ANALYTICAL
BIOCHEMISTRY
207,
go-93
Spectrophotometric of 2’,5’-Oligoadenylate Just
Justesenl
Department
Received
June
and Niels
of Molecular
(19%)
Pyrophosphate Synthetase
Ole Kjeldgaard
Biology, Aarhus
University,
DK-8000
.&hus
C, Denmark
15,1992
A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2’,5’-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The assay is at least as sensitive for measurements of 2’,5’-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH. o 1992 Academic Press.
Assay
Inc.
triphosphates as substrates, which, however, all are rather laborious and imprecise (2,3), as the 2’-5’ oligoadenylates are difficult to separate from ATP. The production of inorganic pyrophosphate offers the possibility to establish an assay system independent of radioactively labeled nucleotides. Using a modification of the method of Johnson et al. (4) for the determination Of PP; we designed a 2,5A synthetase assay adapted for determinations of multiple samples in an ELISA spectrophotometer. The method makes use of a coupled enzymatic reaction resulting in the formation of NADPH (Fig. 1) which can be measured spectrophotometrically at 340 nm or with even higher sensitivity as a fluorometric response (5). MATERIALS
AND
METHODS
Reagents
In vertebrate cells, interferons induce the doublestranded RNA (dsRNA)’ dependent enzyme 2’,5’-oligoadenylate synthetase (2,5A synthetase) catalyzing the reaction ATP + ATP + ppp5’A2’p5’A + PPi and the subsequent dissipative elongation reaction, with the predominance of n = 2 according to the general scheme
Enzymes, ATP, UDP-Glc, glucose 1,6-diphosphate, and NADP+ used in the assay were from BoehringerMannheim. The poly(I) - poly(C) was from Pharmacia (Uppsala, Sweden) and flat-bottom 96-well microtiter plates were from NUNC (Roskilde). Assay
(n + l)ATP
+ ppp5’A(2’p5’A),
+ nPPi.
ill
The 2,5A synthetase activity can be assayed by a number of different methods using radioactive nucleoside-
1 To whom correspondence should be addressed at: Department of Molecular Biology, Aarhus University, C. F. Mellers All;, Building 130,800O Arhus C, Denmark. Telefax: +45 86 19 65 00. * Abbreviations used: dsRNA, double-stranded RNA; 2,5A, 2’,5’oligoadenylate; PP,, inorganic pyrophosphate; poly(1) * poly(C), double-stranded polyinosine:polycytosine; HuIFNa, human interferon cu; TM, triethanolamine-acetate buffer mixture; AM, ATP mixture; EM, enzyme mixture; DTT, dithiothreitoh ELISA, enzyme-linked immunosorbent assay.
Solutions
The assay makes use of three triethanolamine reaction mixtures buffered at pH 7.6: The triethanolamineacetate buffer mixture (TM) contains 7.0 ml of 1 M triethanolamine, 245 ~1 glacial acetic acid, 2.0 ml of 1 M Mg acetate, 2.0 ml of 1 M K acetate, 400 ~1 of 500 mM EDTA, Na,, 200 ~1 of 1 M dithiothreitol (DDT), and 88 ml H,O. The ATP mixture (AM) contains 151 mg ATP, Na,, 3H,O, 8.75 ml H,O, 750 ~1 of 1 M triethanolamine, 500 ~1 of 1 M Mg acetate, and 40 ~1 of 500 mM EDTA, Na,. The enzyme mixture (EM) contains 3.00 ml TM, 6.50 ml H,O, 300 ~1 of 100 mM NADP+, 200 ~1of 100 mM UDP-Glc, 30 ~1 of 10 mM glucose 1,6diphosphate, 7.5 U UDP-Glc pyrophosphorylase (beef liver), 5 U phos-
90 Copyright 0 1992 All rights of reproduction
0003-2697/92 $5.00 by Academic Press, Inc. in any form reserved.
