163
Biochimica et Biophysica Acta, 1160 (1992) 163-170
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34326
Purine nucleoside phosphorylase from bovine lens: purification and properties Daniela Barsacchi a, Mario Cappiello a, Maria Grazia Tozzi a Antonella Del Corso Mario Peccatori b, Marcella Camici a, Pier Luigi Ipata a and Umberto Mura c
a,
a Dipartimento di Fisiologia e Biochimica, Laboratorio di Biochimica, Universit~ di Pisa, Pisa (Italy), b CRESAM, Sezione Radiopatologia e Radiotossicologia, San Piero a Grado (Italy) and c Istituto di Chimica Biologica, Facolt~ di Medicina, Universitd di Modena, Modena (Italy)
(Received 22 April 1992)
Key words: Protein purification; Protein characterization; Purine nucleoside phosphorylase; Lens; (Calf)
Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was purified 38 750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97 000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15 250 and 14 650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.
Introduction
Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) catalyzes the phosphorolytic cleavage of the ribose moiety of inosine, guanosine and their 2'-deoxy derivatives. The enzyme has been purified from several mammalian and avian sources [1-9], as well as from microorganisms [10-13]. Despite the extensive characterization, some of the properties of PNP are not fully stated and some differences have been reported depending on both the source, as well as the experimental conditions employed. Thus, the enzyme has been described as a monomer [14,15], a dimer [5,16], a trimer [6,7,17] or a hexamer [11]; the mechanism of the enzyme is clearly sequential, but discrepancies exist on the type of sequential mechanism: ordered Bi-Bi, Theorell-Chance and random Bi-Bi mechanisms have
Correspondence to: U. Mura, Dipartimento di Fisiologia e Biochimica, Laboratorio di Biochimica, Via S. Maria 55, 56100 Pisa, Italy. Abbreviations: PNP, purine nucleoside phosphorylase; Rib l-P, ribose 1-phosphate; dRib l-P, 2'-deoxyribose 1-phosphate; DTI', dithiothreitol; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid).
been suggested for human erythrocyte [18], bovine brain [5] and chicken liver [3,8] purine nucleoside phosphorylases, respectively. Kinetic studies with purine nucleoside phosphorylases from various sources have frequently reported a non-hyperbolic kinetic behaviour which gives rise to an apparent negative cooperativity with inorganic phosphate [11,19,20] a n d / o r nucleosides [1-3,17,20]; however, in other studies, no cooperativity with phosphate [3-6] or with nucleosides [2,46,19] was observed. Also the question whether this enzyme is capable of allosteric regulation and the existence of metabolic regulators of the enzyme activity are at present discussed [16,21]. It has been proposed that some of the discrepancies observed may be due to the redox state of the enzyme sulfhydryl groups, to subunit interactions a n d / o r to the occurrence of isoenzymatic forms [1,22-24]. In recent work, PNP has been shown to be the most active enzyme of the metabolism of purine derivatives in calf and rat eye lens, where it may function as an energy-saving device, by salvaging both the base and the ribose moiety of nucleosides [25]. This paper describes the purification to homogeneity and the characterization of PNP from calf lens.
164 Materials and Methods
Materials Calf eyes were obtained from freshly slaughtered animals at the local slaughterhouse and the lenses were removed and kept frozen at -20°C until needed. Molecular weight markers for SDS-PAGE, pentyl agarose, phosphorylated sugars, purine nucleosides and bases, DTT, DTNB, milk xanthine oxidase (EC 1.1.3.22) were from Sigma. Hydroxyapatite (Biogel HTP), Bio-Sil SEC 250 column and molecular weight markers for HPLC gel permeation were from Bio-Rad. Sephacryl S-200 was obtained from Pharmacia and DEAE-cellulose (DE-52) was from Whatman. YM30 and YM5 ultrafiltration membranes were from Amicon. All other chemicals were of reagent grade.
Enzyme assays All enzyme assays were performed at 37°C using a Beckman DU-7 spectrophotometer. PNP activity with inosine as substrate was measured by using a coupled enzyme assay utilizing milk xanthine oxidase as ancillary enzyme [26]. The standard incubation mixture (final volume 1 ml) contained 0.5 mM inosine, 40 mM sodium phosphate and 0.015 U of xanthine oxidase in 25 mM Tris-HC1 buffer (pH 7.4). The commercial xanthine oxidase was extensively dialyzed against 50 mM Tris-HC1 buffer (pH 7.4) before use. The reaction was started by the addition of the enzyme sample. The molar extinction coefficient at 293 nm associated to the conversion of inosine to uric acid is 11260 M -1 cm -1. Inosine synthesis from hypoxanthine and Rib 1-P was followed by monitoring the change in absorbance at 280 nm [27]. The molar extinction coefficient associated to the conversion of hypoxanthine to inosine is 1180 M -1 cm -1. The phosphorolysis and synthesis of guanosine catalyzed by PNP were measured by following the change in absorbance at 258 nm [28]. The molar extinction coefficient associated to the synthesis and breakdown of guanosine at pH 7.4 is 5300 M-1 cm-1. The activity of bovine lens PNP towards xanthosine and adenosine was tested in the presence of xanthine oxidase by following the increase in absorbance at 293 and 305 nm, respectively. The molar extinction coefficients at pH 7.4 associated to the formation of 2,8-dihydroxyadenine and uric acid are 15 500 and 9300 M-1 cm- 1, respectively. One unit of PNP is the amount of enzyme which catalyzes the transformation of 1/.~mol of substrate per rain in the assay conditions.
