ANALYTICAL
BIOCHEMISTRY
189,138-141
(1990)
An Ultrafiltration Assay for Nucleotide to Ribonucleotide Reductase’ Mats
Ormij
Department
Received
and Britt-Marie of Molecular
March
Biology,
Sjijberg University
of Stockholm,
S-106 91 Stockholm,
Press,
Inc.
The intricate nucleotide binding properties of ribonucleotide reductase (EC 1.17.4.1.) have since long been an object of great interest to many groups. The enzyme plays a central role in every living cell by catalyzing the formation of DNA precursors from their corresponding ribonucleotides. To support a balanced production of all four types of deoxyribonucleotides, the enzyme is put under strict control by its allosteric effecters (1). Ribonucleotide reductase from Escherichia coli consists of two different proteins, each built up by two identical subunits. The large protein, denoted Bl, has a molecular weight of 171,500. It contains the binding sites for substrates and effecters and at least two different classes of redox-active cysteines (2-4). The small protein, named B2, with a molecular weight of 86,800 contributes the stable tyrosine free radical which participates in the irreversible reduction at the 2’ position in the ribose moiety (5,6).
Besides two binding sites for ribonucleoside diphosphate substrates, protein Bl carries two pairs of allosteric binding sites for nucleoside triphosphate effectors. The allosteric sites are defined as high- or low-affinity sites depending on their affinity for dATP. The high-affinity sites determine the substrate specificity and bind ATP, dATP, dTTP, and dGTP. The low affinity sites regulate the overall enzyme activity and bind ATP and dATP. For recent reviews on the allosteric regulation of ribonucleotide reductase, see Reichard (1) and Eriksson and Sjijberg (7). The complicated, but fascinating regulatory pattern of ribonucleotide reductase was originally unraveled with such techniques as rapid-rate dialysis and gel chromatography (a), affinity chromatography (9), equilibrium dialysis (lo), and nitrocellulose filter binding (11). Although these methods have proveduseful for determination of binding constants, they all have certain limitations: some are time consuming and require low temperatures to avoid protein inactivation, others consume much material, and one is based on chemically active filters that may change protein characteristics and have a high unspecific nucleotide binding. We hereby present a method for the study of nucleotide binding to ribonucleotide reductase based on direct partition through ultrafiltration. The method combines the speed of a centrifugation step with the reliability of separation of bound and unbound ligand by an inert membrane. In this paper the technique has been used to study binding of substrate and effector nucleotides to overproduced protein Bl from E. coli. The results correlate well and extend earlier studies made on the bacterial enzyme from wild-type, thymine-starved, and overproducing cells. MATERIALS
1 This ety. 138
work
Sweden
21. 1990
Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4°C with dissociation constants ranging from 0.1 to 80 PM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25°C. o 1990 Academic
Binding
was supported
by grants
from
the Swedish
Cancer
Soci-
AND
METHODS
Materials. Protein Bl and protein B2 were obtained from an overproducing E. coli strain carrying the nrdA gene or the nrdI3 gene in a runaway vector (12,13). Puri0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ULTRAFILTRATION
ASSAY
fication was according to standard procedures as described elsewhere (2,13,14). [2,8-3H]ATP, [8-3H]dATP, [8-3H]dGTP, [methyl-3H]dTTP, and [8-3H]GDP were purchased from Amersham, have purities of 97.398.4%, and were diluted to a specific activity of 0.5-20 Bq/pmol with more than 98% pure nucleotides from Pharmacia. No further purifications of nucleotides were performed. The Ultrafree-MC filter units with polysulfone PTTK membranes, molecular cutoff 30,000, were obtained from Millipore. Binding experiments were carried out at 25 or 4°C in a buffer containing 50 mM Tris-HCl, pH 7.6, 10 mM MgCl,, and 2 mM dithiothreitol. Nucleotide concentrations were typically 0.1-15 PM. In experiments with nucleotides having high Kd values, e.g., binding of dATP to low-affinity sites, GDP and ATP, the nucleotide concentration was increased to up to 40, 