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Published online before print June 2, 2006, 10.1110/ps.062183206
Protein Science (2006), 15:1794-1800. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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FOR THE RECORD

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy

Anna Modrak-Wójcik1, Katarzyna Stepniak1, Vladimir Akoev2, Michal Zólkiewski2 and Agnieszka Bzowska1

1 Department of Biophysics, Institute of Experimental Physics, University of Warsaw, 02-089 Warsaw, Poland
2 Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA

(RECEIVED February 22, 2006; FINAL REVISION March 16, 2006; ACCEPTED March 16, 2006)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150 ± 10 kDa, regardless of the conditions investigated—protein concentration, 0.18–1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4–20°C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 µg/mL–1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s020,w = 7.7 ± 0.3 and 8.3 ± 0.4 S, respectively. The molecular mass of the sedimenting particle (146 ± 30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.

Keywords: purine nucleoside phosphorylase (PNP); oligomeric state; analytical ultracentrifugation; CD spectroscopy; active enzyme sedimentation


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
The ubiquitous purine nucleoside phosphorylase (PNP, purine nucleoside:ortho-phosphate ribosyl transferase, EC 2.4.2.1.), a key enzyme of the anabolic and catabolic pathways of purine nucleosides, catalyzes the reversible phosphorolytic cleavage of the glycosidic bond of purine nucleosides and their analogs, as follows:

Formula

Inhibitors of PNP are potential immunosuppressive drugs that could be used against the host versus graft reaction in organ transplantation and in T-cell tumors (Stoeckler 1984). PNP inhibitors could also prevent intracellular degradation of some (nucleoside-based) anti-tumor and anti-viral agents (Montgomery et al. 1993). Recently, Escherichia coli PNP has been shown to be a promising candidate for tumor-directed gene therapy (Parker et al. 1997; Deharvengt et al. 2004).

There are two main structural classes of PNPs: homotrimers found mainly in mammals and homohexamers (trimers of dimers) occurring in various micro-organisms (Bzowska et al. 2000). In either class, the phosphorylases contain one complete active site per PNP monomer. In the case of the PNP hexamer, however, the minimal functional unit seems to be a dimer, because each active site contains two residues contributed by the neighboring monomer (Koellner et al. 1998, 2002).

There have been conflicting reports concerning the catalytic activity of monomeric PNP and a possible role of coexistence of various oligomeric species (which might differ in specific activity) in complex kinetic characteristics observed for PNPs from various sources (Bzowska et al. 2000). Jensen and Nygaard (1975) showed that only the hexameric forms of E. coli and S. typhimurium PNP were enzymatically active. In contrast, the analytical ultracentrifugation studies by Nixon et al. (1998) suggested that E. coli PNP exists as an equilibrium mixture of at least two oligomeric forms. Moreover, Ropp and Traut (1991) reported that dilution of the trimeric PNP from calf spleen leads to dissociation of the protein into subunits with a marked increase in specific activity and that phosphate acts as an allosteric regulator of the process. However, recent studies of calf spleen PNP have unequivocally shown that the only active enzyme form is a homotrimer, regardless of the conditions investigated, e.g., the presence of phosphate (Bzowska 2002; Behlke et al. 2005).

In this work, we investigated the oligomeric structure of PNP from E. coli using analytical ultracentrifugation and circular dichroism spectroscopy. We found that hexameric PNP is predominant and stable under a variety of experimental conditions.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
Sedimentation velocity (boundary sedimentation)
Sedimentation velocity experiments were performed to examine the effect of ionic strength, protein concentration, and the presence of phosphate (Pi) on the oligomeric state of PNP in solution. PNP at high (~1.3 mg/mL) and low (~0.03 mg/mL) concentration was centrifuged in Tris or Hepes buffer (pH 7.0) without or with 0.5 M NaCl and 50 mM phosphate.

