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Application of simultaneous determination of 3H, 14C, and 22Na by liquid scintillation counting to the measurement of cellular lon-transport
ANALYTICAL
BIOCHEMISTRY
198, 194-199 (1991)
Application of Simultaneous Determination of 3H, 14C, and **Na by Liquid Scintillation Counting to the Measurement of Cellular Ion-Transport Yuka
Miyazawa,
Naoko
Sakai,
Naoyuki
Murakami,
and Tetsuya
Konishil
Department of Radiochemistry-Biophysics, Niigata Collegeof Pharmacy, Kamishin-ei 5-13-2, Niigata 950-21, Japan
Received
March
29,199l
A liquid scintillation counting method for simultaneous determination of three radioactive nuclides (‘H, 14C, and 22Na) of biological interest was studied. By comparing the @ spectra of the three nuclides, their counting energy ranges, A, B, and C, were determined. “NA was set high enough to avoid any spillover counts from lower-energy nuclides. Region A for ‘H was set to maximize the counting efficiency. A good correlation between the counting efficiency for 22Na in region C and the counting efficiency of other nuclides in all regions was obtained. Prior to 3H and “C dpm calculations, the 22Na counts spilled down in regions A and B were subtracted from the total counts in regions A and B. A simple linear equation was then used to compute ‘H and 14C dpm. Findings show that the method presented is adaptable for highly quenched samples up to quenching indices of tslE = 100. The method is useful for studying the biological transport coupled to Na+. Q 1991 Academic
Press, Inc.
Ion transport across the cell membrane is a basic process of cellular physiology. Bioenergetic conversion of chemical energy into electrical energy or vice versa is essentially coupled to the ion-transport process. These processes are driven by a proton motive force, Abu+ ( 1) . Intrinsically, the analysis of certain ion-transport processesrequires kinetic comparisons of at least three different parameters: membrane potential (A*), transmembrane pH gradient (ApH), and the transport ion itself. A few spectroscopic or electrode methods for determining two parameters at once have been reported (2,3), but the determination of three parameters contains inherent difficulties. 1 To whom
correspondence
should
be addressed.
A radioactive tracer is advantageous in determining the net change of ion or other transport substrates of interest without disturbing the physiological conditions of the experimental system. A radioactive tracer is routinely used to determine cellular uptake or release of ions or transport substrate (4). A* and ApH are also determined by the equilibrium distribution of radiolabeled lipophilic ion and by weak acid (or base), respectively (5,6). 3H- and 14C-labeled membrane probes and transport substrates are available. Simultaneous determination of 3H, 14C, and other nuclide could thus provide a useful methodology for the study of cellular iontransport processes. A triple-tracer radioautographic method for studying cerebral physiological conditions has been reported. With this method the nuclides are distinguished by their half-life difference ( 7,8). However, the method does not forego opportunities for studying kinetics. In the present paper, we studied a liquid scintillation counting ( LSC ) 2 method to simultaneously determine 3H, 14C, and 22Na, in the same sample. The method is also applicable for light-driven Na+/H+ exchange in Halobacterium halobium. MATERIALS
AND
METHODS
Liquid scintillation counting was carried out using a Packard Tri-Carb 2200CA liquid scintillation counter. Manufactured scintillation cocktail, Cleasol-I, was purchased from Nakarai Tesque Co. 14C-salicylate (2.2 GBq / mmol) , 3H-TPP+ ( 1.67 TBq / mmol) , and 22NaC1 (carrier free) were purchased from NEN. Buffers and other reagents were purchased from Wako Chemical 2 Abbreviations used: LX, traphenyl phosphonium salt.
liquid
scintillation
counting;
TPP+,
0003.2697191
194
Copyright All
rights
0 1991 by Academic
of reproduction
in any form
te-
$3.00
Press, Inc. reserved.
TRIPLE-TRACER
TECHNIQUE
FOR
KeV
CELL
ION
TRANSPORT
0
0
KeV
195
STUDY
KeV
500 >
4 p-energy FIG. 1. Quench-dependent addition of increased amounts
changes of fl energy of acetone.
spectra
of 3H, “C,
Co. Halobacterial membrane vesicles were prepared from H. halobium R,M, according to the method previously reported (9). Standard stock solutions of 14C-salicylate and 3HTPP+ were prepared as 10 mM aqueous solutions of salicylate and TPP’, both having specific radioactivities of 200 dpm/pl, respectively. 22NaC1 was prepared as a 100 mM aqueous solution with a specific radioactivity of 100 dpm/pl. For the quench experiments, a series of 10 counting samples containing 5000 dpm was prepared for each of the nuclides. These samples were made in 5 ml of low-K glass vials by adding an aliquot of their radioactive stock solution into 5 ml of scintillation cocktail. By adding increasing volumes of acetone from 10 to 300 ~1 a series of quench standard samples were obtained. The series of standard samples was counted for 10 min to obtain the p spectra. At the same time, counting efficiencies were determined for the samples by the conventional external standard method. The values were correlated to the quenching index, tSIE, obtained for the samples. The quenching index, tSIE, is a transformed spectral index of the external standard used in the TriCarb scintillation system (10). To determine the sensitivity limits of the method, a series of counting samples was prepared using the three
and “Na.