100, and 300 PM, respectively. The amount of protein in the assay was chosen so that more than 10% and less than 80% of total nucleotide concentration was bound, giving protein Bl concentrations ranging from 0.15 to 18.5 PM. With a sample volume of 150 ~1 this resulted in the use of between 4 and 475 pg Bl protein for each point in a Scatchard plot, depending on the dissociation constant for the nucleotide of interest. Procedure for binding experiments. Protein Bl was added to pretempered solutions to a final volume of 150 ~1, followed by an incubation for 5 min at 25°C. Aliquots of 30 ~1 were withdrawn for scintillation counting and the remaining 120 ~1 were transferred to the upper sample reservoirs of the Ultrafree-MC filter units. After centrifugation at 6500 rpm (3300g) for 30-60 s in a MSE Micro-Centaur Eppendorf tube centrifuge, aliquots of 30 ~1 were withdrawn from the filtrate volume (approximately 40-50 ~1) for scintillation counting and calculation of the free nucleotide concentration. Experiments at 4°C were made in a cold room in the same manner as described for experiments made at 25”C, except that the incubation time was increased to 10 min and the centrifugation time to 45-90 s. The amount of bound nucleotide was calculated as the difference between total and filtrate nucleotide concentration. Binding ratios were calculated from a molecular mass for protein Bl of 171,477 Da. A molar absorption index of 180,000 M-lcm-’ (c280-310)was used for protein Bl. Ribonucleotide reductase activity was determined spectrophotometrically as oxidation of NADPH with CDP as substrate (14). The reaction mixture contained 0.5 mM CDP, 1.5 mM ATP, 11 mM MgCl,, 0.4 mM NADPH, 33 mM Hepes, pH 7.6, 13 pM thioredoxin, 0.5 PM thioredoxin reductase, 0.5 PM protein B2, and 70 IIM protein Bl. RESULTS
Protein Bl activity After an incubation
was constant during experiments. for 10 min at 25°C in the assay
FOR
NUCLEOTIDE
139
BINDING
k I> :
0.6 0.4
0-
0
20
40 Filtrate
60 volume
80
100
120
(bl)
FIG. 1. The influence on binding ratio by the filtrate volume. Binding was assayed at different experiments where the amount of filtrate was varied by increasing the centrifugation time up to 160 s. The experiments were carried out at 25°C with a dGTP concentration of 2 pM and a protein Bl concentration of 1.5 KM.
buffer, the specific Bl activity was the same as in the starting material. Thus, no corrections for protein inactivation were necessary. The retention of protein Bl by the filters was total. No ribonucleotide reductase activity could be detected in the filtrate after a 60-s centrifugation at 25°C of a 2.9 FM protein Bl solution. The absence of unspecific nucleotide binding to filters was established in two different experiments. In the first, buffers containing variable concentrations of nucleotides were centrifuged through the filters. In a second experiment, protein B2, which has no nucleotide binding capacity, was used at a concentration of 3.8 PM. There was no detectable unspecific nucleotide binding in either experiment and consequently no corrections for filter binding had to be introduced. Two other types of filters were also tested, PLGC filters from Millipore and YMT filters from Amicon, both with a molecular cutoff of 30,000. Those filters, however, turned out to have some unspecific nucleotide affinity. In the use of these filters corrections for unspecific binding had to be considered, and in the case of Amicon YMT filters differences between batches were considerable and had to be accounted for. The centrifugation time and consequently the amount of sample that was passed through the filter did not change the binding ratio (see Fig. 1). Even in the case where 90% of the sample volume was forced through the filter, no changes were evident. However, to avoid unnecessary molecular crowding at the filter surface and dramatically increased protein concentrations, filtrate volumes were kept to a minimum. The ideal total sample volume of 150 ~1 was chosen after a minimization experiment, in which the volume was varied from 50 to 250 ~1. Volumes below 150 ~1 gave slightly lower binding ratios, were harder to handle, and gave lower accuracy due to the small volumes to be withdrawn for scintillation counting. Figure 2 shows Scatchard plots for the binding of dTTP, dGTP, dATP, and ATP to protein Bl at 25°C.
