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Simple assay of 0.1–1.0 pmol of ATP, ADP, and AMP in single somatic cells using purified luciferin luciferase
ANALYTICAL
BIOCHEMISTRY
Simple
113,172-l
78 (198 1)
Assay of 0.1-l .O pmol of ATP, ADP, and AMP in Single Somatic Cells Using Purified Luciferin Luciferasel
HORST SPIELMANN,URSULAJACOB-MUELLER, AND PETRA SCHULZ Institut
fiir
Toxikologie und Embryonul-Pharmakologie. Garystr. 9, D-3000 Berlin 33, Federal
Republic
Freie Unir~ersitiit of Germany
Berlin,
Received December 19, 1980 The sensitivity of ATP determinations with crude firefly luciferin luciferase is limited by contaminating ATP converting enzymes, which cause a rapid decrease of the ATP level during the assay. Purified luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration. Sensitivity and specificity of the ATP assay are, therefore, considerably increased when purified enzyme is used instead of crude extracts of the enzyme. ATP, 0.1 - 1.O pmol as well as higher amounts can be determined with commercial preparations of purified and stabilized luciferase. In ADP and AMP measurements with the luciferase assay, problems are arising from the enzymes required for the conversion to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides. Exclusion of contaminated enzymes and removal of ammonium sulfate from adenylate kinase were the only prerequisites for determinations of 0. 1- 1.O pmol of ADP and AMP with purified luciferase. The application of the assay in determinations of ATP, ADP, and AMP in single preimplantation mouse embryos is described.
One promising approach to study the energetic state of cells is to determine the concentrations and ratios of the adenine ribonucleotides (ATP, ADP, and AMP), which are involved in stoichiometric energy transduction between most metabolic reactions. Atkinson (1) proposed that the regulation of cellular energy metabolism may be related to the adenylate energy charge defined as ((ATP) + O.S(ADP)/(ATP) + (ADP) + (AMP)), whereby the adenylate energy charge is a measure of the metabolically available energy stored in the adenine ribonucleotides. Investigations on the energy metabolism of mouse embryos homozygous for the t12 mutation, which die prior to implantation, have shown considerable
changes in the ATP and ADP contents of litters of preimplantation embryos containing varying amounts of lethal t12/t12 embryos (2). Such determinations could not be performed on single lethal and normal preimplantation mouse embryos, since the firefly luciferin luciferase assay for ATP and ADP was lacking sensitivity. For the same reason investigations on AMP content and on the adenylate energy charge have not yet been carried out on preimplantation mouse embryos, which contain l-72 cells between fertilization and implantation (3).
The luciferase reaction is highly specific and no other ribonucleotide triphosphate may substitute for ATP (4). Unpurified preparations of luciferase, however, which have generally been used in ATP determinations, contain ATP-converting enzymes that cause a rapid decrease of the ATP level during the assay (5). The sensitivity of ATP
r This investigation was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29 (Embryonale Entwicklung und Differenzierung). We are indebted to Drs. A. Lundin and A. Thore (Sundbyberg, Sweden) for their helpful criticism. 0003-269718 l/O701 72-07S02.00/0 Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
measurements with crude enzyme extracts 172
SENSITIVE
ASSAY FOR ATP, ADP, AND AMP
is particularly limited by the difficulties of reproducibly monitoring the changing light production of the reaction. Purified firefly luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration (5). Sensitivity and specificity of the ATP assay could, therefore, be considerably increased when purified luciferase was used instead of crude enzyme extracts (5,6). Only recently have purified and stabilized firefly luciferase and also photometers specially designed for ATP analyses become commercially available. We used standardized, purified luciferin reagents to determine the absolute concentrations of intracellular ATP, ADP, and AMP in preimplantation mouse embryos at the O.l- to l.O-pmol range. ADP and AMP were enzymatically converted to ATP, which was then assayed by the purified luciferase. Although detailed determinations in bacterial cell extracts have been published before (7,8), several modifications were necessary in order to measure low levels of these metabolites in somatic cells. Problems are particularly arising from the enzymes required for the conversion of ADP and AMP to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides, which can be neglected when determinations are performed in the picomole and nanomole range. The present report describes the simple assay of ATP, ADP, and AMP in somatic cells at the O.l- to l.O-pmol range using the purified firefly luciferase system. MATERIALS
AND METHODS
Reagents. Purified firefly luciferin luciferase was purchased as “ATP Monitoring Reagent” from LKB Instruments (Grgfelfing, Germany). This reagent is a mixture of firefly luciferase and luciferin in magnesium acetate supplemented with bovine serum albumin. Each vial of the reagent was filled up with 10 ml distilled water. This dilution allows the assay of 100 samples using a re-
173
agent volume of 0.1 ml per assay sample and a total reaction volume of 0.5 ml. The reconstituted reagent may be stored at -20°C for several weeks. PKZ (EC 2.7.1.40) from rabbit muscle (type II, 350 to 500 units/mg protein) and AK (EC 2.7.4.3) from pig muscle (grade V, 1000 to 1500 unitslmg protein) were obtained from Sigma GmbH (Munich, Germany). Ammonium sulfate has to be removed from the AK solution, since it inhibits the conversion of AMP to ATP. Purification of AK was achieved by centrifugation of 50 or 100 ~1 of the ammonium sulfate solution for 10 min at 12,OOOg in a Beckman airfuge (Beckman Instruments, Munich, Germany), removal of the ammonium sulfate supernatant and solubilization of the AK crystals in an appropriate volume of Tris-EDTA buffer (0.1 M, pH 7.75). PEP, ATP, ADP, and AMP were obtained from Boehringer (Mannheim, Germany). Standard solutions of the three adenine ribonucleotides were made up with Tris-EDTA buffer (0.1 M, pH 7.75). ATP, ADP, and AMP standard solutions were kept frozen in aliquots at - 20°C for several months. Nucleotide releasing reagent for somatic cells (NRS, Lumac Systems, Basle, Switzerland) diluted 1: 1 in Tris-EDTA buffer as suggested by the manufacturer, was used in a few experiments to extract adenine ribonucleotides from mouse embryos. All other chemicals were of analytical grade and purchased from Merck AG (Darmstadt, Germany). Deionized water from a Milli-QSystem (Millipore GmbH, Neu Isenburg, Germany) was used to make up solutions in all experiments. The assay buffer was 0.1 M Tris-EDTA buffer, pH 7.75. This Tris-EDTA buffer was used for all sample preparations and dilutions and for the extraction of biological material. 2 Abbreviations
used: PK, pyruvate kinase; AK, ad-
enylate kinase; PEP, phosphoenolpyruvate.