Standard
samples
for 3H, W,
and **Na
are quenched
by the
nuclides to form combinations of five different radioactivities ranging from 100 to 5000 dpm. Thus, the standard counting samples consisted of 125 different combinations of three nuclides. Each sample was measured for 5 min and the counts obtained were processed for dpm calculation using the software program “DPM l-2-3,” with which the Tri-Carb scintillation counter ( 10) calculates. Also, the method described in this paper was used to obtain the dpm. The kinetic behavior of light-driven 22Na efflux in the H. halobium membrane vesicles was measured by the membrane filtration method reported previously (9). Briefly, the membrane vesicles ( 10 mg / ml) from H. hulobium R, M, were equilibrated for 3 days in a refrigerator while immersed in a solution of 2.9 M KC1 and 0.1 M 22NaC1 (4.4 X lo3 dpm/pmol) in 5 mM Pipes buffer containing 5 PM 3H-TPP+ (4.3 X lo8 dpm/pmol) and 200 pM “C-salicylate (6.3 X lo6 dpm/pmol) . An aliquot of the vesicle was placed on a membrane filter (0.45~pm pore size) and filtered under reduced pressure at defined time periods before and after illumination with an actinic light source (150-W halogen tungsten lamp). The filter was solubilized in 1 ml of dioxan and the radioactivity was measured after the addition of Cleasol-1. The dpm calculation was carried out by the method presented in this paper. The values of ApH and A\k were
196
MIYAZAWA
1.0
5 5 .E w P P s 8
0.8
0.6
Od
0.2
0.0 1.0
lRegion1. 0.6 0.6
-
0.4
-
100
200
Quenching
300
Index
400
(tSIE)
FIG. 2. Quench-efficiency correlation for 3H, “C, and **Na in regions A, B, and C. Counting efficiencies of the triple nuclides in a quench standard sample were obtained for each of the measuring energy regions by the external standard method. The results were correlated to the values of the external quench index, t,,.
obtained from the counts in the ordinary way using the Nernst equation as described in the previous paper (9). RESULTS The B energy spectra of 3H, 14C, and 22Na and the quench-dependent spectral changes are shown in Fig. 1. From these spectral changes, the counting energy ranges for each nuclide were determined as follows: 3H, O-8 keV, region A, 14C, 8-90 keV, region B; and 22Na, 90-2000 keV, region C. Using standard counting samples containing 5000 dpm of 14C-salicylate, 3H-TPP+,
ET
AL.
or “Na, the counting efficiencies for each nuclide in the A, B, and C counting regions were calculated from the counts obtained at the different quench levels. These were then correlated to the external standard quench index, tsIE (Fig. 2 ) . No spillover counts of 3H and 14C were determined in region C at any quench level. As expected, **Na counts spilled down into regions A and B, under the above conditions used for measuring energy range. The counting efficiencies for each nuclide in regions A and B were obtained at different quench levels and were correlated to the counting efficiency of 22Na in region C at the same quench level (at the same t,,). Results show linear correlations in both regions A and B though the counting efficiency of 22Na in region C is less than 10% (Fig. 3). Therefore, when the counting efficiency of **Na in region C is determined by the external standard method and the value is larger than lo%, the counting efficiencies for the nuclides in other regions can be routinely calculated. Using the counting efficienties thus obtained, dpm of individual nuclides in the triple tracer are computed from cpm values in regions A, B, and C. We next discuss the procedure for computing net 3H, 14C, and **Na dpm values in the sample. The notations used are as follows: dpms for 3H, 14C, and 22N, II,, Dc, and II,,; counting efficiencies for 3H in regions A and B, Et and Ei; counting efficiencies for 14C in regions A and B, EC, EB’CT counting efficiencies for 22Na in regions A, B, and C, E&a, %a, G,; Total counts in regions A, B, and C, C,, Ca, Cc. In our method, the dpm of 22Na can be simply obtained from the following equation, because any spillover contribution from other nuclides is neglected in region C: DNa = Cc I@&. Since the 22Na counting efficiencies in regions A and B are known from the 22Na counting efficiency in region C, the spilled down counts of **Na in regions A and B can be subtracted from the total counts in regions A and B, respectively, to obtain net counts of 3H and 14C in regions A (Ci) and B (CL) according to the equations C:, = CA - DNa X Et,
[21
C;=CB-DNaXEEa.