140
ORMij
AND
FIG. 2. Scatchards plots for binding of nucleotides to protein Bl at 25°C where L is the concentration of free ligand and U is the amount of moles of bound ligand per mole of protein Bl. All curves were calculated by linear least-squares fit. (A) dTTP binding; protein Bl concentration 1.85 pM (Cl). (B) dATP binding; protein Bl concentration, 0.63 pM (0) and 3.7 pM (0). The regression lines for the highand low-affinity sites are calculated from the first five and last three points, respectively. (C) dGTP binding; protein Bl concentration, 1.85 pM (0). (D) ATP binding; protein Bl concentration, 4.5 pM (W), 9.3 pM (A), and 18.5 PM (6). The plots (A), (B), and (C) were obtained from two different experiments, while plot (D) is a compilation of three different experiments.
Figures 2A and 2C show the high-affinity sites binding of dTTP (I& = 1.9 PM) and dGTP (Kd = 0.77 PM). In Fig. 2B, dATP binding displays the two types of binding site. The first part of the curve extrapolates to two binding sites with high affinity (Kd = 0.43 PM) and the second part to two additional binding sites with lower affinity (& = 6 PM). Binding of ATP to protein Bl is depicted in Fig. 2D and shows binding to four sites with a dissociation constant of 80 PM. Binding parameters for nucleotide binding to protein Bl at 25 and 4°C are presented in Table 1. Effector binding at 4°C was generally four to six times stronger than at 25°C. This was also true for dGTP where binding has only been measured at 2°C before. A similar temperature-dependent difference in dissociation constant was not found for the binding of the substrate GDP which, in the presence of the positive effector dTTP, had essentially the same affinity at both temperatures. DISCUSSION
In a study on Mg2+ binding to concavalin Sophianopoulos et al. showed that ultrafiltration is theoretically equivalent to equilibrium dialysis (15). Problems one could meet with ultrafiltration which would invalidate the statement above are: if the molecular crowding at the filter surface changes the binding properties of the
SJijBERG
protein or if the protein being retained at the filter surface acts as a second membrane, thereby changing the properties of the filter. The latter problem can be partially dealt with by centrifugation in a fixed angle rotor which successively removes the protein from the filter surface to one side of the filter. In the present study no such complications were seen. The assay was, on the contrary, very insensitive to such phenomena and the binding ratio did not vary even during prolonged centrifugation (Fig. 1). In a stirred ultrafiltration cell system, recalculations of the protein concentration are carried out consecutively as a part of the assay, depending on the removal of filtrated volume. In the type of assay described here, with a nonstirred ultrafiltration unit and with separation achieved in 30-60 s, this correction is apparently not necessary. Perhaps the increase in protein concentration is initially limited to the filter surface and therefore does not affect the binding ratio in the rest of the volume. Since direct partition through ultrafiltration reduces manipulation times down to a couple of minutes, timedependent complications such as protein inactivation and nucleotide breakdown should be negligible. This allows for experiments at physiological temperatures, which definitely are preferred with ribonucleotide reductase. It has been shown for ribonucleotide reductase from E. coli that the activation energies for the reduction of substrates changes at low temperature (16) and therefore binding experiments at lower temperatures should, if possible, be avoided. The binding constants, obtained here with the ultrafiltration technique at 4°C (Table l), correspond well to earlier studies by Brown and Reichard (8), von Dijbeln
TABLE
1
Binding of Effecters and Substrate to Ribonucleotide Reductasefrom E. coli Temperature 25°C Nucleotide dTTP” dGTP” dATP” h 1 ATP’ GDP’
Kd (PM) 1.9 0.77 0.43 6 80 24
4°C n 1.8 1.9 1.8 3.7b 3.7 2.0
K+ (PM)
n
0.50 0.21
1.7 2.0
0.11 1 13d 26
1.9 3.6* 3.3 2.0
(1 The Kd values come from two or more determinations. * The n value includes binding to the two high affinity sites (h). ’ Kd values were obtained by compiling three different experiments. d From the final slope of the binding curve. c GDP binding was measured as a single experiment in the presence of 40 pM dTTP with three or four different concentrations of GDP.