174
SPIELMANN,
JACOB-MtJLLER,
Equipment. To obtain maximum sensitivity, a specially designed ATP photometer was used, the LKB-Wallac Luminometer 1250 and LKB potentiometric recorder 2210-032 (LKB Instruments, Grafelfing, Germany). Measurements were performed in disposable polystyrol cuvettes from the same manufacturer. Eppendorf micro test tubes (volume 1.5 ml, Eppendorf GmbH, Hamburg, Germany) were used for the incubation of tissue extracts and adenine ribonucleotide standards with PK and AK. Incubation and centrifugation of the micro-test tubes were performed in a thermostat and in a centrifuge from the same company (Eppendorf centrifuge 5412; Eppendorf thermostat 5320). Microliter pipettes with disposable plastic tips were also purchased from the Eppendorf GmbH. These pipettes were used in all determinations. However, preimplantation mouse embryos were taken up and transferred into capillary pipets, calibrated for 10 ~1, which had been drawn from Pasteur pipets. Tissue samples. Preimplantation embryos were flushed from the uterine tubes or uteri of pregnant mice with phosphatebuffered saline according to Whittingham and Wales (9) at appropriate times as described previously (3). One-cell embryos or blastocysts were immediately taken up singly in 10 ~1 of Tris-EDTA buffer, transferred into a micro-test tube, and kept frozen in liquid nitrogen. Assay of ATP, ADP, and AMP. To reduce background luminescence of the ADP and AMP converting enzymes and to obtain the highest sensitivity at the same time, tissue samples or standards were incubated in the smallest but still easy-to-handle volume. Therefore, 10 ~1 of tissue extract or standard adenine ribonucleotide solution was incubated with 10 ~1 of the appropriate enzyme solution in an Eppendorf microtest tube for 30 min, and the ATP content was measured after dilution to 400 ~1.
AND SCHULZ
To determine the three adenine ribonucleotides in somatic cells by the luciferase method, at least three measurements have to be performed on three different samples by endpoint determination of the ATP content: one sample without any ATP generating enzyme (for ATP), another sample containing PK (for ATP + ADP), and the third sample containing both PK and AK (for ATP + ADP + AMP). The amount of ADP is obtained by subtracting the ATP value from the (ATP + ADP) value and the amount of AMP is represented by the difference between the (ATP + ADP + AMP) and the (ATP + ADP) content. In somatic cells each of the three adenine ribonucleotides has to be determined in the presence of the two others. Standard curves have, therefore, to be measured under identical complex conditions. To minimize the effects of ionic interference (8), standard solutions of ATP, ADP, and AMP were prepared in an ionic medium identical to that of the samples. Routinely determinations of the three adenine ribonucleotides were carried out using the following procedure. Ten microliters of Tris-EDTA buffer as blank or 10 ~1 of buffer containing a mixture of equal amounts of ATP, ADP, and AMP, or 10 ~1 of the same buffer containing a single preimplantation mouse embryo is transferred into a micro-test tube and three times rapidly frozen and thawed in liquid nitrogen. Tris-EDTA buffer containing phosphoenolpyruvate (PEP), MgC& , and KC1 at concentrations suggested by earlier investigators (5-8) is used for the conversion of ADP and AMP to ATP. Optimum conditions were achieved by addition of 10 ,ul of reaction buffer (PEP-buffer: 0.3 mrvr PEP, 9 mM MgCl,, 5 mM KC1 in 0.1 M TrisEDTA buffer, pH 7.75) to 10 ~1 of sample, blank or standard. For the determination of ATP + ADP, 10 ~1 PEP-buffer containing PK (20 units/ml) was added and for the measurement of ATP + ADP + AMP, 10
SENSITIVE
175
ASSAY FOR ATP, ADP, AND AMP
b,o,“ml”esce”ce (units) 90.
.* 071 -1 pmol
ATP
80. ,.: (..”
70.
.:’
60. 50. .:’ ,..’“,*’ y” , .:’ ,’ . ..’“, ,*’ ,..’ , ..‘ ,’ . ...‘. *’ ...‘. ,
4030-
20. ,o.
..+ ...‘. ,..‘, ’ .I: I 6
1
,:’
.:’ ,I
,*
,..’
(..’