[31
Therefore, C; = D, x E; + D, x Et
[41
TRIPLE-TRACER
TECHNIQUE
FOR
CELL
ION
TRANSPORT
197
STUDY
0.0 0.0
Efficiency FIG. were
3. Standard curves compared with those
0.2
for ** Na in Region
0.3
0.4
0.5
0.6
C;,=D,xE;+D,XE& Therefore, the net dpm of 3H and 14C can be computed from the following equations, in their respective order: Eg x C; - EC x C;, EE x E; - E; X E:
[61
E”, x C;, - E; x C;, Dc== E;xE;-E$xEg’
[71
Standard samples containing triple nuclides were measured at different quench levels for 5 min, and the dpms computed by the methods presented were compared with those obtained by a conventional method having the software program DPM l-2-3 equipped in a Tri-Carb LSC system under the same measuring conditions. It is clearly shown in Fig. 4 that our method gives a minimal deviation in dpm recovery of the three nuelides even in highly quenched samples up to t,, = 100. On the contrary, conventional methods cause serious deviations, especially in 3H and 22Na dpm calibrations (even in slightly quenched samples, tsIE = 400). The minimum radioactivity required to obtain reliable accuracy in 22Na counting was determined in the presence of other radionuclides. The counting deviation significantly increased as the radioactivity becomes less than 250 dpm (Fig. 5) because of a relative increase in the background contribution to the net 22Na count. This indicates that at least 300 dpm are required for reliable measurement of “Na when the 22Na counting region is set as described above and the counting time is 5 min. The counting sensitivities of 3H and 14C were determined at various 3H /22Na and 14C /22Na dpm ratios. Re-
0.7
C
for efficiency calculation. Counting efficiencies of the triple nuclides in each of measuring are the same as in the text. of **Na in region C at the same tsm value. Notations
and
DH =
0.1
energy
regions
A and B
sults indicated that 40 times more radioactivity is required for 3H than for 22Na and 20 times more radioactivity is needed for 14C than for 22Na to obtain meaningful countings (dpm recovery deviation smaller than 10% for 3H and 14C as shown in Fig. 6). The reliability of the method presented was examined in the halobacterial 22Na transport system. Light-driven 22Na efflux mediated by the Na+ /H+ antiporter in the
Present
2
Ezp cu
Conventional method
method
100
0
34 -100 ZE v -200
f -300
' 100
I 200
Quenching
1 300
I
I
I
400
500
Index (tSIE)
FIG. 4. Quenching effect on dpm calculation. Standard samples containing3H, “C, and *‘Na at different quench levels were measured for 5 min by the Tri-Carb 2200A LSC system using the measuring condition described in the text. The dpm values were computed according to both our method and the conventional “DPM-1-2-3” method. The percentage recoveries of dpm (deviations from the standard dpm) are compared.
198
MIYAZAWA
ET
AL.
. I . t . . ma
-10
0
500
1000
1500
2000
2500
3000
22 Na (dpm)
FIG. 5.
Minimal counting sensitivity for “Na. Standard samples contained various amounts of 3H, W, and “Na at different tsIE and were measured for 5 min as in Fig. 4. The dpm recovery was computed by the method presented in the paper.
100
80
halobacterial membrane vesicles was measured using 14C-salicylate and 3H-TPP+ as ApH and A!# probes, respectively. Results in Fig. 7 clearly show that the 40
8.
I.
8.
8.
I.,
1.
I.
60
I.
30
I
01
-2 20
2
0
TIME
4
6
(min)
FIG. 7.
Kinetic changes of ApH, A*, and intravesicular 22Na in halobacterial membrane vesicle upon illumination. Membrane vesicles ( 10 mg/ml) from H. halobium R,M, were suspendedin 2.9 M KC1 and 0.1 M *‘NaCI in 5 mM Pipes buffer containing5 FM 3H-TPP+, 200 pM 14C-salicylate. After equilibration, the membranes were placed on membrane filter (0.45-pm pore size) under reduced pressure at defined time periods before and after illumination (at the arrow). Radioactivity measurement and dpm calculation were according to the method described in the text. Data transformation into ApH and A* was carried out according to the Nernst equation described previously (9).