ULTRAFILTRATION
ASSAY
FOR
NUCLEOTIDE
141
BINDING
and Reichard (16), and Siiderman and Reichard (11) made at 0 or FZ’C.~ In those experiments, dATP bound to 1.6-1.85 high-affinity sites with dissociation constants of 0.03-0.06 PM. Additional binding to low-affinity sites with dissociation constants between 0.5 and 0.8 pM extrapolated to a total of 3.6 to 3.7 sites. dTTP and dGTP showed, in those studies, 1.4-1.8binding sites with dissociation constants of 0.3-0.4 PM for dTTP and 0.08 PM for dGTP. Brown and Reichard (8) found the binding of ATP to be cooperative at low temperatures (& = 10 PM), but due to the high dissociation constants it was impossible to study its Kd at ambient temperature. With the present method, we observe the same cooperative effects of ATP binding at low temperatures. However, due to the advantages of the present method we have also measured binding of ATP to protein Bl at 25°C and the cooperative effects are much less pronounced. This suggests that the cooperative effects of ATP binding have very little or no physiological importance. Substrate binding to protein Bl was earlier performed in the presence of nucleoside triphosphate analogues to avoid the kinase activity found in the preparations at that time (16). Binding of GDP with a dTTP analogue as effector at 0 and 20°C showed, in those experiments binding to 1.3-1.8 sites with dissociation constants in the range of 22-26 PM. This corresponds well to the data obtained in the present study, which also shows that the binding of substrate (in the presence of effecters), in contrast to the binding of effecters, is not affected by temperature. In summary the rapid and simple ultrafiltration method presented here gives results in excellent agreement with earlier data acquired with other techniques. Since it is based on a quick separation over a chemically inert filter, no corrections have to be made for protein breakdown and unspecific filter binding. This makes the assay straightforward and easy to use, and thereby extends its applications beyond earlier techniques to
the use of physiological temperatures and nucleotide ligands with dissociation constants on the order of 100 pM. Also considering the low amounts of protein needed and the short duration of a binding experiment, the method should be ideal for measuring nucleotide binding to ribonucleotide reductases from other species, which may be less stable and not yet available in an overproduced form. In addition, the method will be of great use in the characterization of substrate and effector binding to catalytically defective mutant proteins, and in the study of the effector binding surfaces of ribonucleotide reductase.
2 The 200,000
16. von Dobeln, 3616-3622.
molecular [Ref. (S)],
weights 160,000
of protein [Ref. (IS)],
Bl used in these studies and 170,000 [Ref. (ll)].
were
REFERENCES 1. Reichard, P. (1987) Biochemistry 26,3245-3248. 2. Aberg, A., Hahne, S., Karlsson, M., Larsson, A., Ormo, M., Ahgren, A., and Sjiiberg, B.-M. (1989) J. Biol. Chem. 264,12,24912,252. 3. Mao, S. S., Johnston, J. M., Bollinger, J. M., and Stubbe, Proc. Natl. Acad. Sci. USA 86, 1485-1489. 4. Stubhe, J. (1990) J. Biol. Chem., 265,5329-5332.