Purification of bovine lens purine nucleoside phosphorylase All operations were carried out at 4°C, unless otherwise stated. About 200 frozen bovine lenses were suspended in 1 litre of 10 mM sodium phosphate buffer
(pH 7.0) containing 2 mM DTT (buffer A) and stirred for 1 h. The suspension was then centrifuged for 30 min at 40000 x g and the ensuing supernatant is referred to as lens extract (Table I). The lens extract was stirred with 0.2 volume of DE-52 for 1 h and then washed on a Buchner funnel firstly with buffer A and then with buffer A supplemented with 50 mM NaCl; each washing was carried out until the absorbance at 280 nm of the eluting buffer reached the baseline value. The DE-52 was then packed in a column and the enzyme was eluted with a linear gradient of NaC1 (50-200 mM) at a flow rate of 55 ml/h. The fractions containing the enzyme activity were pooled, dialyzed through Amicon YM30 membrane against buffer A and subjected to fractional precipitation with ammonium sulphate to a final concentration of 45% of salt saturation. After centrifugation, the ensuing supernatant was brought to 70% of salt saturation, centrifuged again and the sediment resuspended in 50 mM phosphate buffer (pH 7.4) containing 2 mM DTT (buffer B) supplemented with 1.2 M ammonium sulphate. The solution was applied to a pentyl agarose column (2 x 21 cm) equilibrated with buffer B supplemented with 1.2 M ammonium sulphate. The flow rate was 30 m l / h and 7-ml fractions were collected. The column was washed with the equilibrating buffer and then with buffer B supplemented with 1.1 M ammonium sulphate until the absorbance at 280 nm reached the baseline value. The enzyme was eluted stepwise by reducing ammonium sulphate concentration to 0.9 M. The active fractions were pooled, concentrated through Amicon YM5 membrane and then applied to Sephacryl S-200 column (2.8 x 97 cm). The flow rate was 50 m l / h and 7 ml fractions were collected. The active fractions were pooled, concentrated and dialyzed through Amicon YM5 membrane against buffer A and then applied to a Biogel HTP column (1.5 × 7 cm). After washing with buffer A and then with buffer B, the enzyme was eluted by a linear gradient of sodium phosphate (50200 mM) at a flow rate of 20 ml/h. The enzyme was released from the column as a symmetrical peak at a phosphate concentration of approx. 75 mM.
Molecular weight determination The apparent molecular weight of the native enzyme was determined by HPLC gel permeation on Bio-Sil SEC 250 column (Bio-Rad) (300 x 7.8 mm), equilibrated with 100 mM sodium orthophosphate buffer (pH 7.0) containing 100 mM sodium chloride. The proteins used as molecular weight standards were: bovine y-globulin (158000), conalbumin (77000), chicken ovalbumin (45000) and equine myoglobin (17000). The apparent molecular weight of the subunit was determined by SDS-PAGE in 15% polyacrylamide slab gel according to Laemmli [29]. The proteins used as
165 molecular weight standards were: ovalbumin (45000), glyceraldehyde 3-phosphate dehydrogenase (36000), carbonic anhydrase (29 000), trypsinogen (24 000) and myoglobin (17 000).