NONRADIOACTIVE
2’,5’-OLIGOADENYLATE 2-5A
91
ASSAY
synthetase
n+ I ATP UDP-Glc
ppp5’A(Z’p5’A)
n
+ n PPi
pyrophosphorylase
_c_)
PPi + UDP-Glc
SYNTHETASE
UTP
+
glucose-
1-phosphate
phosphoglucomutase
glucose-
1-phosphate
-
glucose-&phosphate
glucose-6-phosphate
+ NADP+
-C
glucose-6-phosphate dehydrogenase
NADPH
+ 6-phosphogluconolactone
FIG. 1. Reaction scheme for the mole to mole conversion of pyrophosphate to NADPH. The K,,, values for the different substrates are UDP-Glc Dvrophosphorvlase, 8 X IO-’ M for PP,; phosphoglucomutase, 6 X 10m5 M for glucose-l-phosphate and glucose-6-phosphate dehydrogenase and2 2 10-j M for glucose 6-phosphate.(i).
phoglucomutase (rabbit muscle), and 4 U glucose-6phosphate dehydrogenase (yeast). 2’,5’-Oligoadenylate Synthetase Assay The assay conditions are essentially analogous to those described by Justesen et al. (2) for measuring the synthesis of radioactive 2,5A oligomers from [(Y3ZP]ATP. The production of PP, can be measured as a multisample assay in an ELBA system or as a single-sample assay in a spectrophotometer. In the microtiter assay system each well contains 30 ~1AM, 100 ~1 EM, 150 ~1 TM, and 20-~1 samples of 2,5A synthetase, giving final concentrations of 50 mM triethanolamine, 12 mM K+, 11 mM Na+, 17 mM MS+, 1.2 mM DTT, 1.4 mM EDTA, 0.7 mM UDP-Glc, 10 PM glucose, 1,6-diphosphate, 1.0 mM NADP+, 2.5 mM ATP. For the standard assays, the TM also contains the dsRNA, poly(1). poly(C) at 1 mg/ml. All solutions are kept and mixed at 0°C. The microtiter plate is subsequently incubated at 37°C and the absorbance at 340 nm recorded in a multiwell scanner at intervals up to 120 min. When very low enzymatic activities are to be measured the 2,5A synthetase samples (20 ~1) are mixed with 30 ~1AM and 150 ~1TM in each microtiter well and incubated overnight at 37°C. The amounts of PPi formed are subsequently measured by the addition to each well of 100 ~1 EM. The absorbance values are measured at 340 nm in a microplate scanner after incubation at 37°C for 30 min.
recorded through an increase in absorbance at 340 nm (c = 6220). The NADPH is formed in a mole to mole ratio compared to PPi and the conversion is hardly limited by the auxiliary enzymes, since the K,,, values for all the relevant substrates of the different enzymes are around 50 PM (6). After addition of various amounts of PPi to the reaction mixture at 37”C, the enzymatic reaction is terminated within 180 s, showing an excellent proportionality between the amount of PPi added and the absorbance within a range of 10 to 200 PM PP, (Fig. 2). The enzyme activity can be measured with an increased sensitivity in a fluorometer using the excitation wavelength of 340 nm of NADPH and the emission wavelength of 460 nm (5). With the very simple and inexpensive
1.000
0.500
0.000 0
RESULTS Measurements
AND
DISCUSSlON of Pyrophosphate
The photometric pyrophosphate assay system is based on a multistep enzymatic coupling (Fig. 1) resulting in the reduction of NADP+ to NADPH, which can be
50
100
150
200 PM
FIG. 2. Different amounts of PP, were added to the assay system at 37°C and the absorbance at 340 nm was recorded. The figure shows the correlation between PP, concentrations and the absorbance at 240 s. The results are strictly in accordance with the theoretical value of A = 0.622 for a NADPH solution at a concentration of 100 WM.
92
JUSTESEN
0.600
AND
J
0
20
40
60
80
minutes FIG. 3. Dependence of the 2,5A synthetase reaction on dsRNA. The 42-kDa human 2,5A synthetase was mixed with the assay components (except for the dsRNA) and the absorbance at 340 nm recorded at regular short intervals. At 15 min poly(1). poly(C) was added to 1 pg/ml and at 60 min the poly(1) * poly(C) concentration was increased to 30 pg/ml.