Figure 1 shows the apparent sedimentation coefficient distributions (g(s020,w)) for two PNP concentrations (0.03 and 1.3 mg/mL) (Fig. 1A) and for the latter concentration, additionally in the presence of 0.5 M NaCl or/and 50 mM Pi (Fig. 1B). All of the distributions in Figure 1 are symmetrical, indicating a single molecular species in solution. Similar results were obtained also in the other experimental conditions mentioned above, leading to the average sedimentation coefficient: Formula = 7.7 ± 0.3S. The molecular mass of the sedimenting particle (146 ± 30 kDa), calculated using the Svedberg equation, is consistent with the single species being a PNP hexamer (predicted molecular weight of 155,000 Da using the monomer molecular mass of 25.8 kDa [Hershfield et al. 1991]). We conclude that hexameric PNP does not dissociate upon ~50-fold dilution, at high ionic strength, or in the presence of phosphate.


Figure 1
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  		Figure 1. Sedimentation coefficient distributions (g(s020,w)) obtained for E. coli PNP. Sedimentation velocity experiments were carried out in (A) 50 mM Tris-HCl (pH 7.0) for 1.3 mg/mL (– –) and 0.03 mg/mL (–) PNP and in 50 mM phosphate buffer (pH 7.0) with 1M GdnHCl for 0.6 mg/mL (- - -) and 0.03 mg/mL ({circ}) PNP and (B) for 1.3 mg/mL PNP in 50 mM Tris-HCl (pH 7.0) without (– –) and with (–) 0.5 M NaCl, with 50 mM sodium phosphate ({circ}), or with 0.5 M NaCl and 50 mM sodium phosphate (- - -). Centrifugation was at 60,000 rpm and 20°C.

 
The crystal structure of E. coli PNP suggested that the assembly of homohexameric PNP could involve association of three dimers (Koellner et al. 2002). Hence, it is possible that under destabilizing conditions PNP can dissociate into dimers that might be catalytically active. To verify this hypothesis, we have examined the effect of guanidine hydrochloride (GdnHCl) on enzymatic activity, circular dichroism (CD) spectra, and sedimentation coefficient of E. coli PNP.

Figure 2A compares CD spectra for PNP (1.75 mg/mL) under the native and the denaturing conditions (0–4 M GdnHCl). Relative values of CD signal at 220 nm and PNP activity toward 0.5 mM m7Guo as a function of GdnHCl concentration are compared in Figure 2B. The correlation between these two parameters is evident—the enzyme activity and secondary structure are lost as the concentration of GdnHCl increases. The transition from the native state of PNP to the random coil is a single-step cooperative process. Figure 2B shows that at 2 M GdnHCl, only ~30% of the enzyme activity remains, whereas PNP is ~98% active at 1 M of GdnHCl. Thus, we have chosen the latter concentration of GdnHCl as the highest concentration of the destabilizing factor at which the enzyme is almost fully active, and we have used these conditions to determine the sedimentation coefficient of PNP.


Figure 2
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  		Figure 2. (A) CD spectra obtained for 1.75 mg/mL PNP in 50 mM phosphate buffer (pH 7.0) without (–) and with 1M (- - -), 2M ({blacktriangleup}), and 4M ({circ}) guanidine hydrochloride (GdnHCl) and for 4 M GdnHCl without PNP (*). (B) Influence of a denaturant (GdnHCl) on enzymatic activity and secondary structure of E. coli PNP. Relative values of CD signal at 220 nm ({square}) and enzymatic activity of PNP with 0.5 mM m7Guo as a nucleoside substrate (•) are presented as a function of GdnHCl concentration.

 
Sedimentation velocity experiments in the presence of 1M GdnHCl were performed for two PNP concentrations (0.63 and 0.03 mg/mL) in 50 mM Pi buffer (pH 7.0). In both cases, sedimentation coefficient distributions (g(s020,w)) are almost symmetrical and their maxima overlap (Fig. 1A). The values of sedimentation coefficient (7.7 ± 0.3 S) agree with those obtained for PNP without the addition of the denaturing factor. This result indicates that even under destabilizing conditions of 1 M GdnHCl, the PNP hexamer remains stable at low protein concentration and is the only catalytically active form of E. coli PNP.