.:’ ,’
.“I
,/” *, , ,’ * - 10 pm,,
ATP
;.’ *.: ”,, 9,’ ,y’ ,* .’ *, ;.’ ,:’ ,,’ .:’ ,’ ” *, ,’
,,;, .:; ..“,a? z ..;fl o-2 2
(x4 3
4
0.6 5
6
0,6 7
8
9
LO : 10
pmol ATP
FIG. 1. Calibration curves for 0. 1 - 1.Oand for 1.O- 10 pmol ATP. Recorder sensitivity in bioluminescence measurements: for the O.l- to 1.O-pmol range 100 units was equal to 10 mV and for the 1.O- to IO-pm01 range 100 units was equal to 100 mV.
~1 of PEP-buffer was used which contained PK (20 units/ml) and AK (150 units/ml). The 20-~1 sample or standard and PEP-buffer were mixed by centrifugation and incubated for 30 min at room temperature. The reaction was terminated by addition of 400 ~1 of Tris-EDTA buffer and the whole volume was pipetted with a Pasteur pipet into a disposable LKB cuvette. One hundred microliters of diluted ATP Monitoring Reagent was added and’after immediate mixing on a Vortex vibrator the ATP content was measured in the luminometer. RESULTS AND DISCUSSION
ATP Determination As demonstrated in Fig. 1, the introduction of purified and stabilized firefly luciferin luciferase and sensitive luminometers for measuring bioluminescence of the luciferin luciferase reaction allows determinations of 0. 1 - 1.O pmol ATP as well as of any higher amounts of ATP in the picomole
range. This assay system is, therefore, more sensitive than any previous method using crude firefly luciferase extracts. ADP Determination In extremely sensitive measurements of ADP and AMP it has been necessary to reduce the level of adenine ribonucleotides contained in the PK and AK enzyme preparation (7,g). Furthermore, it has been reported that the linearity of the ATP standard curve in the presence of PK and PEP was greatly affected by ADP-contaminating crude luciferase preparations and a heat-inactivation step was proposed to allow sensitive measurements (7). We failed to find any indications for traces of ADP in purified luciferase or in PK from different suppliers. Consequently, our assay system allowed ADP determinations in the same range of sensitivity as the ATP measurements. This is illustrated in Fig. 2. In one case, however, a charge of PEP was contaminated by ATP.
176
SPIELMANN,
JACOB-MULLER,
AND SCHULZ
biolummescence (units)
.’.*.i
240 I
: 200 200--4OmV 40mV
I
: :
.’
:
:
.’
.i .* .’ :‘ATP*ADP+AMP (with
PK*MK)
.*
,-‘/ATP+ADP
140. .’
.
.* lOO--2OmV :
:
:
.’
.’
:
60-
0
0.1
0.2
0.4
0.4
oa
.*
r---T---l------
0 p
0
.
/
/
,
/
(
.
0.3
.
.
.,
.
0.6
.
.
.
.
.
/+
/
/
1.0 ----
0.2 .
/
/
,
,.....................*
1.2
/
/
/
/
/
/
/
/
(with
PK)
1.65
pm01 ATP
3.3
pmol
ATP*ADP
. . . . . . . . . . . . . . . . . . . . . . . . ,.. 3>0 5.0
pmol
ATP+ADP l AMP
r----------7-
2x)
FIG. 2. Calibration curves for ATP + ADP + AMP. The IO-p.1 standard was a mixture of equal amounts of ATP, ADP, and AMP. Following incubation with 10 ~1 of PEP-buffer containing no enzymes, the ATP standard curve was obtained. The ATP + ADP calibration curve resulted after incubation of the same standard solutions with PEP-buffer containing PK and the ATP + ADP + AMP values were obtained in the presence of PEP-buffer containing both PK and AK. Since ADP and AMP are determined after conversion to ATP, the 5.0-pmol ATP + ADP + AMP value represents the sum of 1.65 pmol ATP, 1.65 pmol ADP, and 1.65 pmol AMP contained in the standard solution. This type of standard curves allows an easy testing of the activity of PK and AK for determinations of the three adenine ribonucleotides in tissue extracts.
AMP Determination Many problems arose from impurities of AK preparations obtained from different sources. Since the background bioluminescence of the Boehringer enzyme was very strong, we only used the two enzymes, PK and AK, from Sigma. Unfortunately, different charges of AK from this supplier vary considerably in the level of contamination
by several of the adenine ribonucleotides. Therefore, every new charge of AK has to be checked for contaminations before being used in the assay. Reproducible results in the AMP + ADP + ATP measurements could only be obtained when the removal of ammonium sulfate from the AK solution was incorporated in the determination procedure as described above.
SENSITIVE
1
2
3
ASSAY FOR ATP. ADP, AND AMP
4 "umber Of embr"OB per assay
FIG. 3. Determination of ATP. and AMP in one to four preimplantation mouse embryos. ATP was determined in 1-cell embryos (1 -cell) and ATP, ADP and AMP were determined in mouse blastocysts (blast) containing 36-72 cells but lower amounts of the three adenine ribonucleotides than l-cell embryos. ATP, ADP, and AMP were extracted from the embryos by three times freezing and thawing in liquid nitrogen. Means are given + SD and were obtained by 6-10 measurements on different days.