Photoinactivation of the enzyme The photoinactivation of PNP was studied between p H 5.5 and 8.0, using Methylene blue as photosensitizer. Before the irradiation, PNP was extensively dialyzed against 100 m M sodium phosphate buffer to remove Dq-T. Samples containing 60 m U / m l of the enzyme and 0.15 m M Methylene blue in 100 m M sodium phosphate were exposed in a glass ice-cold bath to the light of a 100 W tungsten bulb at a distance of 15 cm. After 6 min irradiation, aliquots of 50 /zl were transferred to 200 /zl of 100 m M sodium phosphate buffer (pH 7.4), containing 5 m M DTT, and the residual activity was measured. Residual activity was also measured both after irradiation of the enzyme in the absence of Methylene blue (light controls) and after incubation of the enzyme-Methylene blue mixtures in the dark (dark controls). Other methods Reduced thiol groups of bovine PNP were measured by the method of Ellman [30]. Protein concentration was estimated by the Comassie blue binding assay [31], with bovine serum albumin as standard. Results and Discussion
Purity and molecular weight PNP of bovine lens was purified approx. 39 000-fold with an overall yield of 21%. Table I summarizes the purification procedure of the enzyme to apparent electrophoretic homogeneity. Physical differences with respect to molecular weight and subunit composition of purine nucleoside phosphorylases from various sources have been reported. Thus, this enzyme has been described as a monomer, dimer or trimer [5-7, 14-17]. A phosphate-dependent subunit dissociation has been reported for the trimeric
origin--
/ 45 J. 36 --29
- 24
,ron,_ ill Fig. 1. Silver-stained SDS-PAGE of purine nucleoside phosphorylase purified from calf lens. The numbers alongside the gel represent apparent molecular weights, divided by 1000, of the polypeptide species used as molecular weight standards. See Materials and Methods for details.
bovine spleen PNP [32], which could explain the apparent heterogeneous structural organization found for this enzyme. The purified bovine lens PNP showed a single band on S D S - P A G E with an apparent molecular weight of 30 000 (Fig. 1). A molecular weight of 97 000 was evaluated for the native enzyme by H P L C gel permeation, thus suggesting a trimeric structure for the enzyme. In contrast to what was observed for bovine spleen PNP [32], no difference in the molecular weight was observed for the lens enzyme in the range of phosphate concentration between 10 and 100 mM. This result indicates that, unlike the spleen enzyme,
1.5 v
©
TABLE I Purification of bovine lenspurine nucleosidephosphorylase Fraction
Extract • DE-52 45-70% Ammonium sulphate Pentyl agarose Sephacryl S-200 Biogel HTP
Total volume (ml) 1060 227 8.5 187 46 16
Total activity (Units) 187 114.5 93.8 71.5 48.5 38.6
0.5
Specific Recovery Purifiactivity (%) cation (U/mg) (fold) 0.002 .100 1 0.041 61 205 1.8 9 30 77.5
50 38 26 21
il
900 4500 15000 38750
s f j~
100
0 I/[S]
( m M ) -1
Fig. 2. Influence of substrate concentration on the activityof bovine lens purine nucleoside phosphorylase. The activitywas measured at different concentrations of either inosine (o), in the presence of 40 mM orthophosphate, or orthophospate (o) in the presence of 0.5 mM inosine.
166 the apparent negative cooperativity behaviour observed for the lens PNP when orthophosphate was used as variable substrate (see below) cannot be ascribed to subunit dissociation.
Stability The enzyme was stable for at least 4 months (85% recovery) when stored at 4°C in 10 mM sodium phosphate buffer (pH 7.0), provided a weekly dialysis with 2 mM DT-I" was performed. When stored at 4°C in the absence of DT-F, the enzyme showed a progressive loss in activity, which was much more pronounced at pH below 6.0 or above 9.0. At neutral pH, the enzyme lost about 50% of its initial activity in 4 weeks. While PNP was stable in intact lenses at -20°C, freezing and
A.
"~_
~ _
150
thawing resulted in the complete loss of the activity of the pure enzyme.
Substrate specificity and kinetic behaviour The kinetic parameters of bovine lens PNP were determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases (Table II). The enzyme catalyzed the phosphorolysis and the synthesis of inosine, guanosine and their deoxyderivatives. Xanthosine was a poor substrate, while adenosine and 5'-deoxymethylthioadenosine were ineffective as substrates. Purine nucleoside phosphorylases from different sources have been reported to display a non-linear double-reciprocal plot when, at a saturating concentration of orthophosphate, inosine was varied
B.
150
Guo e."' 25 ~M
5 100
•
50
100
~f
I
0
I
50
C.
I
0
I
1 1/~
eo
120 100 200
I
I
2 ImM)-I
3
Gua
D.
Ribol.P 50 ~M
e-
"~
OR
75
o~-
~
O~,~.~r~
1~
n
mm
I
--
|
C
""-.........
~ -1.8
3 0
I
n
330
350
T-1 ( K - l x l O 5) Fig. 7. Effect of temperature on the kinetic and thermodynamic parameters of guanosine phosphorolysis catalyzed by bovine lens purine nucleoside phosphorylase. Panel A, plot of log V vs. 1/T for phosphorolysis (©) and synthesis (e) of guanosine; panel B, plot of pK s vs. 1/T for guanosine (e), guanine (o), ribose-l-phosphate (D) and phosphate (•). Ks and V at different temperatures were determined from double-reciprocal plots by interpolation of at least six rate measurements; panel C, plot of log geq vs. 1/T for phosphorolysis of guanosine. Keq was calculated by the Haldane relationship.