Active enzyme centrifugation (band sedimentation)
In order to study the sedimentation behavior of PNP at very low protein concentrations (1–20 µg/mL), we performed the active enzyme sedimentation experiments (Cohen et al. 1967; Cohen and Mire 1971; Kemper and Everse 1973). Only catalytically active forms of an enzyme can be examined with this method, since the observation is based on either the appearance of the product of the reaction or on the disappearance of the substrate. Representative radial absorption distributions of the reaction mixture generated as the enzyme-band sediments inside the centrifuge cell are shown in Figure 3 (for MESG as a nucleoside substrate) and in Figure 4 (for m7Guo). The absorption change (increase in Fig. 3 or decrease in Fig. 4) is a single-step profile, indicating that there is only one active oligomeric form of the enzyme in solution (Cohen and Claverie 1975).


Figure 3
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  		Figure 3. Active enzyme centrifugation experiment for 20 µg/mL PNP and the substrate MESG. Centrifugation was performed at 40,000 rpm and 20°C. Absorbance distribution, corresponding to the concentration of the product of phosphorolysis of MESG, observed while the enzyme layer moves along the cuvette filled with the reaction mixture containing both substrates (300 µM MESG and 50 mM phosphate) was measured at 360 nm. Radial absorption profiles were obtained in 5-min intervals. Inset shows the plot of ln(r) vs. time (r is the position of the midpoint of the sedimenting boundary) ({blacksquare}) and the linear fit of these data (—).

 


Figure 4
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  		Figure 4. Active enzyme centrifugation experiment for 20 µg/mL PNP and substrate m7Guo. Centrifugation was performed at 40,000 rpm and 25°C. Absorbance distribution, corresponding to the concentration of the product of phosphorolysis of m7Guo, observed while the enzyme layer moves along the cuvette filled with the reaction mixture containing both substrates (300 µM m7Guo and 50 mM phosphate) was measured at 260 nm. Radial absorption profiles were obtained in 7.5-min intervals. The inset shows plot of ln(r) vs. time (r is the position of the midpoint of the sedimenting boundary) ({blacksquare}) and the linear fit of these data (—).

 
To directly compare the band and the boundary sedimentation coefficients, i.e., values obtained from the active enzyme method with those from the classical sedimentation velocity studies, a band sedimentation experiment for high PNP concentration (3.4 mg/mL) was performed. In this case, the enzyme was layered on a buffered solution without substrates and the enzyme concentration profiles were measured using protein absorption at 280 nm (Fig. 5).


Figure 5
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  		Figure 5. Band centrifugation of E. coli PNP (3.4 mg/mL). Centrifugation was performed at 40,000 rpm and 20°C. Protein was loaded on 50 mM HEPES buffer (pH 7.0) with 150 mM KCl. Absorbance distribution of protein observed while enzyme layer moves along the cuvette was measured at 280 nm. Radial absorption profiles were obtained in 15-min intervals. The inset shows plot of ln(r) vs. time (r is the maximum of a spectrum) ({blacksquare}) and the fit of these data (—).

 
Table 1 contains the values of sedimentation coefficients of E. coli PNP obtained at 1–20 µg/mL concentration range with the active enzyme centrifugation method. Sedimentation parameters could not be determined for PNP concentrations below 1 µg/mL, due to low absorption changes obtained with such small amounts of the enzyme.


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  		Table 1. Sedimentation coefficients of E. coli PNP obtained in the low protein concentration range (1–20 µg/mL) from active enzyme sedimentation experimentsa

 
Sedimentation coefficients were calculated using the midpoint method for determination of the location of the enzyme band center (r) (see Materials and Methods). Representative plots of ln(r) versus time are shown for MESG substrate (Fig. 3, inset) and for m7Guo (Fig. 4, inset). In all cases, the plots are linear and the values of s20,w are independent of either PNP concentration or nucleoside substrate (Table 1). This result confirms the existence of only one active oligomeric form of the enzyme at very low protein concentration.