AMP + ADP + ATP determinations in the O.l- to l.O-pmol range could very accurately be performed when purified luciferase and partly purified AK were used in the assay, as illustrated in Fig. 2. The standards contained equal amounts of ATP, ADP, and AMP. The ATP standard curve is obtained by incubation with PEP-buffer. If PK is incorporated into the PEP-buffer, a doubling of the ATP values occurs representing the ATP + ADP standard curve. When the PEP-buffer contains both enzymes, PK and AK, the standard curve for ATP + ADP + AMP results. The values of the latter curve are three times as high as the ATP curve. This type of calculating the three standard curves allows an easy testing of the activity of PK and AK in the course of a series of determinations of the adenine ribonucleotides. The rate-limiting step for the conversion of AMP to ATP via the coupled AK/PK reaction is the initial conversion of AMP to
177
ADP (i.e., AMP + ATP = ZADP). The K, of AK for AMP is a function of the concentration of ATP in solution; therefore, the overall reaction rate is dependent upon the concentration of ATP in the reaction mixture (73). To ensure a more efficient conversion of AMP to ATP, some investigators have suggested to add a known amount of ATP to the assay mixture when very low amounts of ATP are expected (7). Our standard curves for ATP + ADP + AMP demonstrate that there was no indication for an inhibition of the conversion of AMP to ADP in the range of 0.1-l .O pmol per assay. However, this reaction was considerably inhibited when AK still contained ammonium sulfate. The earlier investigators did not comment on this problem (73). Determination of ATP, ADP, and AMP in Tissue Samples To prevent any shifting in the concentrations of the adenine ribonucleotides during handling of tissue samples, preimplantation mouse embryos were flushed from the uterus or uterine tube and were immediately taken up in Tris-EDTA buffer and frozen in liquid nitrogen. Previous investigators used heat inactivation (7) or acid extraction (2,10,11) as essential steps of the extraction of adenine ribonucleotides from bacterial and somatic cells. Using very small volumes during the conversion of ADP and AMP to ATP, we tried to avoid the acid extraction and neutralization step, which may also cause ionic interference with the luciferase reaction (8). We compared the determination of ATP from preimplantation embryos after extraction with the detergent NRS, after destruction by heating to 96°C and after destruction and solubilization by freezing and thawing. The amounts of ATP found after extraction with NRS (incubation for 5 and 10 min) and after heat inactivation of the ATP metabolizing enzymes (96°C for 5 and 10 min) were significantly lower than the values measured after freezing and thawing
178
SPIELMANN,
JACOB-MULLER,
the embryos three times in liquid nitrogen. ATP levels determined by this procedure were comparable to data obtained on higher numbers of embryos by earlier investigators using acid extraction and the crude firefly luciferase assay system (10,ll) or other methods (12). ATP extraction by freezing and thawing could not be improved by subsequent or simultaneous addition of NRS or additional heating to 96°C. Similar results were obtained for the ATP + ADP and for the ATP + ADP + AMP determinations in preimplantation embryos. The extraction procedure may have to be modified when the three adenine ribonucieotides are determined in other somatic tissue samples. Typical data for one to four preimplantation embryos at the one-cell to blastocyst stage are given in Fig. 3. The results clearly demonstrate that determinations of the three adenine ribonucleotides can be carried out on single embryos. The amount of the three substrates is higher in one-cell embryos than in later cleavage stage embryos, as, e.g., blastocysts. For this reason, data obtained for ATP, ADP, and AMP in blastocysts are given in Fig. 3 and for comparison purposes the ATP values for one-cell embryos are added. It has to be emphasized that ADP and AMP are not measured as such by the luciferase assay but after conversion to ATP in addition to the amount of ATP that is contained in the cells. The ADP and AMP values of 0.1 pmol per embryo in Fig. 3, which is at the sensitivity limit of the method, have actually not been determined but rather been measured as ATP + ADP and as ATP + ADP + AMP and were obtained after subtraction of ATP for ADP and of ATP and ADP for AMP. Therefore, the lowest value measured by this procedure was the amount of ATP per embryo. Preimplantation mouse embryos, which had been kept frozen in liquid nitrogen for a few weeks, still contained the same amounts of each of the adenine ribonucleo-
AND SCHULZ
tides as freshly collected embryos. The relation between the concentrations of ATP, ADP, and AMP, the ATP/ADP ratio and the adenylate energy charge measured by the present procedure for the first time in preimplantation mouse embryos agree quite well with data obtained in studies on other mammalian tissues (13,14). As demonstrated by the present study, determinations of the three adenine ribonucleotides by the luciferase reaction are no longer limited by the sensitivity of the firefly luciferase but by contaminations in the enzymes converting ADP and AMP to ATP. The method would, therefore, considerably be improved and simplified if the manufacturers of PK and AK could provide enzymes which are particularly purified for bioluminescence studies. REFERENCES 1. Atkinson,
D. E. (1968) Biochemistry
7, 4030-
4034. 2.
Ginsberg, L., and Hillman, N. (1975) J. Embryol. Exp.
Morphol.
33, 715-723.
Schiffner, J., and Spielmann, H. (1976)5. Reprod. Fert. 47, 145-147. 4. McElroy, W. D., and Green, A. (1956) Arch. Bio3.
them. 5.
Biophys.
64, 257-271.
Lundin, A., and Thore, A. (1975) Anal. Biochem. 66, 47-63.
Lundin, A., Rickardson, A., and Thore, A. (1976) Anal. Biochem. 75, 61 l-620. 7. Karl, D. M., and Helm-Hansen, 0. (1978) Marine
6.
Biol.
48, 185-197.
Helm-Hansen, O., and Karl, D. M. (1978) in Methods in Enzymology (DeLuca, M. A., ed.), Vol. 57, pp. 73-85, Academic Press, New York. 9. Whittingham, D. G., and Wales, R. G. (1969) Aust. J. Biol. SC;. 22, 1065-1068. 10. Ginsberg, L., and Hillman, N. (1973) J. Embryol. 8.