Model TKO 100 DNA fluorometer (Hoefer Scientific Instruments, San Francisco, CA) normally used for quantitation of DNA-Hoechst 33258 complexes, the sensitivity of NADPH measurements is increased 5- to lo-fold over that of a spectrophotometer. 2’,-5’-Oligoadenylate
KJELDGAARD
column (Whatman) and fractionation on heparin-sepharose (Pharmacia). Figure 4 gives the results of a microtiter assay of 2,5A synthetase using different amounts of a heparin-Sepharose-purified enzyme fraction from IFNa-treated bovine kidney cells (MDBK). The figure shows that, below the maximum absorbance value, the proportionality between absorbance and the amounts of enzyme is relatively independent of the time of incubation at 37°C. We have confirmed that enzyme activities measured by the spectrophotometric method are in accordance with those determined by the formation of radioactive oligoadenylates (2). Using a heparin-sepharose fraction of an AMA cell extract we measured a 2,5A synthetase activity of 74 nmol min-’ ml-’ for the pyrophosphate assay and 71 nmol min-’ ml-’ for the radioactive oligoadenylate assay. We have observed that the assay is not functioning at pH values below 6; consequently the neutralization of the ATP in the AM is critical. Chloride ions are a noncompetitive inhibitor of UDP-Glc pyrophosphorylase (Ki = 7.3 mM (5)); consequently chlorides have been omitted from all assay solutions. Furthermore, the use of highly purified phosphoglucomutase requires the addition of small amounts of glucose 1,6-diphosphate to the reaction mixture acting as a cofactor for the enzyme. The assay is sensitive to the presence of both NADPH-oxidizing enzymes and pyrophosphatase activities (inorganic pyrophosphatase or glucose 6-phosphatase (9)). Determinations of 2,5A synthetase activities in crude extracts are therefore not possible with the
Synthetase
We have specifically used the PP, assay for measurements of the activity of the interferon-induced enzyme 2’,5’-oligoadenylate synthetase. Depending on the mammalian cell type, 2,5A synthetases with different molecular weights are induced (42,46,69, and 100 kDa) (7). These enzymes catalyze the polymerization of ATP into oligomers, having the unusual 2’,5’-phosphoester bond and liberating one PPi molecule for each 2’,5’ linkage formed. All 2,5A synthetases have a K,,, value for ATP of around 2 mM and require the presence of dsRNA to be active, although at various optimum concentrations. Figure 3 shows the increase in absorbance with time of incubation at 37°C using a human 2,5A synthetase. No dsRNA was present during the first 10 min of incubation, poly(1) . poly(C) was then added to 1 pg/ml, and after 30 min the concentration was increased to 30 pgl ml. The 2,5A synthetase used was a 42-kDa human enzyme expressed in Sf9 insect cells from a 1.6-kb cDNA fragment (8) subcloned through the pAcC4 transfer vector into a recombinant Autographa californica multiple-nuclear polyhedrosis virus (N. Din, J. Justesen, and N. Hoogenraad, unpublished results). The enzyme was partially purified by passage through a DE-52 cellulose
1 .ooo
0.000 0
20
40
FIG. 4. Measurements of various amounts of 2,5A synthetase from MDBK cells. A culture of MDBK cells was induced by HuIFNa for 24 h. The cells were washed and lysed by the addition of buffer (TM) with 1% NP-40 and the cell extract was applied to a column of DE-52 cellulose. The run-through solution was subsequently fractionated on a heparin-Sepharose column through elution by a gradient of potassium acetate, yielding two separate peaks of 2,5A syntbetase. Aliquots of the first peak (eluted at 0.6 M K acetate) were added to the reaction mixtures in a microtiter plate and the absorbance at 340 nm was recorded at different times: (0) 30 min, (+) 40 min. (W 60 min, and (0) 90 min.