Sedimentation coefficients obtained using active enzyme centrifugation are slightly higher (Formula = 8.3 ± 0.4S) (see Table 1) than those from the classical sedimentation velocity method (Formula = 7.7 ± 0.3S) (see Fig. 1), although only the latter coefficients were corrected to values obtained in the limit of a dilute protein solution (which leads to an increase of s). Overestimation of s could be due to too high a concentration of the enzyme (Kemper and Everse 1973), but the data in Table 1 do not show any correlation between PNP concentration and the value of s. In addition, different values of sedimentation coefficient were obtained for the same enzyme concentration examined with both methods. Moreover, the sedimentation coefficient determined from band sedimentation for high concentration of PNP (3.4 mg/mL) was also greater (s20,w = 8.8 ± 0.5 S) than the values obtained from the sedimentation velocity method.

The active enzyme centrifugation method is prone to artifacts and has some limitations resulting from indirect observation of the enzyme sedimentation. To analyze the data, we used an approximate method of midpoints for calculation of sedimentation coefficient, which give precise values of s for enzymes with sedimentation coefficient above 3 S (Llewellyn and Smith 1978). The existence of an enzyme as rapid (cf. sedimentation velocity) equilibrium of two oligomeric forms complicates interpretation of results. In such cases, however, the values of s are lowered, whereas we observe reverse tendency. In our opinion, the active enzyme sedimentation is less accurate than the boundary sedimentation method, which could be associated with the time-dependent values of density and viscosity of the medium around the moving protein layer (Ralston 1993).

Overall, the results in Table 1 are consistent with the sedimentation of a single particle not smaller than the hexamer at very low PNP concentration. Furthermore, sedimentation equilibrium studies (see below) confirm that the enzyme does not dissociate into inactive subunits (which are invisible with active enzyme centrifugation).

Sedimentation equilibrium
Sedimentation equilibrium studies were performed to directly determine the molecular mass and the oligomeric state of PNP. Nixon et al.'s (1998) ultracentrifugation study suggested that E. coli PNP exists in solution as an equilibrium mixture of either trimer and hexamer or tetramer and octamer. Those models have been controversial in the light of crystallographic data—the crystal structure of E. coli PNP shows an apparent trimer of dimers (Koellner et al. 1998, 2002).

We performed sedimentation equilibrium experiments at various conditions, including those applied by Nixon et al. (1998). Experiments were conducted in HEPES (pH 7.0) (with or without Pi) and in Tris (pH 7.5), and covered the range of enzyme concentrations between 0.18 and 1.7 mg/mL. In all cases, temperature was either 4°C or 20°C.

The sedimentation equilibrium data for PNP in Tris at 20°C (Fig. 6), as well as the data obtained with other conditions (data not shown), are consistent with a single-species model. The values of molecular mass obtained from the sedimentation equilibrium data (150 ± 10 kDa) correspond to the molecular mass of the PNP hexamer (155 kDa).


Figure 6
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  		Figure 6. Sedimentation equilibrium experiment of E. coli PNP in 50 mM Tris-HCl buffer (pH 7.5) with 100 mM KCl and 0.1 mM DTT at 20°C. Centrifugation was performed at 10,200 rpm. The enzyme was loaded at 0.46 mg/mL (A), 1.08 mg/mL (B), and 1.7 mg/mL (C). Protein concentration gradient in equilibrium measured at 280 nm (shown as open circles at bottom) is shown together with the single-species model fit (line). (Top) Residuals (Aexp–Amod) for this model (fitted simultaneously to all three data sets). Similar curves and residual plots were obtained for the self-association model (i.e., coexistence of monomers and N-mer oligomer). The following parameters for these two models were obtained: single-species model, M = 150,746 Da; self-association model, Mmonomer = 25,988 Da, N = 5.8, Ka = 4.0 x 1087 M–5.