10
0
0
20
40
60
60
dpm
100
ratio
120
of
140
160
160
200
3~1 ,,Na
60 40 30
---.t--M.----------07
20
_.--__---.---__-
______.__.____.___ -~-___ I_--_-
10
e
l -
Oz
--.--?lL--
l
--
-106ow -20
1 0
I 20
40
dpm
FIG. 6.
__--
-___8-
ratio
60
of
60
method presented in this paper is useful for following the kinetic changes of intravesicular “Na contents, ApH, and A\k when these modes are simultaneously affected by illumination. “Na extruded coincidentally with ApH formation. A%, on the other hand, increased to the maximum level immediately after illumination, then gradually decreased as intravesicular NaC depleted. The profiles of the kinetic changes of these three parameters are essentially the same as those obtained previously with the use of electrodes (9).
“C122Na
Effect of dpm ratio of 3H and 14C to 22Na on dpm recovery. The standard samples and the counting conditions are the same as those given in the legend to Fig. 5.
DISCUSSION A triple-nuclide
tracer is optionally carried out using a software program equipped in the Tri-Carb LSC sys-
TRIPLE-TRACER
TECHNIQUE
FOR
tern, such as DPM l-2-3. The latter system employs the Gauss-Jordan matrix elimination method to compute individual radionuclide dpm from the cpm and the counting efficiency of each nuclide in regions A, B, and C as used for the double-tracer calculation ( 10). However, the method has an inherent disadvantage. That is, the dpm recovery significantly deviates in the lowest and highest energy nuclides as the quench level of the samples become high (as shown in Fig. 4). This disadvantage is overcome by an adequate setting of the counting regions for the three nuclides and by a simple subtraction of the “Na (the highest energy nuclide) spilldown counts from the gross counts in regions A and B. The adjustments are made prior to 3H and 14C dpm calculation. Bioenergetic parameters such as A* and ApH are routinely obtained from the equilibrium distribution of membrane probes. The equilibrium distribution of membrane probes is determined by a radiotracer or fluorescence or electrode techniques ( 11). Experimental conditions such as probe concentration differ depending on the method used. Therefore, it is generally difficult to adapt the data obtained by different techniques to the same membrane preparation, For example, using data from different techniques makes it difficult to evaluate the quantitative relationship between the thermodynamic parameters and the net change of transport substrate in the transport system being analyzed. The LSC method presented for determining triple tracers makes it possible to quantitatively discuss the above parameters in the same biological preparation, even in highly quenched samples. Therefore, the method would be quite useful for studying the energy coupling mecha-
CELL
ION
TRANSPORT
199
STUDY
nism of certain cellular transport systems, the Na+-coupled transport. Although the present studies were carried the Tri-Carb 2200A LSC system, the method can be employed with any LSC apparatus. method can be used to study other nuclides in ing fl spectra that are of interest.
especially out using presented Also, the determin-
REFERENCES 1. Mitchell, P. ( 1977) Ann. Reu. Biochem. 46,996-1005. 2. Konishi, T., Murakami, N., Hatano, Y., and Nakazato, K. (1986) Biochim. Biophys. Acta. 862, 278-284. 3. Kamo, N., Racanell, T., and Packer, L. (1982) in Methods in Enzymology (Packer, L., Ed.), Vol. 88, pp. 356-360, Academic Press, San Diego. 4. Kessler, M., and Toggenburger, G. ( 1979) in Membrane Biochemistry (Carafoli, E., and Semenza, G., Eds.), pp. l-24, SpringerVerlag, New York /Berlin. 5. Bally, M. B., Hope, M. J., VanEchteld, C. JA., and Cullis, P. R. ( 1985) Biochim. Biophys. Actu 812,66-76. 6. Ramos, S., Schuldiner, S., and Kaback, H. R. (1979) in Enzymology (Fleiscber, S., and Packer, L., Eds.), 680-688, Academic Press, San Diego.
in Methods Vol. 55, pp.
7. Mies, G., Bodsch, W., Paschen, W., and Hossmann, K. A. (1986) J. Cereb. Blood Flow Metab. 6, 59-70. 8. (a) Nakai, H., Diksic, M., and Yamamoto, Y. L. (1988) Stroke 19, 758-763; (b) Nakai, H., Yamamoto, Y. L., Diksic, M., Worsley, K. J., and Takara, E. (1988) Stroke 19, 764-772. 9. Murakami, 231-236.