NONRADIOACTIVE
2’,5’-OLIGOADENYLATE
present assay system unless some preliminary purification steps of the enzyme are instituted. As mentioned above, the sensitivity of the assay can be increased by taking advantage of the strong fluorescence of NADPH. We have routinely used the very simple Hoefer TKO 100 DNA fluorometer for single-sample measurements of 2,5A synthetase activity with good results; however, it should be pointed out that in our experience several spectrofluorometers and multiwell fluorescence readers have an optical design such that the potential advantage of the high sensitivity of NADPH fluorescence is lost. In the 1968 paper of Johnson et al. (4) the inorganic pyrophosphate determination was used as an assay for RNA polymerase. It is obvious that the multiwell assay presented in this paper can also be adapted for use as a convenient assay procedure for other enzymes yielding inorganic pyrophosphate such as DNA polymerase and reverse transcriptase.
SYNTHETASE
93
ASSAY
REFERENCES 1. Justesen, Acad.
Sci.
J., Ferbus, USA
D.,
and Thang,
M.
N. (1980)
Proc.
2. Justesen, J., Ferbus, D., and Thang, M. N. (1980) Nucleic Res. 8.3073-3085. 3. Johnston, M. I., and Torrence, P. F. (1984) in Interferon man,
R. M., Ed.),
Nutl.
77,4618-4622.
Vol.
3, pp. 189-298,
Eisevier,
Acids
(Fried-
Amsterdam.
4. Johnson, 5.
J. C., Shanoff, M., Bass, S. T., Boezi, J. A., and Hansen, R. G. (1968) Anal. Biochem. 26,137-145. Cartier, P. H., and Thuiller, L. (1974) Anal. Biochem. 61, 416-
428. 6. Barman,
T. E. (1969)
Enzyme
Handbook,
Springer-Verlag,
Ber-
lin. Chebath, J., Benech, P., Hovanessian, M. (1987) J. Biol. Chem. 262,3852-3857. Rysiecki, G., Gewert, feron Res. 9, 649-657.
D., and Williams,
A., Galabru,
J., and Revel,
B. R. G. (1989)
Nordlie, R. C. (1971) in The Enzymes (Bayer, pp. 543-610, Academic Press, New York.
P. D., Ed.),
J. Zntervol. IV,
BIOCHEMISTRY
207,
go-93
Spectrophotometric of 2’,5’-Oligoadenylate Just
Justesenl
Department
Received
June
and Niels
of Molecular
(19%)
Pyrophosphate Synthetase
Ole Kjeldgaard
Biology, Aarhus
University,
DK-8000
.&hus
C, Denmark
15,1992
A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2’,5’-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The assay is at least as sensitive for measurements of 2’,5’-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH. o 1992 Academic Press.
Assay
Inc.
triphosphates as substrates, which, however, all are rather laborious and imprecise (2,3), as the 2’-5’ oligoadenylates are difficult to separate from ATP. The production of inorganic pyrophosphate offers the possibility to establish an assay system independent of radioactively labeled nucleotides. Using a modification of the method of Johnson et al. (4) for the determination Of PP; we designed a 2,5A synthetase assay adapted for determinations of multiple samples in an ELISA spectrophotometer. The method makes use of a coupled enzymatic reaction resulting in the formation of NADPH (Fig. 1) which can be measured spectrophotometrically at 340 nm or with even higher sensitivity as a fluorometric response (5). MATERIALS
AND
METHODS
Reagents
In vertebrate cells, interferons induce the doublestranded RNA (dsRNA)’ dependent enzyme 2’,5’-oligoadenylate synthetase (2,5A synthetase) catalyzing the reaction ATP + ATP + ppp5’A2’p5’A + PPi and the subsequent dissipative elongation reaction, with the predominance of n = 2 according to the general scheme
Enzymes, ATP, UDP-Glc, glucose 1,6-diphosphate, and NADP+ used in the assay were from BoehringerMannheim. The poly(I) - poly(C) was from Pharmacia (Uppsala, Sweden) and flat-bottom 96-well microtiter plates were from NUNC (Roskilde). Assay
(n + l)ATP
+ ppp5’A(2’p5’A),
+ nPPi.