J. (1989)
5. Sjiiberg, B.-M., Griislund, A., and Eckstein, F. (1983) J. Biol. Chem. 258,8060-8067. 6. Stubbe, J. (1989) in Advances in Enzymology (Meister, A., Ed.), Vol. 63, pp. 349-417, Academic Press, New York. 7. Eriksson, S., and SjBberg, B.-M. (1989) in Allosteric Enzymes (He&, G., Ed.), pp. 189-215, CRC Press, Florida. a. Brown, N. C., and Reichard, P. (1969) J. Mol. Biol. 9. von Dobeln, U. (1977) Biochemistry 16,4368-4371. 10. Eriksson, S. (1983) J. Biol. Chem. 258,5674-5678. 11. Soderman, K., and Reichard, P. (1986) 93. 12. Larsson, A. (1984) Acta Chem. Stand., 13. Sjoberg, B.-M., Hahne, S., Karlsson, M., and Uhlin, B. E. (1986) J. Biol. 14. Thelander, L., Sjoberg, B.-M., and ods Enzymology (Hoffee, P. A., and pp. 227-237, Academic Press, New
Anal.
46,
Biochem.
152,89-
Ser. B 38,905-907.
M., Jiirnvall, H., Giiransson, &em. 261,5658-5662. Eriksson, S. (1978) in MethJones, M. E., Eds.), Vol. 51, York.
15. Sophianopoulos, J. A., Durham, S. J., Sophianopoulos, Ragsdale, H. L., and Cropper, W. P., Jr. (1978) Arch. Biophys. 187, 132-137. U., and
Reichard,
39-55.
P. (1976)
J. Biol.
A. J., Biochem.
&em.
261,
BIOCHEMISTRY
189,138-141
(1990)
An Ultrafiltration Assay for Nucleotide to Ribonucleotide Reductase’ Mats
Ormij
Department
Received
and Britt-Marie of Molecular
March
Biology,
Sjijberg University
of Stockholm,
S-106 91 Stockholm,
Press,
Inc.
The intricate nucleotide binding properties of ribonucleotide reductase (EC 1.17.4.1.) have since long been an object of great interest to many groups. The enzyme plays a central role in every living cell by catalyzing the formation of DNA precursors from their corresponding ribonucleotides. To support a balanced production of all four types of deoxyribonucleotides, the enzyme is put under strict control by its allosteric effecters (1). Ribonucleotide reductase from Escherichia coli consists of two different proteins, each built up by two identical subunits. The large protein, denoted Bl, has a molecular weight of 171,500. It contains the binding sites for substrates and effecters and at least two different classes of redox-active cysteines (2-4). The small protein, named B2, with a molecular weight of 86,800 contributes the stable tyrosine free radical which participates in the irreversible reduction at the 2’ position in the ribose moiety (5,6).
Besides two binding sites for ribonucleoside diphosphate substrates, protein Bl carries two pairs of allosteric binding sites for nucleoside triphosphate effectors. The allosteric sites are defined as high- or low-affinity sites depending on their affinity for dATP. The high-affinity sites determine the substrate specificity and bind ATP, dATP, dTTP, and dGTP. The low affinity sites regulate the overall enzyme activity and bind ATP and dATP. For recent reviews on the allosteric regulation of ribonucleotide reductase, see Reichard (1) and Eriksson and Sjijberg (7). The complicated, but fascinating regulatory pattern of ribonucleotide reductase was originally unraveled with such techniques as rapid-rate dialysis and gel chromatography (a), affinity chromatography (9), equilibrium dialysis (lo), and nitrocellulose filter binding (11). Although these methods have proveduseful for determination of binding constants, they all have certain limitations: some are time consuming and require low temperatures to avoid protein inactivation, others consume much material, and one is based on chemically active filters that may change protein characteristics and have a high unspecific nucleotide binding. We hereby present a method for the study of nucleotide binding to ribonucleotide reductase based on direct partition through ultrafiltration. The method combines the speed of a centrifugation step with the reliability of separation of bound and unbound ligand by an inert membrane. In this paper the technique has been used to study binding of substrate and effector nucleotides to overproduced protein Bl from E. coli. The results correlate well and extend earlier studies made on the bacterial enzyme from wild-type, thymine-starved, and overproducing cells. MATERIALS