 
The self-association model was also considered in data analysis with no improvement of the fits. Moreover, the parameters of the latter model also describe the hexameric form of the enzyme since the mass of associating species is equal to the mass of PNP monomer (~25 kDa), the stoichiometry of self-association is 6, and the equilibrium constant is very high (~10100 M–5). These results indicate that PNP is a stable hexamer.

Conclusion
The results of this work show unequivocally that irrespective of protein concentration, ionic strength, temperature, presence of substrates, or destabilizing factors, the only active form of E. coli PNP is a hexamer.

As mentioned in the introduction, all PNPs, both trimeric and hexameric, show complex kinetic characteristics, which suggests possible coexistence of different active oligomeric enzyme forms (e.g., Ropp and Traut 1991). The results of this study, together with the results of similar experiments conducted for trimeric calf spleen PNP (Behlke et al. 2005), disprove that hypothesis by showing that the only active enzyme forms are hexamer and trimer for the bacterial and the mammalian enzyme, respectively. We have previously outlined the molecular phenomena responsible for the complex kinetics observed for phosphorolytic reaction catalyzed by calf-spleen trimeric PNP (Bzowska 2002). These are (1) sequential but random, not ordered, binding of substrates; (2) unusually potent binding of some purine bases (e.g., hypoxanthine); and (3) a dual role of phosphate acting as a substrate but also as the reaction modifier that stimulates a release of the tightly bound purine base.

In the case of hexameric PNPs that structurally could be assembled as a trimer of dimers, complex kinetic characteristic arises from other molecular reasons. First, it is an interaction between monomers in a dimer that leads to negative cooperativity in the binding of ground state ligands—phosphate (Kierdaszuk et al. 1997) and nucleosides, the latter in the presence of phosphate (Koellner et al. 2002). Two conformations of active sites in every dimer are possible: an open one, with low substrate binding affinity, and a closed one, binding substrates tightly (Koellner et al. 2002). Moreover, the crystal structure has shown that two monomers in the catalytic dimer are not independent and that only one of them can adopt the closed tight-binding conformation at a time. The alternate arrangement of monomers with open and closed active-site conformations within the hexamer implies that communication must also occur between adjacent catalytic dimers. It seems that if one binding pocket is closed, it determines whether the remaining five may be (stably) closed or not. This mechanism underlies the significance of maintaining the hexameric structure in E. coli PNP. Indeed, the results described in this study show that the only active and stable form of this enzyme is a hexamer.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
HEPES, NaOH, KOH, NaCl, KCl, and Tris were from Fisher Chemicals; mono- and dibasic-sodium phosphates were from Research Organics. Inosine (Ino), N(7)-methylguanosine (m7Guo), and xanthine oxidase from buttermilk (28 U/mg) were products of Sigma Chemical Co. 2-Amino-6-mercapto-7-methylpurine ribonucleoside (MESG) was obtained from Molecular Probes as a part of EnzChek Phosphate Assay Kit. Guanidine chloride (GdnHCl) was purchased from ROTH.

Partially purified PNP from E. coli (~60% pure, about 60 U/mg), a gift of Dr. G. Koszalka (Wellcome Research Labs) was further purified to apparent homogeneity as described by Bzowska et al. (1998), concentrated, and stored at –80°C. Before the experiments, the sample of PNP was thawed, diluted, and dialyzed against an appropriate buffer. Protein concentrations were determined from absorbance measurements with {varepsilon}1%(278) = 2.7 (Bzowska et al. 1998).

Enzyme activity was monitored spectrophotometrically at ~30°C in 50 mM phosphate buffer (pH 7.0), by the coupled xantine oxidase procedure with 0.5 mM Ino as substrate (Kalckar 1947) or by following the changes in absorption at 260 nm of m7Guo as substrate (Kulikowska et al. 1986).