ill
The 2,5A synthetase activity can be assayed by a number of different methods using radioactive nucleoside-
1 To whom correspondence should be addressed at: Department of Molecular Biology, Aarhus University, C. F. Mellers All;, Building 130,800O Arhus C, Denmark. Telefax: +45 86 19 65 00. * Abbreviations used: dsRNA, double-stranded RNA; 2,5A, 2’,5’oligoadenylate; PP,, inorganic pyrophosphate; poly(1) * poly(C), double-stranded polyinosine:polycytosine; HuIFNa, human interferon cu; TM, triethanolamine-acetate buffer mixture; AM, ATP mixture; EM, enzyme mixture; DTT, dithiothreitoh ELISA, enzyme-linked immunosorbent assay.
Solutions
The assay makes use of three triethanolamine reaction mixtures buffered at pH 7.6: The triethanolamineacetate buffer mixture (TM) contains 7.0 ml of 1 M triethanolamine, 245 ~1 glacial acetic acid, 2.0 ml of 1 M Mg acetate, 2.0 ml of 1 M K acetate, 400 ~1 of 500 mM EDTA, Na,, 200 ~1 of 1 M dithiothreitol (DDT), and 88 ml H,O. The ATP mixture (AM) contains 151 mg ATP, Na,, 3H,O, 8.75 ml H,O, 750 ~1 of 1 M triethanolamine, 500 ~1 of 1 M Mg acetate, and 40 ~1 of 500 mM EDTA, Na,. The enzyme mixture (EM) contains 3.00 ml TM, 6.50 ml H,O, 300 ~1 of 100 mM NADP+, 200 ~1of 100 mM UDP-Glc, 30 ~1 of 10 mM glucose 1,6diphosphate, 7.5 U UDP-Glc pyrophosphorylase (beef liver), 5 U phos-
90 Copyright 0 1992 All rights of reproduction
0003-2697/92 $5.00 by Academic Press, Inc. in any form reserved.
NONRADIOACTIVE
2’,5’-OLIGOADENYLATE 2-5A
91
ASSAY
synthetase
n+ I ATP UDP-Glc
ppp5’A(Z’p5’A)
n
+ n PPi
pyrophosphorylase
_c_)
PPi + UDP-Glc
SYNTHETASE
UTP
+
glucose-
1-phosphate
phosphoglucomutase
glucose-
1-phosphate
-
glucose-&phosphate
glucose-6-phosphate
+ NADP+
-C
glucose-6-phosphate dehydrogenase
NADPH
+ 6-phosphogluconolactone
FIG. 1. Reaction scheme for the mole to mole conversion of pyrophosphate to NADPH. The K,,, values for the different substrates are UDP-Glc Dvrophosphorvlase, 8 X IO-’ M for PP,; phosphoglucomutase, 6 X 10m5 M for glucose-l-phosphate and glucose-6-phosphate dehydrogenase and2 2 10-j M for glucose 6-phosphate.(i).
phoglucomutase (rabbit muscle), and 4 U glucose-6phosphate dehydrogenase (yeast). 2’,5’-Oligoadenylate Synthetase Assay The assay conditions are essentially analogous to those described by Justesen et al. (2) for measuring the synthesis of radioactive 2,5A oligomers from [(Y3ZP]ATP. The production of PP, can be measured as a multisample assay in an ELBA system or as a single-sample assay in a spectrophotometer. In the microtiter assay system each well contains 30 ~1AM, 100 ~1 EM, 150 ~1 TM, and 20-~1 samples of 2,5A synthetase, giving final concentrations of 50 mM triethanolamine, 12 mM K+, 11 mM Na+, 17 mM MS+, 1.2 mM DTT, 1.4 mM EDTA, 0.7 mM UDP-Glc, 10 PM glucose, 1,6-diphosphate, 1.0 mM NADP+, 2.5 mM ATP. For the standard assays, the TM also contains the dsRNA, poly(1). poly(C) at 1 mg/ml. All solutions are kept and mixed at 0°C. The microtiter plate is subsequently incubated at 37°C and the absorbance at 340 nm recorded in a multiwell scanner at intervals up to 120 min. When very low enzymatic activities are to be measured the 2,5A synthetase samples (20 ~1) are mixed with 30 ~1AM and 150 ~1TM in each microtiter well and incubated overnight at 37°C. The amounts of PPi formed are subsequently measured by the addition to each well of 100 ~1 EM. The absorbance values are measured at 340 nm in a microplate scanner after incubation at 37°C for 30 min.