Exp.
Morphol.
30, 267-282.
11. Quinn, P., and Wales, R. G. (1973) J. Reprod. Fert. 32, 231-241. 12. Clegg, K. B., and Piko, L. (1977) Develop. Biol. 58, 76-95.
13. Beis, I., and Newsholme,
E. A. (1975) Biochem.
J. 152, 23-32.
14. Iles, R. A., Baron, P. G., and Cohen, R. D. (1979) Biochem.
J. 184,635-642.
BIOCHEMISTRY
Simple
113,172-l
78 (198 1)
Assay of 0.1-l .O pmol of ATP, ADP, and AMP in Single Somatic Cells Using Purified Luciferin Luciferasel
HORST SPIELMANN,URSULAJACOB-MUELLER, AND PETRA SCHULZ Institut
fiir
Toxikologie und Embryonul-Pharmakologie. Garystr. 9, D-3000 Berlin 33, Federal
Republic
Freie Unir~ersitiit of Germany
Berlin,
Received December 19, 1980 The sensitivity of ATP determinations with crude firefly luciferin luciferase is limited by contaminating ATP converting enzymes, which cause a rapid decrease of the ATP level during the assay. Purified luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration. Sensitivity and specificity of the ATP assay are, therefore, considerably increased when purified enzyme is used instead of crude extracts of the enzyme. ATP, 0.1 - 1.O pmol as well as higher amounts can be determined with commercial preparations of purified and stabilized luciferase. In ADP and AMP measurements with the luciferase assay, problems are arising from the enzymes required for the conversion to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides. Exclusion of contaminated enzymes and removal of ammonium sulfate from adenylate kinase were the only prerequisites for determinations of 0. 1- 1.O pmol of ADP and AMP with purified luciferase. The application of the assay in determinations of ATP, ADP, and AMP in single preimplantation mouse embryos is described.
One promising approach to study the energetic state of cells is to determine the concentrations and ratios of the adenine ribonucleotides (ATP, ADP, and AMP), which are involved in stoichiometric energy transduction between most metabolic reactions. Atkinson (1) proposed that the regulation of cellular energy metabolism may be related to the adenylate energy charge defined as ((ATP) + O.S(ADP)/(ATP) + (ADP) + (AMP)), whereby the adenylate energy charge is a measure of the metabolically available energy stored in the adenine ribonucleotides. Investigations on the energy metabolism of mouse embryos homozygous for the t12 mutation, which die prior to implantation, have shown considerable
changes in the ATP and ADP contents of litters of preimplantation embryos containing varying amounts of lethal t12/t12 embryos (2). Such determinations could not be performed on single lethal and normal preimplantation mouse embryos, since the firefly luciferin luciferase assay for ATP and ADP was lacking sensitivity. For the same reason investigations on AMP content and on the adenylate energy charge have not yet been carried out on preimplantation mouse embryos, which contain l-72 cells between fertilization and implantation (3).
The luciferase reaction is highly specific and no other ribonucleotide triphosphate may substitute for ATP (4). Unpurified preparations of luciferase, however, which have generally been used in ATP determinations, contain ATP-converting enzymes that cause a rapid decrease of the ATP level during the assay (5). The sensitivity of ATP
r This investigation was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29 (Embryonale Entwicklung und Differenzierung). We are indebted to Drs. A. Lundin and A. Thore (Sundbyberg, Sweden) for their helpful criticism. 0003-269718 l/O701 72-07S02.00/0 Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
measurements with crude enzyme extracts 172
SENSITIVE
ASSAY FOR ATP, ADP, AND AMP
is particularly limited by the difficulties of reproducibly monitoring the changing light production of the reaction. Purified firefly luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration (5). Sensitivity and specificity of the ATP assay could, therefore, be considerably increased when purified luciferase was used instead of crude enzyme extracts (5,6). Only recently have purified and stabilized firefly luciferase and also photometers specially designed for ATP analyses become commercially available. We used standardized, purified luciferin reagents to determine the absolute concentrations of intracellular ATP, ADP, and AMP in preimplantation mouse embryos at the O.l- to l.O-pmol range. ADP and AMP were enzymatically converted to ATP, which was then assayed by the purified luciferase. Although detailed determinations in bacterial cell extracts have been published before (7,8), several modifications were necessary in order to measure low levels of these metabolites in somatic cells. Problems are particularly arising from the enzymes required for the conversion of ADP and AMP to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides, which can be neglected when determinations are performed in the picomole and nanomole range. The present report describes the simple assay of ATP, ADP, and AMP in somatic cells at the O.l- to l.O-pmol range using the purified firefly luciferase system. MATERIALS
AND METHODS
Reagents. Purified firefly luciferin luciferase was purchased as “ATP Monitoring Reagent” from LKB Instruments (Grgfelfing, Germany). This reagent is a mixture of firefly luciferase and luciferin in magnesium acetate supplemented with bovine serum albumin. Each vial of the reagent was filled up with 10 ml distilled water. This dilution allows the assay of 100 samples using a re-
173