1 This ety. 138
work
Sweden
21. 1990
Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4°C with dissociation constants ranging from 0.1 to 80 PM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25°C. o 1990 Academic
Binding
was supported
by grants
from
the Swedish
Cancer
Soci-
AND
METHODS
Materials. Protein Bl and protein B2 were obtained from an overproducing E. coli strain carrying the nrdA gene or the nrdI3 gene in a runaway vector (12,13). Puri0003-2697/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ULTRAFILTRATION
ASSAY
fication was according to standard procedures as described elsewhere (2,13,14). [2,8-3H]ATP, [8-3H]dATP, [8-3H]dGTP, [methyl-3H]dTTP, and [8-3H]GDP were purchased from Amersham, have purities of 97.398.4%, and were diluted to a specific activity of 0.5-20 Bq/pmol with more than 98% pure nucleotides from Pharmacia. No further purifications of nucleotides were performed. The Ultrafree-MC filter units with polysulfone PTTK membranes, molecular cutoff 30,000, were obtained from Millipore. Binding experiments were carried out at 25 or 4°C in a buffer containing 50 mM Tris-HCl, pH 7.6, 10 mM MgCl,, and 2 mM dithiothreitol. Nucleotide concentrations were typically 0.1-15 PM. In experiments with nucleotides having high Kd values, e.g., binding of dATP to low-affinity sites, GDP and ATP, the nucleotide concentration was increased to up to 40, 100, and 300 PM, respectively. The amount of protein in the assay was chosen so that more than 10% and less than 80% of total nucleotide concentration was bound, giving protein Bl concentrations ranging from 0.15 to 18.5 PM. With a sample volume of 150 ~1 this resulted in the use of between 4 and 475 pg Bl protein for each point in a Scatchard plot, depending on the dissociation constant for the nucleotide of interest. Procedure for binding experiments. Protein Bl was added to pretempered solutions to a final volume of 150 ~1, followed by an incubation for 5 min at 25°C. Aliquots of 30 ~1 were withdrawn for scintillation counting and the remaining 120 ~1 were transferred to the upper sample reservoirs of the Ultrafree-MC filter units. After centrifugation at 6500 rpm (3300g) for 30-60 s in a MSE Micro-Centaur Eppendorf tube centrifuge, aliquots of 30 ~1 were withdrawn from the filtrate volume (approximately 40-50 ~1) for scintillation counting and calculation of the free nucleotide concentration. Experiments at 4°C were made in a cold room in the same manner as described for experiments made at 25”C, except that the incubation time was increased to 10 min and the centrifugation time to 45-90 s. The amount of bound nucleotide was calculated as the difference between total and filtrate nucleotide concentration. Binding ratios were calculated from a molecular mass for protein Bl of 171,477 Da. A molar absorption index of 180,000 M-lcm-’ (c280-310)was used for protein Bl. Ribonucleotide reductase activity was determined spectrophotometrically as oxidation of NADPH with CDP as substrate (14). The reaction mixture contained 0.5 mM CDP, 1.5 mM ATP, 11 mM MgCl,, 0.4 mM NADPH, 33 mM Hepes, pH 7.6, 13 pM thioredoxin, 0.5 PM thioredoxin reductase, 0.5 PM protein B2, and 70 IIM protein Bl. RESULTS
Protein Bl activity After an incubation
was constant during experiments. for 10 min at 25°C in the assay
FOR
NUCLEOTIDE
139
BINDING
k I> :
0.6 0.4
0-
0
20
40 Filtrate
60 volume
80
100
120
(bl)
FIG. 1. The influence on binding ratio by the filtrate volume. Binding was assayed at different experiments where the amount of filtrate was varied by increasing the centrifugation time up to 160 s. The experiments were carried out at 25°C with a dGTP concentration of 2 pM and a protein Bl concentration of 1.5 KM.