N., and Konishi,
10. Packerd Instrument DPM l-2-3 Option. 11. Creamer, Biological
T. (1988)
Co., Inc. ( 1987)
J. Biochem. Packard
W. A., and Knaff, D. B. (1989) Membranes, Springer-Verlag,
103,
(Tokyo)
Operation
Manual,
Energy Transduction New York /Berlin.
in
BIOCHEMISTRY
198, 194-199 (1991)
Application of Simultaneous Determination of 3H, 14C, and **Na by Liquid Scintillation Counting to the Measurement of Cellular Ion-Transport Yuka
Miyazawa,
Naoko
Sakai,
Naoyuki
Murakami,
and Tetsuya
Konishil
Department of Radiochemistry-Biophysics, Niigata Collegeof Pharmacy, Kamishin-ei 5-13-2, Niigata 950-21, Japan
Received
March
29,199l
A liquid scintillation counting method for simultaneous determination of three radioactive nuclides (‘H, 14C, and 22Na) of biological interest was studied. By comparing the @ spectra of the three nuclides, their counting energy ranges, A, B, and C, were determined. “NA was set high enough to avoid any spillover counts from lower-energy nuclides. Region A for ‘H was set to maximize the counting efficiency. A good correlation between the counting efficiency for 22Na in region C and the counting efficiency of other nuclides in all regions was obtained. Prior to 3H and “C dpm calculations, the 22Na counts spilled down in regions A and B were subtracted from the total counts in regions A and B. A simple linear equation was then used to compute ‘H and 14C dpm. Findings show that the method presented is adaptable for highly quenched samples up to quenching indices of tslE = 100. The method is useful for studying the biological transport coupled to Na+. Q 1991 Academic
Press, Inc.
Ion transport across the cell membrane is a basic process of cellular physiology. Bioenergetic conversion of chemical energy into electrical energy or vice versa is essentially coupled to the ion-transport process. These processes are driven by a proton motive force, Abu+ ( 1) . Intrinsically, the analysis of certain ion-transport processesrequires kinetic comparisons of at least three different parameters: membrane potential (A*), transmembrane pH gradient (ApH), and the transport ion itself. A few spectroscopic or electrode methods for determining two parameters at once have been reported (2,3), but the determination of three parameters contains inherent difficulties. 1 To whom
correspondence
should
be addressed.
A radioactive tracer is advantageous in determining the net change of ion or other transport substrates of interest without disturbing the physiological conditions of the experimental system. A radioactive tracer is routinely used to determine cellular uptake or release of ions or transport substrate (4). A* and ApH are also determined by the equilibrium distribution of radiolabeled lipophilic ion and by weak acid (or base), respectively (5,6). 3H- and 14C-labeled membrane probes and transport substrates are available. Simultaneous determination of 3H, 14C, and other nuclide could thus provide a useful methodology for the study of cellular iontransport processes. A triple-tracer radioautographic method for studying cerebral physiological conditions has been reported. With this method the nuclides are distinguished by their half-life difference ( 7,8). However, the method does not forego opportunities for studying kinetics. In the present paper, we studied a liquid scintillation counting ( LSC ) 2 method to simultaneously determine 3H, 14C, and 22Na, in the same sample. The method is also applicable for light-driven Na+/H+ exchange in Halobacterium halobium. MATERIALS
AND
METHODS
Liquid scintillation counting was carried out using a Packard Tri-Carb 2200CA liquid scintillation counter. Manufactured scintillation cocktail, Cleasol-I, was purchased from Nakarai Tesque Co. 14C-salicylate (2.2 GBq / mmol) , 3H-TPP+ ( 1.67 TBq / mmol) , and 22NaC1 (carrier free) were purchased from NEN. Buffers and other reagents were purchased from Wako Chemical 2 Abbreviations used: LX, traphenyl phosphonium salt.
liquid
scintillation
counting;
TPP+,
0003.2697191
194
Copyright All
rights
0 1991 by Academic
of reproduction
in any form
te-
$3.00
Press, Inc. reserved.
TRIPLE-TRACER
TECHNIQUE
FOR
KeV
CELL
ION
TRANSPORT
0
0
KeV
195
STUDY
KeV
500 >
4 p-energy FIG. 1. Quench-dependent addition of increased amounts
changes of fl energy of acetone.
spectra
of 3H, “C,
Co. Halobacterial membrane vesicles were prepared from H. halobium R,M, according to the method previously reported (9). Standard stock solutions of 14C-salicylate and 3HTPP+ were prepared as 10 mM aqueous solutions of salicylate and TPP’, both having specific radioactivities of 200 dpm/pl, respectively. 22NaC1 was prepared as a 100 mM aqueous solution with a specific radioactivity of 100 dpm/pl. For the quench experiments, a series of 10 counting samples containing 5000 dpm was prepared for each of the nuclides. These samples were made in 5 ml of low-K glass vials by adding an aliquot of their radioactive stock solution into 5 ml of scintillation cocktail. By adding increasing volumes of acetone from 10 to 300 ~1 a series of quench standard samples were obtained. The series of standard samples was counted for 10 min to obtain the p spectra. At the same time, counting efficiencies were determined for the samples by the conventional external standard method. The values were correlated to the quenching index, tSIE, obtained for the samples. The quenching index, tSIE, is a transformed spectral index of the external standard used in the TriCarb scintillation system (10). To determine the sensitivity limits of the method, a series of counting samples was prepared using the three
and “Na.