"~) 40J~
o
I
mm
-1.4
-2
,,¢ r< -r O.
I
oI c ~ B
~- -1.6
100
I
l'S
I-Ivp (nrrl°')
>
.i
(1)
50
n u-1"
Effect of temperature on the enzyme kinetics
I
I
I
6
7
8
pH Fig. 6. Photoinactivation of purine nucleoside phosphorylase. After 6 min irradiation in the presence of Methylene blue at different pH values the residual activitywas measured (e). ( • ) and ( [] ) are light and dark controls, respectively. See Materials and Methods for further details. Inset: residual activity after irradiation at pH 7.4 in the presence of hypoxanthine.
Kinetic parameters for the phosphorolysis and synthesis of guanosine were determined at different temperatures ranging from 15 to 45°C. The effect of the t e m p e r a t u r e on the maximal rate gives rise to linear Arrehnius plots from which very similar activation energies of 15 250 and 14 650 c a l / m o l were evaluated for phosphorolysis and synthesis, respectively (Fig. 7, panel A). Due to the identity of K m with the dissociation constant K s, the plots of p K a vs. 1 / T for the four substrates analyzed (Fig. 7, panel B) allowed an estimation of the heats of formation of the enzyme-substrate complexes which are reported in the diagrammatic
170
E+ G u a + Rib-1
.P
..! Fig. 8. Diagrammatic representation of A H ° values for the guanosine-guanine interconversion catalyzed by bovine lens purine nucleoside phosphorylase. r e p r e s e n t a t i o n of Fig. 8. A A H ° of - 4 4 5 0 c a l / m o l evaluated by p r o p e r c o m b i n a t i o n of A H ° values determ i n e d for each step of the enzymatic r e a c t i o n is essentially the same value ( - 4250 c a l / m o l ) arising from the analysis of the effect of the t e m p e r a t u r e o n the phosphorolysis r e a c t i o n e q u i l i b r i u m (Fig. 7, p a n e l C). I n this regard the e q u i l i b r i u m c o n s t a n t s (Keq) calculated b e t w e e n 25 a n d 45°C by the H a l d a n e r e l a t i o n s h i p [38] are consistent with those previously r e p o r t e d for p u r i n e nucleoside phosphorolysis [3,39,40].
Acknowledgements This work was s u p p o r t e d by N a t i o n a l I n s t i t u t e of H e a l t h G r a n t R01-EY07832. W e are i n d e b t e d with Mr. G.L. Dall'Olio, B E C A of P r u n a r o di Budrio, for the kind supply of b o v i n e lenses a n d to Dr. T. Mazza ( B E C A ) for his cooperation.
References 1 Kim, B.K., Cha, S. and Parks, R.E., Jr. (1968) J. Biol. Chem. 243, 1763-1770. 2 Agarwal, R.P. and Parks, R.E. (1969) J. Biol. Chem. 244, 644-647. 3 Murakami, K. and Tsushima, K. (1975) Biochim. Biophys. Acta 384, 390-398. 4 Lewis, A.S. and Glantz, M.D. (1976) J. Biol. Chem. 251, 407-413. 5 Lewis, A.S. and Glantz, M.D. (1976) Biochemistry 15, 4451-4456. 6 Milman, G., Anton, D.L. and Weber, J.L. (1976) Biochemistry 15, 4967-4972.