buffer, the specific Bl activity was the same as in the starting material. Thus, no corrections for protein inactivation were necessary. The retention of protein Bl by the filters was total. No ribonucleotide reductase activity could be detected in the filtrate after a 60-s centrifugation at 25°C of a 2.9 FM protein Bl solution. The absence of unspecific nucleotide binding to filters was established in two different experiments. In the first, buffers containing variable concentrations of nucleotides were centrifuged through the filters. In a second experiment, protein B2, which has no nucleotide binding capacity, was used at a concentration of 3.8 PM. There was no detectable unspecific nucleotide binding in either experiment and consequently no corrections for filter binding had to be introduced. Two other types of filters were also tested, PLGC filters from Millipore and YMT filters from Amicon, both with a molecular cutoff of 30,000. Those filters, however, turned out to have some unspecific nucleotide affinity. In the use of these filters corrections for unspecific binding had to be considered, and in the case of Amicon YMT filters differences between batches were considerable and had to be accounted for. The centrifugation time and consequently the amount of sample that was passed through the filter did not change the binding ratio (see Fig. 1). Even in the case where 90% of the sample volume was forced through the filter, no changes were evident. However, to avoid unnecessary molecular crowding at the filter surface and dramatically increased protein concentrations, filtrate volumes were kept to a minimum. The ideal total sample volume of 150 ~1 was chosen after a minimization experiment, in which the volume was varied from 50 to 250 ~1. Volumes below 150 ~1 gave slightly lower binding ratios, were harder to handle, and gave lower accuracy due to the small volumes to be withdrawn for scintillation counting. Figure 2 shows Scatchard plots for the binding of dTTP, dGTP, dATP, and ATP to protein Bl at 25°C.
140
ORMij
AND
FIG. 2. Scatchards plots for binding of nucleotides to protein Bl at 25°C where L is the concentration of free ligand and U is the amount of moles of bound ligand per mole of protein Bl. All curves were calculated by linear least-squares fit. (A) dTTP binding; protein Bl concentration 1.85 pM (Cl). (B) dATP binding; protein Bl concentration, 0.63 pM (0) and 3.7 pM (0). The regression lines for the highand low-affinity sites are calculated from the first five and last three points, respectively. (C) dGTP binding; protein Bl concentration, 1.85 pM (0). (D) ATP binding; protein Bl concentration, 4.5 pM (W), 9.3 pM (A), and 18.5 PM (6). The plots (A), (B), and (C) were obtained from two different experiments, while plot (D) is a compilation of three different experiments.
Figures 2A and 2C show the high-affinity sites binding of dTTP (I& = 1.9 PM) and dGTP (Kd = 0.77 PM). In Fig. 2B, dATP binding displays the two types of binding site. The first part of the curve extrapolates to two binding sites with high affinity (Kd = 0.43 PM) and the second part to two additional binding sites with lower affinity (& = 6 PM). Binding of ATP to protein Bl is depicted in Fig. 2D and shows binding to four sites with a dissociation constant of 80 PM. Binding parameters for nucleotide binding to protein Bl at 25 and 4°C are presented in Table 1. Effector binding at 4°C was generally four to six times stronger than at 25°C. This was also true for dGTP where binding has only been measured at 2°C before. A similar temperature-dependent difference in dissociation constant was not found for the binding of the substrate GDP which, in the presence of the positive effector dTTP, had essentially the same affinity at both temperatures. DISCUSSION
In a study on Mg2+ binding to concavalin Sophianopoulos et al. showed that ultrafiltration is theoretically equivalent to equilibrium dialysis (15). Problems one could meet with ultrafiltration which would invalidate the statement above are: if the molecular crowding at the filter surface changes the binding properties of the
SJijBERG