agent volume of 0.1 ml per assay sample and a total reaction volume of 0.5 ml. The reconstituted reagent may be stored at -20°C for several weeks. PKZ (EC 2.7.1.40) from rabbit muscle (type II, 350 to 500 units/mg protein) and AK (EC 2.7.4.3) from pig muscle (grade V, 1000 to 1500 unitslmg protein) were obtained from Sigma GmbH (Munich, Germany). Ammonium sulfate has to be removed from the AK solution, since it inhibits the conversion of AMP to ATP. Purification of AK was achieved by centrifugation of 50 or 100 ~1 of the ammonium sulfate solution for 10 min at 12,OOOg in a Beckman airfuge (Beckman Instruments, Munich, Germany), removal of the ammonium sulfate supernatant and solubilization of the AK crystals in an appropriate volume of Tris-EDTA buffer (0.1 M, pH 7.75). PEP, ATP, ADP, and AMP were obtained from Boehringer (Mannheim, Germany). Standard solutions of the three adenine ribonucleotides were made up with Tris-EDTA buffer (0.1 M, pH 7.75). ATP, ADP, and AMP standard solutions were kept frozen in aliquots at - 20°C for several months. Nucleotide releasing reagent for somatic cells (NRS, Lumac Systems, Basle, Switzerland) diluted 1: 1 in Tris-EDTA buffer as suggested by the manufacturer, was used in a few experiments to extract adenine ribonucleotides from mouse embryos. All other chemicals were of analytical grade and purchased from Merck AG (Darmstadt, Germany). Deionized water from a Milli-QSystem (Millipore GmbH, Neu Isenburg, Germany) was used to make up solutions in all experiments. The assay buffer was 0.1 M Tris-EDTA buffer, pH 7.75. This Tris-EDTA buffer was used for all sample preparations and dilutions and for the extraction of biological material. 2 Abbreviations
used: PK, pyruvate kinase; AK, ad-
enylate kinase; PEP, phosphoenolpyruvate.
174
SPIELMANN,
JACOB-MtJLLER,
Equipment. To obtain maximum sensitivity, a specially designed ATP photometer was used, the LKB-Wallac Luminometer 1250 and LKB potentiometric recorder 2210-032 (LKB Instruments, Grafelfing, Germany). Measurements were performed in disposable polystyrol cuvettes from the same manufacturer. Eppendorf micro test tubes (volume 1.5 ml, Eppendorf GmbH, Hamburg, Germany) were used for the incubation of tissue extracts and adenine ribonucleotide standards with PK and AK. Incubation and centrifugation of the micro-test tubes were performed in a thermostat and in a centrifuge from the same company (Eppendorf centrifuge 5412; Eppendorf thermostat 5320). Microliter pipettes with disposable plastic tips were also purchased from the Eppendorf GmbH. These pipettes were used in all determinations. However, preimplantation mouse embryos were taken up and transferred into capillary pipets, calibrated for 10 ~1, which had been drawn from Pasteur pipets. Tissue samples. Preimplantation embryos were flushed from the uterine tubes or uteri of pregnant mice with phosphatebuffered saline according to Whittingham and Wales (9) at appropriate times as described previously (3). One-cell embryos or blastocysts were immediately taken up singly in 10 ~1 of Tris-EDTA buffer, transferred into a micro-test tube, and kept frozen in liquid nitrogen. Assay of ATP, ADP, and AMP. To reduce background luminescence of the ADP and AMP converting enzymes and to obtain the highest sensitivity at the same time, tissue samples or standards were incubated in the smallest but still easy-to-handle volume. Therefore, 10 ~1 of tissue extract or standard adenine ribonucleotide solution was incubated with 10 ~1 of the appropriate enzyme solution in an Eppendorf microtest tube for 30 min, and the ATP content was measured after dilution to 400 ~1.
AND SCHULZ
To determine the three adenine ribonucleotides in somatic cells by the luciferase method, at least three measurements have to be performed on three different samples by endpoint determination of the ATP content: one sample without any ATP generating enzyme (for ATP), another sample containing PK (for ATP + ADP), and the third sample containing both PK and AK (for ATP + ADP + AMP). The amount of ADP is obtained by subtracting the ATP value from the (ATP + ADP) value and the amount of AMP is represented by the difference between the (ATP + ADP + AMP) and the (ATP + ADP) content. In somatic cells each of the three adenine ribonucleotides has to be determined in the presence of the two others. Standard curves have, therefore, to be measured under identical complex conditions. To minimize the effects of ionic interference (8), standard solutions of ATP, ADP, and AMP were prepared in an ionic medium identical to that of the samples. Routinely determinations of the three adenine ribonucleotides were carried out using the following procedure. Ten microliters of Tris-EDTA buffer as blank or 10 ~1 of buffer containing a mixture of equal amounts of ATP, ADP, and AMP, or 10 ~1 of the same buffer containing a single preimplantation mouse embryo is transferred into a micro-test tube and three times rapidly frozen and thawed in liquid nitrogen. Tris-EDTA buffer containing phosphoenolpyruvate (PEP), MgC& , and KC1 at concentrations suggested by earlier investigators (5-8) is used for the conversion of ADP and AMP to ATP. Optimum conditions were achieved by addition of 10 ,ul of reaction buffer (PEP-buffer: 0.3 mrvr PEP, 9 mM MgCl,, 5 mM KC1 in 0.1 M TrisEDTA buffer, pH 7.75) to 10 ~1 of sample, blank or standard. For the determination of ATP + ADP, 10 ~1 PEP-buffer containing PK (20 units/ml) was added and for the measurement of ATP + ADP + AMP, 10
SENSITIVE
175
ASSAY FOR ATP, ADP, AND AMP
b,o,“ml”esce”ce (units) 90.
.* 071 -1 pmol
ATP
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70.