7 Wiginton, D.A., Coleman, M.S. and Hutton, J.J. (1980) J. Biol. Chem. 255, 6663-6669. 8 Mora, M. and Bozal, J. (1985) Comp. Biochem. Physiol. 82B, 805-813. 90sman, A.M., Del Corso, A., Mohamed A.S., Ipata, P.L. and Mura, U. (1990) Comp. Biochem. Physiol. 97B, 177-182. 10 Engelbrecht, A.L. and Sadoff, H.L. (1969) J. Biol. Chem. 244, 6228-6232. 11 Jensen, K.F. and Nygaard, P. (1975) Eur. J. Biochem. 51,253-265. 12 Jensen, K.F. (1976) Eur. J. Biochem. 61, 377-386. 13 Koszalka, G.W., Vanhooke, J., Short, S.A. and Hall, W.W. (1988) J. Bacteriol. 170, 3493-3498. 14 Manzanero, J.C., Mora, M. and Bozal, J. (1986) Rev. Esp. Fisiol. 42, 29-36. 15 Mora, M., Manzanero, J.C. and Bozal, J. (1987) Comp. Biochem. Physiol. 88B, 1143-1149. 16 Lewis, A.S. and Lowy, B.A. (1979) J. Biol. Chem. 254, 9927-9932. 17 Savage, B. and Spencer, N. (1977) Biochem. J. 167, 703-710. 18 Kim, B.K., Cha, S. and Parks, R.E., Jr. (1968) J. Biol. Chem. 243, 1771-1776. 19 Moyer, T.P. and Fischer, A.G. (1976) Arch. Biochem. Biophys. 174, 622-629. 20 Savage, B. and Spencer, N. (1979) Biochem. J. 179, 21-27. 21 Ropp, P.A. and Traut, T.W. (1991) Arch. Biochem. Biophys. 288, 614-620. 22 Turner, B.M., Fisher, R.A. and Harris, H. (1971) Eur. J. Biochem. 24, 288-295. 23 Zannis, V., Doyle, D. and Martin, D.W., Jr. (1978) J. Biol. Chem. 253, 504-510. 24 Mora, M., Manzanero, J.C. and Bozal, J. (1987) Comp. Biochem. Physiol. 87B, 403-409. 25 Cappiello, M., Barsacchi, D., Del Corso, A., Tozzi, M.G., Camici, M., Mura, U. and Ipata, P.L. (1992) Curr. Eye Res. 11, 435-444. 26 Kalckar, H.M. (1947) J. Biol. Chem. 167, 423-443. 27 Mura, U., Sgarrella, F., Felicioli, R.A., Senesi, S. and Ipata, P.L. (1976) Bull. Mol. Biol. Med. 1, 129-139. 28 Stoeckler, J.D., Cambor, C. and Parks, R.E., Jr. (1980) Biochemistry 19, 102-107. 29 Laemmli, U.K. (1970) Nature 227, 680-685. 30 Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70-77. 31 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 32 Ropp, P.A. and Traut, T.W. (1991) J. Biol. Chem. 266, 7682-7687. 33 Rudolph, F.B. and Fromm, H.J. (1979) Methods Enzymol. 63, 138-159. 34 Rudolph, F.B. (1979) Methods Enzymol. 63, 411-436. 35 Lueck, J.D., Ellison, W.R. and Fromm, H.J. (1973) FEBS Lett. 30, 321-324. 36 Carlson, J.D. and Fischer, A.G. (1979) Biochim. Biophys. Acta 571, 21-34. 37 Well. L. (1965) Arch. Biochem. Biophys. 110, 57-68. 38 Dixon, M. and Webb, E.C. (1979) in Enzymes, 3rd Edn., pp. 109-110, Longman, London. 39 Yamada, E.H. (1961) J. Biol. Chem. 236, 3043-3046. 40 De Verdier, C.-H. and Gould, B.J. (1963) Biochim. Biophys. Acta 68, 333-341.
Biochimica et Biophysica Acta, 1160 (1992) 163-170
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34326
Purine nucleoside phosphorylase from bovine lens: purification and properties Daniela Barsacchi a, Mario Cappiello a, Maria Grazia Tozzi a Antonella Del Corso Mario Peccatori b, Marcella Camici a, Pier Luigi Ipata a and Umberto Mura c
a,
a Dipartimento di Fisiologia e Biochimica, Laboratorio di Biochimica, Universit~ di Pisa, Pisa (Italy), b CRESAM, Sezione Radiopatologia e Radiotossicologia, San Piero a Grado (Italy) and c Istituto di Chimica Biologica, Facolt~ di Medicina, Universitd di Modena, Modena (Italy)
(Received 22 April 1992)
Key words: Protein purification; Protein characterization; Purine nucleoside phosphorylase; Lens; (Calf)
Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was purified 38 750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97 000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15 250 and 14 650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.
Introduction
Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) catalyzes the phosphorolytic cleavage of the ribose moiety of inosine, guanosine and their 2'-deoxy derivatives. The enzyme has been purified from several mammalian and avian sources [1-9], as well as from microorganisms [10-13]. Despite the extensive characterization, some of the properties of PNP are not fully stated and some differences have been reported depending on both the source, as well as the experimental conditions employed. Thus, the enzyme has been described as a monomer [14,15], a dimer [5,16], a trimer [6,7,17] or a hexamer [11]; the mechanism of the enzyme is clearly sequential, but discrepancies exist on the type of sequential mechanism: ordered Bi-Bi, Theorell-Chance and random Bi-Bi mechanisms have
Correspondence to: U. Mura, Dipartimento di Fisiologia e Biochimica, Laboratorio di Biochimica, Via S. Maria 55, 56100 Pisa, Italy. Abbreviations: PNP, purine nucleoside phosphorylase; Rib l-P, ribose 1-phosphate; dRib l-P, 2'-deoxyribose 1-phosphate; DTI', dithiothreitol; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid).
