keppler, D., Rudigier, J., and Decker, K. (1970) Anal. Biochem. 38, 105-114.
BIOCHEMISTRY
83,52-56
(1977)
A Rapid Quantitative Method for Measuring UTP, UDP, and UMP in the Picomole Rangel CHI P. CHEUNG Department
of Biochemistry, Philadelphia,
AND ROBERT
J. SUHADOLNIK
Temple University School Pennsylvania 19140
of Medicine,
Received January 17, 1977; accepted June 20, 1977 A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [Wlglucose lphosphate catalyzed by uridine 5’-diphosphoglucose pyrophosphorylase yielding UDP-[Wlglucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 M Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.
A direct method for the determination of uracil nucleotides is desirable for a study of the changes of these components in biological systems. Accurate UTP analysis is also required to determine the specific activity of RNA precursor pools during measurement of the rates of RNA synthesis and turnover. Enzymatic assay procedures have been described for the determination of uracil nucleotides (2-4). The first method is determined spectrophotometrically, and the sensitivity is limited to nanomole amounts (2). The second one is based on the dilution method with [3H]UTP and the separation of reaction products on a onedimensional PEI-cellulose thin-layer chromatogram (3). RNA polymerase is used in the method described in Ref. (4). However, the conditions required to determine UDP or UMP by RNA polymerase are not established. METHODS
Uracil nucleotides, glucose l-phosphate, Trizma Base, and enzymes were obtained from Sigma Chemical Co. [14C]Glucose l-phosphate was purchased from New England Nuclear Corp. Uridine 5’-diphosphoglucose pyrophosphorylase (yeast), inorganic pyrophosphatase (yeast), and nu* This research was supported by Grant No. PCM 75-01615 A01 from the National Science Foundation and Grant No. AI 12066 from the National Institutes of Health. 52 Copyright All rights
Q 1977 by Academic press, Inc. of reproduction in any form reserved.
ISSN 0003-2697
URACIL
NUCLEOTIDE
53
ASSAY
/ / ,
50 pm&s
FIG.
of
100 UTP
pmoles
510 of
UDP
1. Analysis of UTP. The assay is that described in Methods.
cleoside monophosphate kinase (beef liver) were dissolved in 20% glycerol, 20 mM KCl, 2 mM Tris-HCl (pH 7.6), 1 mM MgCl, and were maintained at 1°C while nucleoside monophosphate kinase was stored at -20°C. Trichloroacetic acid extracts (5%) of germinated wheat embryos were prepared as previously described (5). Assays UTP. A reaction mixture, in a volume of 100 ~1, containing 7 mM MgC&, 125 mM KCl, 88 mM Tris-HCl (pH 7.6), 0.1 pmol of CDP, 0.1 &i of [14C]glucose l-phosphate (specific activity, 222 mCi/mmol), 0.25 unit of uridine 5’-diphosphoglucose pyrophosphorylase, 1.25 units of inorganic pyrophosphatase, and between 25 and 100 pmol of UTP is incubated for 30 min at 37°C. The reaction is terminated by the addition of 1 ml of Trizma Base, and 0.2 ml of 2% charcoal (Merck) well suspended in water are added. The mixture is kept in ice, vortexed three times during a lo-min period, diluted with 5 ml of cold water, again kept in ice for 5 min, and finally filtered through glass fiber filters (Whatman GF/C). The filters are washed four times with 5 ml of cold water, dried, and counted in 10 ml of toluenebased scintillation fluid as previously described (1). UDP. The reaction mixture is identical to that described for the UTP assay, except for the omission of CDP and the addition of ATP (0.01 pmol) and 0.72 unit of nucleoside diphosphate kinase (bovine liver). The amount of UDP is calculated by subtracting the UTP content as determined in the UTP assay. UMP. The reaction mixture is that described for UDP with the addition
54
CHEUNG
AND SUHADOLNIK
,nl%
of UMP
100
FIG. 2. Analysis of UMP. The assay is that described in Methods.