recorded through an increase in absorbance at 340 nm (c = 6220). The NADPH is formed in a mole to mole ratio compared to PPi and the conversion is hardly limited by the auxiliary enzymes, since the K,,, values for all the relevant substrates of the different enzymes are around 50 PM (6). After addition of various amounts of PPi to the reaction mixture at 37”C, the enzymatic reaction is terminated within 180 s, showing an excellent proportionality between the amount of PPi added and the absorbance within a range of 10 to 200 PM PP, (Fig. 2). The enzyme activity can be measured with an increased sensitivity in a fluorometer using the excitation wavelength of 340 nm of NADPH and the emission wavelength of 460 nm (5). With the very simple and inexpensive
1.000
0.500
0.000 0
RESULTS Measurements
AND
DISCUSSlON of Pyrophosphate
The photometric pyrophosphate assay system is based on a multistep enzymatic coupling (Fig. 1) resulting in the reduction of NADP+ to NADPH, which can be
50
100
150
200 PM
FIG. 2. Different amounts of PP, were added to the assay system at 37°C and the absorbance at 340 nm was recorded. The figure shows the correlation between PP, concentrations and the absorbance at 240 s. The results are strictly in accordance with the theoretical value of A = 0.622 for a NADPH solution at a concentration of 100 WM.
92
JUSTESEN
0.600
AND
J
0
20
40
60
80
minutes FIG. 3. Dependence of the 2,5A synthetase reaction on dsRNA. The 42-kDa human 2,5A synthetase was mixed with the assay components (except for the dsRNA) and the absorbance at 340 nm recorded at regular short intervals. At 15 min poly(1). poly(C) was added to 1 pg/ml and at 60 min the poly(1) * poly(C) concentration was increased to 30 pg/ml.
Model TKO 100 DNA fluorometer (Hoefer Scientific Instruments, San Francisco, CA) normally used for quantitation of DNA-Hoechst 33258 complexes, the sensitivity of NADPH measurements is increased 5- to lo-fold over that of a spectrophotometer. 2’,-5’-Oligoadenylate
KJELDGAARD
column (Whatman) and fractionation on heparin-sepharose (Pharmacia). Figure 4 gives the results of a microtiter assay of 2,5A synthetase using different amounts of a heparin-Sepharose-purified enzyme fraction from IFNa-treated bovine kidney cells (MDBK). The figure shows that, below the maximum absorbance value, the proportionality between absorbance and the amounts of enzyme is relatively independent of the time of incubation at 37°C. We have confirmed that enzyme activities measured by the spectrophotometric method are in accordance with those determined by the formation of radioactive oligoadenylates (2). Using a heparin-sepharose fraction of an AMA cell extract we measured a 2,5A synthetase activity of 74 nmol min-’ ml-’ for the pyrophosphate assay and 71 nmol min-’ ml-’ for the radioactive oligoadenylate assay. We have observed that the assay is not functioning at pH values below 6; consequently the neutralization of the ATP in the AM is critical. Chloride ions are a noncompetitive inhibitor of UDP-Glc pyrophosphorylase (Ki = 7.3 mM (5)); consequently chlorides have been omitted from all assay solutions. Furthermore, the use of highly purified phosphoglucomutase requires the addition of small amounts of glucose 1,6-diphosphate to the reaction mixture acting as a cofactor for the enzyme. The assay is sensitive to the presence of both NADPH-oxidizing enzymes and pyrophosphatase activities (inorganic pyrophosphatase or glucose 6-phosphatase (9)). Determinations of 2,5A synthetase activities in crude extracts are therefore not possible with the
Synthetase