been suggested for human erythrocyte [18], bovine brain [5] and chicken liver [3,8] purine nucleoside phosphorylases, respectively. Kinetic studies with purine nucleoside phosphorylases from various sources have frequently reported a non-hyperbolic kinetic behaviour which gives rise to an apparent negative cooperativity with inorganic phosphate [11,19,20] a n d / o r nucleosides [1-3,17,20]; however, in other studies, no cooperativity with phosphate [3-6] or with nucleosides [2,46,19] was observed. Also the question whether this enzyme is capable of allosteric regulation and the existence of metabolic regulators of the enzyme activity are at present discussed [16,21]. It has been proposed that some of the discrepancies observed may be due to the redox state of the enzyme sulfhydryl groups, to subunit interactions a n d / o r to the occurrence of isoenzymatic forms [1,22-24]. In recent work, PNP has been shown to be the most active enzyme of the metabolism of purine derivatives in calf and rat eye lens, where it may function as an energy-saving device, by salvaging both the base and the ribose moiety of nucleosides [25]. This paper describes the purification to homogeneity and the characterization of PNP from calf lens.
164 Materials and Methods
Materials Calf eyes were obtained from freshly slaughtered animals at the local slaughterhouse and the lenses were removed and kept frozen at -20°C until needed. Molecular weight markers for SDS-PAGE, pentyl agarose, phosphorylated sugars, purine nucleosides and bases, DTT, DTNB, milk xanthine oxidase (EC 1.1.3.22) were from Sigma. Hydroxyapatite (Biogel HTP), Bio-Sil SEC 250 column and molecular weight markers for HPLC gel permeation were from Bio-Rad. Sephacryl S-200 was obtained from Pharmacia and DEAE-cellulose (DE-52) was from Whatman. YM30 and YM5 ultrafiltration membranes were from Amicon. All other chemicals were of reagent grade.
Enzyme assays All enzyme assays were performed at 37°C using a Beckman DU-7 spectrophotometer. PNP activity with inosine as substrate was measured by using a coupled enzyme assay utilizing milk xanthine oxidase as ancillary enzyme [26]. The standard incubation mixture (final volume 1 ml) contained 0.5 mM inosine, 40 mM sodium phosphate and 0.015 U of xanthine oxidase in 25 mM Tris-HC1 buffer (pH 7.4). The commercial xanthine oxidase was extensively dialyzed against 50 mM Tris-HC1 buffer (pH 7.4) before use. The reaction was started by the addition of the enzyme sample. The molar extinction coefficient at 293 nm associated to the conversion of inosine to uric acid is 11260 M -1 cm -1. Inosine synthesis from hypoxanthine and Rib 1-P was followed by monitoring the change in absorbance at 280 nm [27]. The molar extinction coefficient associated to the conversion of hypoxanthine to inosine is 1180 M -1 cm -1. The phosphorolysis and synthesis of guanosine catalyzed by PNP were measured by following the change in absorbance at 258 nm [28]. The molar extinction coefficient associated to the synthesis and breakdown of guanosine at pH 7.4 is 5300 M-1 cm-1. The activity of bovine lens PNP towards xanthosine and adenosine was tested in the presence of xanthine oxidase by following the increase in absorbance at 293 and 305 nm, respectively. The molar extinction coefficients at pH 7.4 associated to the formation of 2,8-dihydroxyadenine and uric acid are 15 500 and 9300 M-1 cm- 1, respectively. One unit of PNP is the amount of enzyme which catalyzes the transformation of 1/.~mol of substrate per rain in the assay conditions.
Purification of bovine lens purine nucleoside phosphorylase All operations were carried out at 4°C, unless otherwise stated. About 200 frozen bovine lenses were suspended in 1 litre of 10 mM sodium phosphate buffer
(pH 7.0) containing 2 mM DTT (buffer A) and stirred for 1 h. The suspension was then centrifuged for 30 min at 40000 x g and the ensuing supernatant is referred to as lens extract (Table I). The lens extract was stirred with 0.2 volume of DE-52 for 1 h and then washed on a Buchner funnel firstly with buffer A and then with buffer A supplemented with 50 mM NaCl; each washing was carried out until the absorbance at 280 nm of the eluting buffer reached the baseline value. The DE-52 was then packed in a column and the enzyme was eluted with a linear gradient of NaC1 (50-200 mM) at a flow rate of 55 ml/h. The fractions containing the enzyme activity were pooled, dialyzed through Amicon YM30 membrane against buffer A and subjected to fractional precipitation with ammonium sulphate to a final concentration of 45% of salt saturation. After centrifugation, the ensuing supernatant was brought to 70% of salt saturation, centrifuged again and the sediment resuspended in 50 mM phosphate buffer (pH 7.4) containing 2 mM DTT (buffer B) supplemented with 1.2 M ammonium sulphate. The solution was applied to a pentyl agarose column (2 x 21 cm) equilibrated with buffer B supplemented with 1.2 M ammonium sulphate. The flow rate was 30 m l / h and 7-ml fractions were collected. The column was washed with the equilibrating buffer and then with buffer B supplemented with 1.1 M ammonium sulphate until the absorbance at 280 nm reached the baseline value. The enzyme was eluted stepwise by reducing ammonium sulphate concentration to 0.9 M. The active fractions were pooled, concentrated through Amicon YM5 membrane and then applied to Sephacryl S-200 column (2.8 x 97 cm). The flow rate was 50 m l / h and 7 ml fractions were collected. The active fractions were pooled, concentrated and dialyzed through Amicon YM5 membrane against buffer A and then applied to a Biogel HTP column (1.5 × 7 cm). After washing with buffer A and then with buffer B, the enzyme was eluted by a linear gradient of sodium phosphate (50200 mM) at a flow rate of 20 ml/h. The enzyme was released from the column as a symmetrical peak at a phosphate concentration of approx. 75 mM.