Ultraviolet absorption was monitored with a Shimadzu UV-VIS Recording Spectrophotometer (UV-2401PC) at room temperature. Circular dichroism (CD) spectra were measured with a Jasco J-720 spectrometer using a 0.01-cm cylindrical cell. Analytical ultracentrifugation was carried out using a Beckman Optima XL-I analytical ultracentrifuge with four-position AN-Ti rotor and UV absorption detection system.

Sedimentation velocity experiments
Protein solution (340 or 350 µL) and a reference buffer (350 or 380 µL) were loaded into the right and the left sector, respectively, of a double-sector 1.2-cm cell with an aluminum centerpiece and quartz windows. After equilibration at 3000 rpm at 20°C, the rotor was accelerated to a desired speed (40,000 or 60,000 rpm) and radial absorption scans of protein-concentration profile in the cell was measured (at 3- or 3.5-min intervals). Enzyme activity was not affected by centrifugation, as determined by activity measurements before and after experiments. The sedimentation velocity data were analyzed with a time-derivative method (Stafford 1992). Apparent sedimentation coefficient distributions g(s020,w) and sedimentation coefficient s020,w (as the maximum of g(s020,w)) were calculated using the DCDT+ software (http://www.jphilo.mailway.com/). The program corrects observed sedimentation coefficients to values corresponding to the viscosity and density of water in the limit of a dilute protein solution. Molecular mass was calculated from Svedberg equation, Formula , where Formula is partial specific volume of PNP and {rho} is the density of sedimentation buffer. The diffusion coefficient (D) is related to the standard deviation ({sigma}) of Gaussian functions fitted to sedimentation coefficient distributions g(s020,w) by the following relationship (Stafford 1997):Formula , where t is the sedimentation time, {omega} is the angular velocity of the rotor, and rm the radius of the meniscus. Partial specific volume of PNP (from amino acid composition) as well as densities and viscosities of buffers were calculated using Sednterp program (http://www.rasmb.bbri.org/rasmb/).

Sedimentation equilibrium experiments
PNP solutions (110 or 120 µL) and a reference buffer (120 or 130 µL) were placed in 1.2-cm six-channel cells with quartz windows. The samples were equilibrated at a desired speed at 4° or 20°C for ~20–60 h, and then the cells were scanned at 280 nm. Enzyme activity decreased no more than 15% during centrifugation, as determined by activity measurements before and after experiments. The experiments were performed simultaneously for three concentrations of PNP (~0.18, ~0.36, and ~0.73 mg/mL) in 50 mM HEPES-KOH (pH 7.0) with (0.24 and 10 mM) and without phosphate at 4°C (at 12,000 rpm) and 20°C (1 h of overspeed at 15,000–17,000 rpm, then at 8600–11,000 rpm for ~20 h). Additional experiments were carried out (at 7200, 10,200, and 12,000 rpm) in the same conditions as described by Nixon et al. (1998)—in 50 mM Tris-HCl (pH 7.5), 100 mM KCl, and 0.1 mM DTT at 4° and 20°C for 0.46, 1.08, and 1.7 mg/mL PNP. The data were analyzed using the software supplied with instrument (Beckman) by fitting a single species model or a self-association model.

Active enzyme centrifugation
Active enzyme sedimentation experiments were carried out using a 1.2-cm cell with the Vinograd-type band-forming centerpiece and quartz windows. The sample and reference sectors were filled, respectively, with 350 and 400 µL solution containing 0.2 M NaCl and both substrates of the PNP catalyzed reaction—50 mM phosphate buffer (pH 7.0) and MESG (300 µM) or m7Guo (150 or 300 µM). Concentrations of substrates were much higher then their Km values, which is a necessary condition for the correct active enzyme centrifugation experiment. PNP solutions (20 µL) in 50 mM phosphate buffer (pH 7.0) were loaded into the sample well. Experiments were carried out for 20, 5, 2.5 and 1 µg/mL protein concentrations and for both nucleoside substrates (MESG and m7Guo). After equilibration at 3000 rpm at 20° or 25°C, the rotor was accelerated to 40,000 rpm, and movement of the enzyme band was followed by absorption changes of reaction mixture due to the appearance of products in place of substrates. The cells were scanned at 280 nm for m7Guo and at 355 or 360 nm for MESG (in 1- or 1.75-min intervals). MESG is slightly unstable in alkaline solutions (Webb 1992), but no more than 20% of MESG molecules undergoes decomposition during the time necessary for the active enzyme centrifugation experiment (~4–5 h).