of 0.03 unit of nucleoside monophosphate kinase to the initial reaction mixture. The level of UMP is calculated by subtracting the amounts of UDP and UTP as determined in the UDP assay. RESULTS AND DISCUSSION
The reaction catalyzed by uridine 5’-diphosphoglucose pyrophosphorylase is used to determine the content of UTP. The key step in this analysis is the selective adsorption of the reaction product UDP-[14C]glucose onto activated charcoal in the presence of 0.8 M Trizma Base. Figure 1 shows a standard curve for UTP, indicating a sensitivity of about 5 pmol. The assay is specific since the enzyme, uridine 5’-diphosphoglucose pyrophosphorylase, reacts only with uracil nucleotides 6). The commercially available pyrophosphorylase enzyme is contaminated with nucleoside diphosphate kinase and thus converts UDP to UTP in a biological extract which has endogenous ATP. In the described procedure this difficulty is solved by the addition of excess CDP (0.1 pmol). This suppresses the phosphorylation of UDP by removing endogenous ATP. UDP is assayed by its conversion to UTP in an incubation with excess ATP and nucleoside diphosphate kinase. A basic requirement of the assay is that UDP be completely converted to UTP. Since nucleoside diphosphate kinase is not specific for UDP, other nucleoside diphosphates can be converted to their corresponding nucleoside 5’-triphosphates. However, this does not affect the specificity of the assay for UDP since only UTP is active in the reaction with glucose l-phosphate.
URACIL NUCLEOTIDE TABLE SPECIFICITY
OF THE
URACIL
55
ASSAY
I NUCLEOTIDE
ASSAY”
14Cadsorbed onto charcoal (cpm) Nucleotide (100 pmol each) UTP UDP UMP dUTP ATP + UDP CTP GTP
UTP assay
UDP assay
UMP assay
497 13,750
1,020 16,030 16,000 1,060
1,360 16,150 16,540 17,970
1,330 803 510 512
a The assays are as described in Methods.
The assay for UMP is shown in Fig. 2. The enzyme, nucleoside monophosphate kinase, used in these determinations is unstable and must be stored at -20°C in 20% glycerol buffer. Table 1 demonstrates the specificity of the uracil nucleotide analyses. UTP is the only substrate active in the reaction catalyzed by UDPG-pyrophosphorylase. In the UDP assay, UDP is converted quantitatively to UTP, and UMP does not interfere. Similarly, in the UMP assay, quantitative conversion to UTP is obtained. The application of the procedure to the analysis of trichloroacetic acid extracts of germinated wheat embryos in shown in Table 2. When crude TABLE ANALYSIS
Crude extract a (PI)
2
OF URACIL NUCLEOTIDES OF CRUDE EXTRACT EMBRYOS AFTER 40 MIN OF GERMINATIONS
Nucleotide added (25 pmol)
-
-
2 2 2 2 -
UTP UTP UDP UDP UMP UMP
UTP assay cm 497 1960 5230 3630
Recovery
FROM
WHEAT
UDP assay cm
Recovery
1020 2350
UMP assay cm
Recovery
1360 3420
(104%) 5970 4730
(96%) 7430 5620
(94%)
a The assays are as described in Methods. * Wheat embryos, 125 mg, germinated for 40 min were extracted with 4.5 ml of 5% cold TCA. After ether extraction and neutralization with 1 M Trizma Base, an aliquot was assayed as shown.
56
CHEUNG
AND SUHADOLNIK
biological extracts are measured for the content of uracil nucleotide, an internal standard is always added. This precaution avoids any change in the specific activity of [14C]glucose l-phosphate due to different concentrations of endogenous glucose l-phosphate present in extracts. The present paper describes a condition in which such a precaution could be avoided. The amount of [14C]glucose l-phosphate used in the assay is in large excess, as compared with the endogenous level of the crude extract from isolated wheat embryos. ACKNOWLEDGMENT The authors wish to thank Dr. Abraham Marcus for his valuable assistance and advice in the preparation of this manuscript.
REFERENCES 1. Cheung, C. P., and Marcus, A. (1975)AnaZ. Biochem. 69, 131-139. 2. Kalckar, H. M., and Anderson, E. P. (1957)Zn Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, p. 975, Academic Press, New York. 3. Wiegeos, U., Kalaproth, K., and Hilz, H. (1974) FEES Letr. 47, 307-313. 4. Maxson, R. E., Jr., and Wu, R. S. (1976) Eur. J. Biochem. 62, 551-554. 5. Cheung, C. P., and Marcus, A. (1976) FEBS Left. 70, 141-144. 6. Keppler, D., Rudigier, J., and Decker, K. (1970) Anal. Biochem. 38, 105-114.