We have specifically used the PP, assay for measurements of the activity of the interferon-induced enzyme 2’,5’-oligoadenylate synthetase. Depending on the mammalian cell type, 2,5A synthetases with different molecular weights are induced (42,46,69, and 100 kDa) (7). These enzymes catalyze the polymerization of ATP into oligomers, having the unusual 2’,5’-phosphoester bond and liberating one PPi molecule for each 2’,5’ linkage formed. All 2,5A synthetases have a K,,, value for ATP of around 2 mM and require the presence of dsRNA to be active, although at various optimum concentrations. Figure 3 shows the increase in absorbance with time of incubation at 37°C using a human 2,5A synthetase. No dsRNA was present during the first 10 min of incubation, poly(1) . poly(C) was then added to 1 pg/ml, and after 30 min the concentration was increased to 30 pgl ml. The 2,5A synthetase used was a 42-kDa human enzyme expressed in Sf9 insect cells from a 1.6-kb cDNA fragment (8) subcloned through the pAcC4 transfer vector into a recombinant Autographa californica multiple-nuclear polyhedrosis virus (N. Din, J. Justesen, and N. Hoogenraad, unpublished results). The enzyme was partially purified by passage through a DE-52 cellulose
1 .ooo
0.000 0
20
40
FIG. 4. Measurements of various amounts of 2,5A synthetase from MDBK cells. A culture of MDBK cells was induced by HuIFNa for 24 h. The cells were washed and lysed by the addition of buffer (TM) with 1% NP-40 and the cell extract was applied to a column of DE-52 cellulose. The run-through solution was subsequently fractionated on a heparin-Sepharose column through elution by a gradient of potassium acetate, yielding two separate peaks of 2,5A syntbetase. Aliquots of the first peak (eluted at 0.6 M K acetate) were added to the reaction mixtures in a microtiter plate and the absorbance at 340 nm was recorded at different times: (0) 30 min, (+) 40 min. (W 60 min, and (0) 90 min.
NONRADIOACTIVE
2’,5’-OLIGOADENYLATE
present assay system unless some preliminary purification steps of the enzyme are instituted. As mentioned above, the sensitivity of the assay can be increased by taking advantage of the strong fluorescence of NADPH. We have routinely used the very simple Hoefer TKO 100 DNA fluorometer for single-sample measurements of 2,5A synthetase activity with good results; however, it should be pointed out that in our experience several spectrofluorometers and multiwell fluorescence readers have an optical design such that the potential advantage of the high sensitivity of NADPH fluorescence is lost. In the 1968 paper of Johnson et al. (4) the inorganic pyrophosphate determination was used as an assay for RNA polymerase. It is obvious that the multiwell assay presented in this paper can also be adapted for use as a convenient assay procedure for other enzymes yielding inorganic pyrophosphate such as DNA polymerase and reverse transcriptase.
SYNTHETASE
93
ASSAY
REFERENCES 1. Justesen, Acad.
Sci.
J., Ferbus, USA
D.,
and Thang,
M.
N. (1980)
Proc.
2. Justesen, J., Ferbus, D., and Thang, M. N. (1980) Nucleic Res. 8.3073-3085. 3. Johnston, M. I., and Torrence, P. F. (1984) in Interferon man,
R. M., Ed.),
Nutl.
77,4618-4622.
Vol.
3, pp. 189-298,
Eisevier,
Acids
(Fried-
Amsterdam.
4. Johnson, 5.
J. C., Shanoff, M., Bass, S. T., Boezi, J. A., and Hansen, R. G. (1968) Anal. Biochem. 26,137-145. Cartier, P. H., and Thuiller, L. (1974) Anal. Biochem. 61, 416-
428. 6. Barman,
T. E. (1969)
Enzyme
Handbook,
Springer-Verlag,
Ber-
lin. Chebath, J., Benech, P., Hovanessian, M. (1987) J. Biol. Chem. 262,3852-3857. Rysiecki, G., Gewert, feron Res. 9, 649-657.
D., and Williams,
A., Galabru,
J., and Revel,
B. R. G. (1989)
Nordlie, R. C. (1971) in The Enzymes (Bayer, pp. 543-610, Academic Press, New York.
P. D., Ed.),
J. Zntervol. IV,