Molecular weight determination The apparent molecular weight of the native enzyme was determined by HPLC gel permeation on Bio-Sil SEC 250 column (Bio-Rad) (300 x 7.8 mm), equilibrated with 100 mM sodium orthophosphate buffer (pH 7.0) containing 100 mM sodium chloride. The proteins used as molecular weight standards were: bovine y-globulin (158000), conalbumin (77000), chicken ovalbumin (45000) and equine myoglobin (17000). The apparent molecular weight of the subunit was determined by SDS-PAGE in 15% polyacrylamide slab gel according to Laemmli [29]. The proteins used as
165 molecular weight standards were: ovalbumin (45000), glyceraldehyde 3-phosphate dehydrogenase (36000), carbonic anhydrase (29 000), trypsinogen (24 000) and myoglobin (17 000).
Photoinactivation of the enzyme The photoinactivation of PNP was studied between p H 5.5 and 8.0, using Methylene blue as photosensitizer. Before the irradiation, PNP was extensively dialyzed against 100 m M sodium phosphate buffer to remove Dq-T. Samples containing 60 m U / m l of the enzyme and 0.15 m M Methylene blue in 100 m M sodium phosphate were exposed in a glass ice-cold bath to the light of a 100 W tungsten bulb at a distance of 15 cm. After 6 min irradiation, aliquots of 50 /zl were transferred to 200 /zl of 100 m M sodium phosphate buffer (pH 7.4), containing 5 m M DTT, and the residual activity was measured. Residual activity was also measured both after irradiation of the enzyme in the absence of Methylene blue (light controls) and after incubation of the enzyme-Methylene blue mixtures in the dark (dark controls). Other methods Reduced thiol groups of bovine PNP were measured by the method of Ellman [30]. Protein concentration was estimated by the Comassie blue binding assay [31], with bovine serum albumin as standard. Results and Discussion
Purity and molecular weight PNP of bovine lens was purified approx. 39 000-fold with an overall yield of 21%. Table I summarizes the purification procedure of the enzyme to apparent electrophoretic homogeneity. Physical differences with respect to molecular weight and subunit composition of purine nucleoside phosphorylases from various sources have been reported. Thus, this enzyme has been described as a monomer, dimer or trimer [5-7, 14-17]. A phosphate-dependent subunit dissociation has been reported for the trimeric
origin--
/ 45 J. 36 --29
- 24
,ron,_ ill Fig. 1. Silver-stained SDS-PAGE of purine nucleoside phosphorylase purified from calf lens. The numbers alongside the gel represent apparent molecular weights, divided by 1000, of the polypeptide species used as molecular weight standards. See Materials and Methods for details.
bovine spleen PNP [32], which could explain the apparent heterogeneous structural organization found for this enzyme. The purified bovine lens PNP showed a single band on S D S - P A G E with an apparent molecular weight of 30 000 (Fig. 1). A molecular weight of 97 000 was evaluated for the native enzyme by H P L C gel permeation, thus suggesting a trimeric structure for the enzyme. In contrast to what was observed for bovine spleen PNP [32], no difference in the molecular weight was observed for the lens enzyme in the range of phosphate concentration between 10 and 100 mM. This result indicates that, unlike the spleen enzyme,
1.5 v
©
TABLE I Purification of bovine lenspurine nucleosidephosphorylase Fraction
Extract • DE-52 45-70% Ammonium sulphate Pentyl agarose Sephacryl S-200 Biogel HTP
Total volume (ml) 1060 227 8.5 187 46 16
Total activity (Units) 187 114.5 93.8 71.5 48.5 38.6
0.5
Specific Recovery Purifiactivity (%) cation (U/mg) (fold) 0.002 .100 1 0.041 61 205 1.8 9 30 77.5
50 38 26 21
il
900 4500 15000 38750
s f j~
100
0 I/[S]
( m M ) -1
Fig. 2. Influence of substrate concentration on the activityof bovine lens purine nucleoside phosphorylase. The activitywas measured at different concentrations of either inosine (o), in the presence of 40 mM orthophosphate, or orthophospate (o) in the presence of 0.5 mM inosine.