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4030-
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pmol ATP
FIG. 1. Calibration curves for 0. 1 - 1.Oand for 1.O- 10 pmol ATP. Recorder sensitivity in bioluminescence measurements: for the O.l- to 1.O-pmol range 100 units was equal to 10 mV and for the 1.O- to IO-pm01 range 100 units was equal to 100 mV.
~1 of PEP-buffer was used which contained PK (20 units/ml) and AK (150 units/ml). The 20-~1 sample or standard and PEP-buffer were mixed by centrifugation and incubated for 30 min at room temperature. The reaction was terminated by addition of 400 ~1 of Tris-EDTA buffer and the whole volume was pipetted with a Pasteur pipet into a disposable LKB cuvette. One hundred microliters of diluted ATP Monitoring Reagent was added and’after immediate mixing on a Vortex vibrator the ATP content was measured in the luminometer. RESULTS AND DISCUSSION
ATP Determination As demonstrated in Fig. 1, the introduction of purified and stabilized firefly luciferin luciferase and sensitive luminometers for measuring bioluminescence of the luciferin luciferase reaction allows determinations of 0. 1 - 1.O pmol ATP as well as of any higher amounts of ATP in the picomole
range. This assay system is, therefore, more sensitive than any previous method using crude firefly luciferase extracts. ADP Determination In extremely sensitive measurements of ADP and AMP it has been necessary to reduce the level of adenine ribonucleotides contained in the PK and AK enzyme preparation (7,g). Furthermore, it has been reported that the linearity of the ATP standard curve in the presence of PK and PEP was greatly affected by ADP-contaminating crude luciferase preparations and a heat-inactivation step was proposed to allow sensitive measurements (7). We failed to find any indications for traces of ADP in purified luciferase or in PK from different suppliers. Consequently, our assay system allowed ADP determinations in the same range of sensitivity as the ATP measurements. This is illustrated in Fig. 2. In one case, however, a charge of PEP was contaminated by ATP.
176
SPIELMANN,
JACOB-MULLER,
AND SCHULZ
biolummescence (units)
.’.*.i
240 I
: 200 200--4OmV 40mV
I
: :
.’
:
:
.’
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PK*MK)
.*
,-‘/ATP+ADP
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.
.* lOO--2OmV :
:
:
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.’
:
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0
0.1
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,
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.
.
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.
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.
.
.
.
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/
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/
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/
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/
/
(with
PK)
1.65
pm01 ATP
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. . . . . . . . . . . . . . . . . . . . . . . . ,.. 3>0 5.0
pmol
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FIG. 2. Calibration curves for ATP + ADP + AMP. The IO-p.1 standard was a mixture of equal amounts of ATP, ADP, and AMP. Following incubation with 10 ~1 of PEP-buffer containing no enzymes, the ATP standard curve was obtained. The ATP + ADP calibration curve resulted after incubation of the same standard solutions with PEP-buffer containing PK and the ATP + ADP + AMP values were obtained in the presence of PEP-buffer containing both PK and AK. Since ADP and AMP are determined after conversion to ATP, the 5.0-pmol ATP + ADP + AMP value represents the sum of 1.65 pmol ATP, 1.65 pmol ADP, and 1.65 pmol AMP contained in the standard solution. This type of standard curves allows an easy testing of the activity of PK and AK for determinations of the three adenine ribonucleotides in tissue extracts.
AMP Determination Many problems arose from impurities of AK preparations obtained from different sources. Since the background bioluminescence of the Boehringer enzyme was very strong, we only used the two enzymes, PK and AK, from Sigma. Unfortunately, different charges of AK from this supplier vary considerably in the level of contamination
by several of the adenine ribonucleotides. Therefore, every new charge of AK has to be checked for contaminations before being used in the assay. Reproducible results in the AMP + ADP + ATP measurements could only be obtained when the removal of ammonium sulfate from the AK solution was incorporated in the determination procedure as described above.
SENSITIVE
1
2
3
ASSAY FOR ATP. ADP, AND AMP
4 "umber Of embr"OB per assay
FIG. 3. Determination of ATP. and AMP in one to four preimplantation mouse embryos. ATP was determined in 1-cell embryos (1 -cell) and ATP, ADP and AMP were determined in mouse blastocysts (blast) containing 36-72 cells but lower amounts of the three adenine ribonucleotides than l-cell embryos. ATP, ADP, and AMP were extracted from the embryos by three times freezing and thawing in liquid nitrogen. Means are given + SD and were obtained by 6-10 measurements on different days.