Standard
samples
for 3H, W,
and **Na
are quenched
by the
nuclides to form combinations of five different radioactivities ranging from 100 to 5000 dpm. Thus, the standard counting samples consisted of 125 different combinations of three nuclides. Each sample was measured for 5 min and the counts obtained were processed for dpm calculation using the software program “DPM l-2-3,” with which the Tri-Carb scintillation counter ( 10) calculates. Also, the method described in this paper was used to obtain the dpm. The kinetic behavior of light-driven 22Na efflux in the H. halobium membrane vesicles was measured by the membrane filtration method reported previously (9). Briefly, the membrane vesicles ( 10 mg / ml) from H. hulobium R, M, were equilibrated for 3 days in a refrigerator while immersed in a solution of 2.9 M KC1 and 0.1 M 22NaC1 (4.4 X lo3 dpm/pmol) in 5 mM Pipes buffer containing 5 PM 3H-TPP+ (4.3 X lo8 dpm/pmol) and 200 pM “C-salicylate (6.3 X lo6 dpm/pmol) . An aliquot of the vesicle was placed on a membrane filter (0.45~pm pore size) and filtered under reduced pressure at defined time periods before and after illumination with an actinic light source (150-W halogen tungsten lamp). The filter was solubilized in 1 ml of dioxan and the radioactivity was measured after the addition of Cleasol-1. The dpm calculation was carried out by the method presented in this paper. The values of ApH and A\k were
196
MIYAZAWA
1.0
5 5 .E w P P s 8
0.8
0.6
Od
0.2
0.0 1.0
lRegion1. 0.6 0.6
-
0.4
-
100
200
Quenching
300
Index
400
(tSIE)
FIG. 2. Quench-efficiency correlation for 3H, “C, and **Na in regions A, B, and C. Counting efficiencies of the triple nuclides in a quench standard sample were obtained for each of the measuring energy regions by the external standard method. The results were correlated to the values of the external quench index, t,,.
obtained from the counts in the ordinary way using the Nernst equation as described in the previous paper (9). RESULTS The B energy spectra of 3H, 14C, and 22Na and the quench-dependent spectral changes are shown in Fig. 1. From these spectral changes, the counting energy ranges for each nuclide were determined as follows: 3H, O-8 keV, region A, 14C, 8-90 keV, region B; and 22Na, 90-2000 keV, region C. Using standard counting samples containing 5000 dpm of 14C-salicylate, 3H-TPP+,
ET
AL.
or “Na, the counting efficiencies for each nuclide in the A, B, and C counting regions were calculated from the counts obtained at the different quench levels. These were then correlated to the external standard quench index, tsIE (Fig. 2 ) . No spillover counts of 3H and 14C were determined in region C at any quench level. As expected, **Na counts spilled down into regions A and B, under the above conditions used for measuring energy range. The counting efficiencies for each nuclide in regions A and B were obtained at different quench levels and were correlated to the counting efficiency of 22Na in region C at the same quench level (at the same t,,). Results show linear correlations in both regions A and B though the counting efficiency of 22Na in region C is less than 10% (Fig. 3). Therefore, when the counting efficiency of **Na in region C is determined by the external standard method and the value is larger than lo%, the counting efficiencies for the nuclides in other regions can be routinely calculated. Using the counting efficienties thus obtained, dpm of individual nuclides in the triple tracer are computed from cpm values in regions A, B, and C. We next discuss the procedure for computing net 3H, 14C, and **Na dpm values in the sample. The notations used are as follows: dpms for 3H, 14C, and 22N, II,, Dc, and II,,; counting efficiencies for 3H in regions A and B, Et and Ei; counting efficiencies for 14C in regions A and B, EC, EB’CT counting efficiencies for 22Na in regions A, B, and C, E&a, %a, G,; Total counts in regions A, B, and C, C,, Ca, Cc. In our method, the dpm of 22Na can be simply obtained from the following equation, because any spillover contribution from other nuclides is neglected in region C: DNa = Cc I@&. Since the 22Na counting efficiencies in regions A and B are known from the 22Na counting efficiency in region C, the spilled down counts of **Na in regions A and B can be subtracted from the total counts in regions A and B, respectively, to obtain net counts of 3H and 14C in regions A (Ci) and B (CL) according to the equations C:, = CA - DNa X Et,
[21
C;=CB-DNaXEEa.