protein or if the protein being retained at the filter surface acts as a second membrane, thereby changing the properties of the filter. The latter problem can be partially dealt with by centrifugation in a fixed angle rotor which successively removes the protein from the filter surface to one side of the filter. In the present study no such complications were seen. The assay was, on the contrary, very insensitive to such phenomena and the binding ratio did not vary even during prolonged centrifugation (Fig. 1). In a stirred ultrafiltration cell system, recalculations of the protein concentration are carried out consecutively as a part of the assay, depending on the removal of filtrated volume. In the type of assay described here, with a nonstirred ultrafiltration unit and with separation achieved in 30-60 s, this correction is apparently not necessary. Perhaps the increase in protein concentration is initially limited to the filter surface and therefore does not affect the binding ratio in the rest of the volume. Since direct partition through ultrafiltration reduces manipulation times down to a couple of minutes, timedependent complications such as protein inactivation and nucleotide breakdown should be negligible. This allows for experiments at physiological temperatures, which definitely are preferred with ribonucleotide reductase. It has been shown for ribonucleotide reductase from E. coli that the activation energies for the reduction of substrates changes at low temperature (16) and therefore binding experiments at lower temperatures should, if possible, be avoided. The binding constants, obtained here with the ultrafiltration technique at 4°C (Table l), correspond well to earlier studies by Brown and Reichard (8), von Dijbeln
TABLE
1
Binding of Effecters and Substrate to Ribonucleotide Reductasefrom E. coli Temperature 25°C Nucleotide dTTP” dGTP” dATP” h 1 ATP’ GDP’
Kd (PM) 1.9 0.77 0.43 6 80 24
4°C n 1.8 1.9 1.8 3.7b 3.7 2.0
K+ (PM)
n
0.50 0.21
1.7 2.0
0.11 1 13d 26
1.9 3.6* 3.3 2.0
(1 The Kd values come from two or more determinations. * The n value includes binding to the two high affinity sites (h). ’ Kd values were obtained by compiling three different experiments. d From the final slope of the binding curve. c GDP binding was measured as a single experiment in the presence of 40 pM dTTP with three or four different concentrations of GDP.
ULTRAFILTRATION
ASSAY
FOR
NUCLEOTIDE
141
BINDING
and Reichard (16), and Siiderman and Reichard (11) made at 0 or FZ’C.~ In those experiments, dATP bound to 1.6-1.85 high-affinity sites with dissociation constants of 0.03-0.06 PM. Additional binding to low-affinity sites with dissociation constants between 0.5 and 0.8 pM extrapolated to a total of 3.6 to 3.7 sites. dTTP and dGTP showed, in those studies, 1.4-1.8binding sites with dissociation constants of 0.3-0.4 PM for dTTP and 0.08 PM for dGTP. Brown and Reichard (8) found the binding of ATP to be cooperative at low temperatures (& = 10 PM), but due to the high dissociation constants it was impossible to study its Kd at ambient temperature. With the present method, we observe the same cooperative effects of ATP binding at low temperatures. However, due to the advantages of the present method we have also measured binding of ATP to protein Bl at 25°C and the cooperative effects are much less pronounced. This suggests that the cooperative effects of ATP binding have very little or no physiological importance. Substrate binding to protein Bl was earlier performed in the presence of nucleoside triphosphate analogues to avoid the kinase activity found in the preparations at that time (16). Binding of GDP with a dTTP analogue as effector at 0 and 20°C showed, in those experiments binding to 1.3-1.8 sites with dissociation constants in the range of 22-26 PM. This corresponds well to the data obtained in the present study, which also shows that the binding of substrate (in the presence of effecters), in contrast to the binding of effecters, is not affected by temperature. In summary the rapid and simple ultrafiltration method presented here gives results in excellent agreement with earlier data acquired with other techniques. Since it is based on a quick separation over a chemically inert filter, no corrections have to be made for protein breakdown and unspecific filter binding. This makes the assay straightforward and easy to use, and thereby extends its applications beyond earlier techniques to
the use of physiological temperatures and nucleotide ligands with dissociation constants on the order of 100 pM. Also considering the low amounts of protein needed and the short duration of a binding experiment, the method should be ideal for measuring nucleotide binding to ribonucleotide reductases from other species, which may be less stable and not yet available in an overproduced form. In addition, the method will be of great use in the characterization of substrate and effector binding to catalytically defective mutant proteins, and in the study of the effector binding surfaces of ribonucleotide reductase.
2 The 200,000
16. von Dobeln, 3616-3622.
molecular [Ref. (S)],
weights 160,000
of protein [Ref. (IS)],
Bl used in these studies and 170,000 [Ref. (ll)].
were
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