For the comparison of the band and boundary sedimentation methods, an experiment with high PNP concentration (3.4 mg/mL) was performed. In this case, the enzyme was layered on a buffered solution without substrates (50 mM HEPES/KOH at pH 7.0 and 0.15 M KCl). After equilibration at 3000 rpm at 20°C, the rotor was accelerated to 40,000 rpm, and then the cells were scanned at 280 nm (in 3-min intervals).

Sedimentation coefficients (s) were calculated using the mid-point method (Kemper and Everse 1973) for determination of the location of the enzyme band center (r). The plot of the lnr versus time should yield a straight line with the slope equal to s{omega}2. The sedimentation coefficients (s) obtained with this approach were corrected to the viscosity and density of water at 20°C, as follows: Formula . The index T,b designates the values of partial specific volume of PNP (Formula ), density ({rho}), and viscosity ({eta}) of buffer at experimental temperature; 20,w indicates standard conditions (water at 20°C).

For the high PNP concentration experiment (3.4 mg/mL), the maxima of recorded scans were used to determine the location of the center of the enzyme band, and then the sedimentation coefficient was calculated as described above.


   Footnotes
 
Reprint requests to: Agnieszka Bzowska, Department of Biophysics, Institute of Experimental Physics, University of Warsaw, 93 Zwirki & Wigury, 02-089, Warsaw, Poland; e-mail: abzowska{at}biogeo.uw.edu.pl; fax: +48-22-5540771.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062183206.


   Acknowledgments
 
This work was supported by the Polish Ministry of Education and Science, grant no. 3P04A 035 24 (formerly Polish State Committee for Scientific Research, KBN) and by the BST 833/BF project from the Warsaw University.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 References
 
Behlke J., Koellner G., Bzowska A. 2005. Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation. Z. Naturforsch. 60C: 927–931.

Bzowska A. 2002. Calf spleen purine nucleoside phosphorylase: Complex kinetic mechanism, hydrolysis of 7-methylguanosine, and oligomeric state in solution. Biochim. Biophys. Acta 1596: 293–317.[CrossRef][Medline]

Bzowska A., Kazimierczuk Z., Seela F. 1998. 7-Deazapurine 2'-deoxyribofuranosides are noncleavable competitive inhibitors of Escherichia coli purine nucleoside phosphorylase (PNP). Acta Biochim. Pol. 45: 755–768.[Medline]

Bzowska A., Kulikowska E., Shugar D. 2000. Purine nucleoside phosphorylases: Properties, functions, and clinical aspects. Pharmacol. Ther. 88: 349–425.[CrossRef][Medline]

Cohen R. and Claverie J.-M. 1975. Sedimentation of generalized systems of interacting particles. II. Active enzyme centrifugation—theory and extensions of its validity range. Biopolymers 14: 1701–1716.[Medline]

Cohen R. and Mire M. 1971. Analytical-band centrifugation of an active enzyme-substrate complex. 1. Principle and practice of the centrifugation. Eur. J. Biochem. 23: 267–275.[Medline]

Cohen R., Giraud B., Messiah A. 1967. Theory and practice of the analytical centrifugation of an active substrate-enzyme complex. Biopolymers 5: 203–225.[CrossRef][Medline]

Deharvengt S., Wach S., Uhring M., Aprahamian M., Hajri A. 2004. Suicide gene/prodrug therapy for pancreatic adenocarcinoma by E. coli purine nucleoside phosphorylase and 6-methylpurine 2'-deoxyriboside. Pancreas 28: E54–E64.[Medline]