[31
Therefore, C; = D, x E; + D, x Et
[41
TRIPLE-TRACER
TECHNIQUE
FOR
CELL
ION
TRANSPORT
197
STUDY
0.0 0.0
Efficiency FIG. were
3. Standard curves compared with those
0.2
for ** Na in Region
0.3
0.4
0.5
0.6
C;,=D,xE;+D,XE& Therefore, the net dpm of 3H and 14C can be computed from the following equations, in their respective order: Eg x C; - EC x C;, EE x E; - E; X E:
[61
E”, x C;, - E; x C;, Dc== E;xE;-E$xEg’
[71
Standard samples containing triple nuclides were measured at different quench levels for 5 min, and the dpms computed by the methods presented were compared with those obtained by a conventional method having the software program DPM l-2-3 equipped in a Tri-Carb LSC system under the same measuring conditions. It is clearly shown in Fig. 4 that our method gives a minimal deviation in dpm recovery of the three nuelides even in highly quenched samples up to t,, = 100. On the contrary, conventional methods cause serious deviations, especially in 3H and 22Na dpm calibrations (even in slightly quenched samples, tsIE = 400). The minimum radioactivity required to obtain reliable accuracy in 22Na counting was determined in the presence of other radionuclides. The counting deviation significantly increased as the radioactivity becomes less than 250 dpm (Fig. 5) because of a relative increase in the background contribution to the net 22Na count. This indicates that at least 300 dpm are required for reliable measurement of “Na when the 22Na counting region is set as described above and the counting time is 5 min. The counting sensitivities of 3H and 14C were determined at various 3H /22Na and 14C /22Na dpm ratios. Re-
0.7
C
for efficiency calculation. Counting efficiencies of the triple nuclides in each of measuring are the same as in the text. of **Na in region C at the same tsm value. Notations
and
DH =
0.1
energy
regions
A and B
sults indicated that 40 times more radioactivity is required for 3H than for 22Na and 20 times more radioactivity is needed for 14C than for 22Na to obtain meaningful countings (dpm recovery deviation smaller than 10% for 3H and 14C as shown in Fig. 6). The reliability of the method presented was examined in the halobacterial 22Na transport system. Light-driven 22Na efflux mediated by the Na+ /H+ antiporter in the
Present
2
Ezp cu
Conventional method
method
100
0
34 -100 ZE v -200
f -300
' 100
I 200
Quenching
1 300
I
I
I
400
500
Index (tSIE)
FIG. 4. Quenching effect on dpm calculation. Standard samples containing3H, “C, and *‘Na at different quench levels were measured for 5 min by the Tri-Carb 2200A LSC system using the measuring condition described in the text. The dpm values were computed according to both our method and the conventional “DPM-1-2-3” method. The percentage recoveries of dpm (deviations from the standard dpm) are compared.
198
MIYAZAWA
ET
AL.
. I . t . . ma
-10
0
500
1000
1500
2000
2500
3000
22 Na (dpm)
FIG. 5.
Minimal counting sensitivity for “Na. Standard samples contained various amounts of 3H, W, and “Na at different tsIE and were measured for 5 min as in Fig. 4. The dpm recovery was computed by the method presented in the paper.
100
80
halobacterial membrane vesicles was measured using 14C-salicylate and 3H-TPP+ as ApH and A!# probes, respectively. Results in Fig. 7 clearly show that the 40
8.
I.
8.
8.
I.,
1.
I.
60
I.
30
I
01
-2 20
2
0
TIME
4
6
(min)
FIG. 7.
Kinetic changes of ApH, A*, and intravesicular 22Na in halobacterial membrane vesicle upon illumination. Membrane vesicles ( 10 mg/ml) from H. halobium R,M, were suspendedin 2.9 M KC1 and 0.1 M *‘NaCI in 5 mM Pipes buffer containing5 FM 3H-TPP+, 200 pM 14C-salicylate. After equilibration, the membranes were placed on membrane filter (0.45-pm pore size) under reduced pressure at defined time periods before and after illumination (at the arrow). Radioactivity measurement and dpm calculation were according to the method described in the text. Data transformation into ApH and A* was carried out according to the Nernst equation described previously (9).