AMP + ADP + ATP determinations in the O.l- to l.O-pmol range could very accurately be performed when purified luciferase and partly purified AK were used in the assay, as illustrated in Fig. 2. The standards contained equal amounts of ATP, ADP, and AMP. The ATP standard curve is obtained by incubation with PEP-buffer. If PK is incorporated into the PEP-buffer, a doubling of the ATP values occurs representing the ATP + ADP standard curve. When the PEP-buffer contains both enzymes, PK and AK, the standard curve for ATP + ADP + AMP results. The values of the latter curve are three times as high as the ATP curve. This type of calculating the three standard curves allows an easy testing of the activity of PK and AK in the course of a series of determinations of the adenine ribonucleotides. The rate-limiting step for the conversion of AMP to ATP via the coupled AK/PK reaction is the initial conversion of AMP to
177
ADP (i.e., AMP + ATP = ZADP). The K, of AK for AMP is a function of the concentration of ATP in solution; therefore, the overall reaction rate is dependent upon the concentration of ATP in the reaction mixture (73). To ensure a more efficient conversion of AMP to ATP, some investigators have suggested to add a known amount of ATP to the assay mixture when very low amounts of ATP are expected (7). Our standard curves for ATP + ADP + AMP demonstrate that there was no indication for an inhibition of the conversion of AMP to ADP in the range of 0.1-l .O pmol per assay. However, this reaction was considerably inhibited when AK still contained ammonium sulfate. The earlier investigators did not comment on this problem (73). Determination of ATP, ADP, and AMP in Tissue Samples To prevent any shifting in the concentrations of the adenine ribonucleotides during handling of tissue samples, preimplantation mouse embryos were flushed from the uterus or uterine tube and were immediately taken up in Tris-EDTA buffer and frozen in liquid nitrogen. Previous investigators used heat inactivation (7) or acid extraction (2,10,11) as essential steps of the extraction of adenine ribonucleotides from bacterial and somatic cells. Using very small volumes during the conversion of ADP and AMP to ATP, we tried to avoid the acid extraction and neutralization step, which may also cause ionic interference with the luciferase reaction (8). We compared the determination of ATP from preimplantation embryos after extraction with the detergent NRS, after destruction by heating to 96°C and after destruction and solubilization by freezing and thawing. The amounts of ATP found after extraction with NRS (incubation for 5 and 10 min) and after heat inactivation of the ATP metabolizing enzymes (96°C for 5 and 10 min) were significantly lower than the values measured after freezing and thawing
178
SPIELMANN,
JACOB-MULLER,
the embryos three times in liquid nitrogen. ATP levels determined by this procedure were comparable to data obtained on higher numbers of embryos by earlier investigators using acid extraction and the crude firefly luciferase assay system (10,ll) or other methods (12). ATP extraction by freezing and thawing could not be improved by subsequent or simultaneous addition of NRS or additional heating to 96°C. Similar results were obtained for the ATP + ADP and for the ATP + ADP + AMP determinations in preimplantation embryos. The extraction procedure may have to be modified when the three adenine ribonucieotides are determined in other somatic tissue samples. Typical data for one to four preimplantation embryos at the one-cell to blastocyst stage are given in Fig. 3. The results clearly demonstrate that determinations of the three adenine ribonucleotides can be carried out on single embryos. The amount of the three substrates is higher in one-cell embryos than in later cleavage stage embryos, as, e.g., blastocysts. For this reason, data obtained for ATP, ADP, and AMP in blastocysts are given in Fig. 3 and for comparison purposes the ATP values for one-cell embryos are added. It has to be emphasized that ADP and AMP are not measured as such by the luciferase assay but after conversion to ATP in addition to the amount of ATP that is contained in the cells. The ADP and AMP values of 0.1 pmol per embryo in Fig. 3, which is at the sensitivity limit of the method, have actually not been determined but rather been measured as ATP + ADP and as ATP + ADP + AMP and were obtained after subtraction of ATP for ADP and of ATP and ADP for AMP. Therefore, the lowest value measured by this procedure was the amount of ATP per embryo. Preimplantation mouse embryos, which had been kept frozen in liquid nitrogen for a few weeks, still contained the same amounts of each of the adenine ribonucleo-
AND SCHULZ
tides as freshly collected embryos. The relation between the concentrations of ATP, ADP, and AMP, the ATP/ADP ratio and the adenylate energy charge measured by the present procedure for the first time in preimplantation mouse embryos agree quite well with data obtained in studies on other mammalian tissues (13,14). As demonstrated by the present study, determinations of the three adenine ribonucleotides by the luciferase reaction are no longer limited by the sensitivity of the firefly luciferase but by contaminations in the enzymes converting ADP and AMP to ATP. The method would, therefore, considerably be improved and simplified if the manufacturers of PK and AK could provide enzymes which are particularly purified for bioluminescence studies. REFERENCES 1. Atkinson,
D. E. (1968) Biochemistry
7, 4030-
4034. 2.
Ginsberg, L., and Hillman, N. (1975) J. Embryol. Exp.
Morphol.
33, 715-723.
Schiffner, J., and Spielmann, H. (1976)5. Reprod. Fert. 47, 145-147. 4. McElroy, W. D., and Green, A. (1956) Arch. Bio3.
them. 5.
Biophys.
64, 257-271.
Lundin, A., and Thore, A. (1975) Anal. Biochem. 66, 47-63.
Lundin, A., Rickardson, A., and Thore, A. (1976) Anal. Biochem. 75, 61 l-620. 7. Karl, D. M., and Helm-Hansen, 0. (1978) Marine
6.
Biol.
48, 185-197.
Helm-Hansen, O., and Karl, D. M. (1978) in Methods in Enzymology (DeLuca, M. A., ed.), Vol. 57, pp. 73-85, Academic Press, New York. 9. Whittingham, D. G., and Wales, R. G. (1969) Aust. J. Biol. SC;. 22, 1065-1068. 10. Ginsberg, L., and Hillman, N. (1973) J. Embryol. 8.
Exp.
Morphol.
30, 267-282.
11. Quinn, P., and Wales, R. G. (1973) J. Reprod. Fert. 32, 231-241. 12. Clegg, K. B., and Piko, L. (1977) Develop. Biol. 58, 76-95.
13. Beis, I., and Newsholme,
E. A. (1975) Biochem.
J. 152, 23-32.
14. Iles, R. A., Baron, P. G., and Cohen, R. D. (1979) Biochem.
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