Hershfield M.S., Chaffee S., Koro-Johnson L., Mary A., Smith A.A., Short S.A. 1991. Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol. Proc. Natl. Acad. Sci. 88: 7185–7189.[Abstract/Free Full Text]

Jensen K.F. and Nygaard P. 1975. Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Purification and some properties. Eur. J. Biochem. 51: 253–265.[CrossRef][Medline]

Kalckar H.M. 1947. Differential spectrophotometry of purine compounds by means of specific enzymes. I. Determination of hydroxypurines. J. Biol. Chem. 167: 429–442.[Free Full Text]

Kemper D. and Everse J. 1973. Active enzyme centrifugation. Methods Enzymol. 27: 67–83.[Medline]

Kierdaszuk B., Modrak-Wójcik A., Shugar D. 1997. Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E. coli: Ligand-dependent quenching of enzyme intrinsic fluorescence. Biophys. Chem. 63: 107–118.[CrossRef][Medline]

Koellner G., Luic M., Shugar D., Saenger W., Bzowska A. 1998. Crystal structure of the ternary complex of E. coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, and phosphate (sulphate) at 2.1 Å resolution. J. Mol. Biol. 280: 153–166.[CrossRef][Medline]

Koellner G., Bzowska A., Wielgus-Kutrowska B., Luic M., Steiner T., Saenger W., Stepinski J. 2002. Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism. J. Mol. Biol. 315: 351–371.[CrossRef][Medline]

Kulikowska E., Bzowska A., Wierzchowski J., Shugar D. 1986. Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-Methylguanosine and 7-methylinosine. Biochim. Biophys. Acta 874: 355–363.[CrossRef][Medline]

Llewellyn D.J. and Smith G.D. 1978. An evaluation of active enzyme centrifugation as a zonal and boundary technique by the analysis of simulated data. Arch. Biochem. Biophys. 190: 483–494.[CrossRef][Medline]

Montgomery J.A., Niwas S., Rose J.D., Secrist J.A. III, Babu Y.S., Bugg C.E., Erion M.D., Guida W.C., Ealick S.E. 1993. Structure-based design of inhibitors of purine nucleoside phosphorylase.1. 9-(arylmethyl) derivatives of 9-deazaguanine. J. Med. Chem. 36: 55–69.[CrossRef][Medline]

Nixon A.E., Hunter J.L., Bonifacio G., Eccleston J.F., Webb M.R. 1998. Purine nucleoside phosphorylase: Its use in a spectroscopic assay for inorganic phosphate and for removing inorganic phosphate with the aid of phosphodeoxyribomutase. Anal. Biochem. 265: 299–307.[CrossRef][Medline]

Parker W.B., King S.A., Allan P.A., Bennett L.L. Jr., Secrist J.A. III, Montgomery J.A., Gilbert K.S., Waud W.A., Wells A.H., Gillespie G.Y. et al. 1997. In vivo gene therapy of cancer with E. coli purine nucleoside phosphorylase. Hum. Gene Ther. 8: 1637–1644.[Medline]

Ralston G. Introduction to analytical ultracentrifugation 1993. Beckman Instruments, Fullerton, CA.

Ropp P.A. and Traut T.W. 1991. Purine nucleoside phosphorylase. Allosteric regulation of a dissociating enzyme. J. Biol. Chem. 266: 7682–7687.[Abstract/Free Full Text]

Stafford W.F. III. 1992. Boundary analysis in sedimentation transport experiments: A procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal. Biochem. 203: 295–301.[CrossRef][Medline]

Stafford W.F. III. 1997. Sedimentation velocity spins a new weave for an old fabric. Curr. Opin. Biotechnol. 8: 14–24.[CrossRef][Medline]

Stoeckler J.D. 1984. Purine nucleoside phosphorylase: A target for chemiotherapy. In Development in cancer chemotherapy (ed. Glazer R.E.) . pp. 35–60. CRC Press, Boca Raton, FL.

Webb M.R. 1992. A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc. Natl. Acad. Sci. 89: 4884–4887.[Abstract/Free Full Text]


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