10
0
0
20
40
60
60
dpm
100
ratio
120
of
140
160
160
200
3~1 ,,Na
60 40 30
---.t--M.----------07
20
_.--__---.---__-
______.__.____.___ -~-___ I_--_-
10
e
l -
Oz
--.--?lL--
l
--
-106ow -20
1 0
I 20
40
dpm
FIG. 6.
__--
-___8-
ratio
60
of
60
method presented in this paper is useful for following the kinetic changes of intravesicular “Na contents, ApH, and A\k when these modes are simultaneously affected by illumination. “Na extruded coincidentally with ApH formation. A%, on the other hand, increased to the maximum level immediately after illumination, then gradually decreased as intravesicular NaC depleted. The profiles of the kinetic changes of these three parameters are essentially the same as those obtained previously with the use of electrodes (9).
“C122Na
Effect of dpm ratio of 3H and 14C to 22Na on dpm recovery. The standard samples and the counting conditions are the same as those given in the legend to Fig. 5.
DISCUSSION A triple-nuclide
tracer is optionally carried out using a software program equipped in the Tri-Carb LSC sys-
TRIPLE-TRACER
TECHNIQUE
FOR
tern, such as DPM l-2-3. The latter system employs the Gauss-Jordan matrix elimination method to compute individual radionuclide dpm from the cpm and the counting efficiency of each nuclide in regions A, B, and C as used for the double-tracer calculation ( 10). However, the method has an inherent disadvantage. That is, the dpm recovery significantly deviates in the lowest and highest energy nuclides as the quench level of the samples become high (as shown in Fig. 4). This disadvantage is overcome by an adequate setting of the counting regions for the three nuclides and by a simple subtraction of the “Na (the highest energy nuclide) spilldown counts from the gross counts in regions A and B. The adjustments are made prior to 3H and 14C dpm calculation. Bioenergetic parameters such as A* and ApH are routinely obtained from the equilibrium distribution of membrane probes. The equilibrium distribution of membrane probes is determined by a radiotracer or fluorescence or electrode techniques ( 11). Experimental conditions such as probe concentration differ depending on the method used. Therefore, it is generally difficult to adapt the data obtained by different techniques to the same membrane preparation, For example, using data from different techniques makes it difficult to evaluate the quantitative relationship between the thermodynamic parameters and the net change of transport substrate in the transport system being analyzed. The LSC method presented for determining triple tracers makes it possible to quantitatively discuss the above parameters in the same biological preparation, even in highly quenched samples. Therefore, the method would be quite useful for studying the energy coupling mecha-
CELL
ION
TRANSPORT
199
STUDY
nism of certain cellular transport systems, the Na+-coupled transport. Although the present studies were carried the Tri-Carb 2200A LSC system, the method can be employed with any LSC apparatus. method can be used to study other nuclides in ing fl spectra that are of interest.
especially out using presented Also, the determin-
REFERENCES 1. Mitchell, P. ( 1977) Ann. Reu. Biochem. 46,996-1005. 2. Konishi, T., Murakami, N., Hatano, Y., and Nakazato, K. (1986) Biochim. Biophys. Acta. 862, 278-284. 3. Kamo, N., Racanell, T., and Packer, L. (1982) in Methods in Enzymology (Packer, L., Ed.), Vol. 88, pp. 356-360, Academic Press, San Diego. 4. Kessler, M., and Toggenburger, G. ( 1979) in Membrane Biochemistry (Carafoli, E., and Semenza, G., Eds.), pp. l-24, SpringerVerlag, New York /Berlin. 5. Bally, M. B., Hope, M. J., VanEchteld, C. JA., and Cullis, P. R. ( 1985) Biochim. Biophys. Actu 812,66-76. 6. Ramos, S., Schuldiner, S., and Kaback, H. R. (1979) in Enzymology (Fleiscber, S., and Packer, L., Eds.), 680-688, Academic Press, San Diego.
in Methods Vol. 55, pp.
7. Mies, G., Bodsch, W., Paschen, W., and Hossmann, K. A. (1986) J. Cereb. Blood Flow Metab. 6, 59-70. 8. (a) Nakai, H., Diksic, M., and Yamamoto, Y. L. (1988) Stroke 19, 758-763; (b) Nakai, H., Yamamoto, Y. L., Diksic, M., Worsley, K. J., and Takara, E. (1988) Stroke 19, 764-772. 9. Murakami, 231-236.
N., and Konishi,
10. Packerd Instrument DPM l-2-3 Option. 11. Creamer, Biological
T. (1988)
Co., Inc. ( 1987)
J. Biochem. Packard
W. A., and Knaff, D. B. (1989) Membranes, Springer-Verlag,
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Operation
Manual,
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in