166 the apparent negative cooperativity behaviour observed for the lens PNP when orthophosphate was used as variable substrate (see below) cannot be ascribed to subunit dissociation.
Stability The enzyme was stable for at least 4 months (85% recovery) when stored at 4°C in 10 mM sodium phosphate buffer (pH 7.0), provided a weekly dialysis with 2 mM DT-I" was performed. When stored at 4°C in the absence of DT-F, the enzyme showed a progressive loss in activity, which was much more pronounced at pH below 6.0 or above 9.0. At neutral pH, the enzyme lost about 50% of its initial activity in 4 weeks. While PNP was stable in intact lenses at -20°C, freezing and
A.
"~_
~ _
150
thawing resulted in the complete loss of the activity of the pure enzyme.
Substrate specificity and kinetic behaviour The kinetic parameters of bovine lens PNP were determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases (Table II). The enzyme catalyzed the phosphorolysis and the synthesis of inosine, guanosine and their deoxyderivatives. Xanthosine was a poor substrate, while adenosine and 5'-deoxymethylthioadenosine were ineffective as substrates. Purine nucleoside phosphorylases from different sources have been reported to display a non-linear double-reciprocal plot when, at a saturating concentration of orthophosphate, inosine was varied
B.
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120 100 200
I
I
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--
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~ -1.8
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T-1 ( K - l x l O 5) Fig. 7. Effect of temperature on the kinetic and thermodynamic parameters of guanosine phosphorolysis catalyzed by bovine lens purine nucleoside phosphorylase. Panel A, plot of log V vs. 1/T for phosphorolysis (©) and synthesis (e) of guanosine; panel B, plot of pK s vs. 1/T for guanosine (e), guanine (o), ribose-l-phosphate (D) and phosphate (•). Ks and V at different temperatures were determined from double-reciprocal plots by interpolation of at least six rate measurements; panel C, plot of log geq vs. 1/T for phosphorolysis of guanosine. Keq was calculated by the Haldane relationship.
"~) 40J~
o
I
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-1.4
-2
,,¢ r< -r O.
I
oI c ~ B
~- -1.6
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>
.i
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Effect of temperature on the enzyme kinetics
I
I
I
6
7
8
pH Fig. 6. Photoinactivation of purine nucleoside phosphorylase. After 6 min irradiation in the presence of Methylene blue at different pH values the residual activitywas measured (e). ( • ) and ( [] ) are light and dark controls, respectively. See Materials and Methods for further details. Inset: residual activity after irradiation at pH 7.4 in the presence of hypoxanthine.
Kinetic parameters for the phosphorolysis and synthesis of guanosine were determined at different temperatures ranging from 15 to 45°C. The effect of the t e m p e r a t u r e on the maximal rate gives rise to linear Arrehnius plots from which very similar activation energies of 15 250 and 14 650 c a l / m o l were evaluated for phosphorolysis and synthesis, respectively (Fig. 7, panel A). Due to the identity of K m with the dissociation constant K s, the plots of p K a vs. 1 / T for the four substrates analyzed (Fig. 7, panel B) allowed an estimation of the heats of formation of the enzyme-substrate complexes which are reported in the diagrammatic
170
E+ G u a + Rib-1
.P
..! Fig. 8. Diagrammatic representation of A H ° values for the guanosine-guanine interconversion catalyzed by bovine lens purine nucleoside phosphorylase. r e p r e s e n t a t i o n of Fig. 8. A A H ° of - 4 4 5 0 c a l / m o l evaluated by p r o p e r c o m b i n a t i o n of A H ° values determ i n e d for each step of the enzymatic r e a c t i o n is essentially the same value ( - 4250 c a l / m o l ) arising from the analysis of the effect of the t e m p e r a t u r e o n the phosphorolysis r e a c t i o n e q u i l i b r i u m (Fig. 7, p a n e l C). I n this regard the e q u i l i b r i u m c o n s t a n t s (Keq) calculated b e t w e e n 25 a n d 45°C by the H a l d a n e r e l a t i o n s h i p [38] are consistent with those previously r e p o r t e d for p u r i n e nucleoside phosphorolysis [3,39,40].
Acknowledgements This work was s u p p o r t e d by N a t i o n a l I n s t i t u t e of H e a l t h G r a n t R01-EY07832. W e are i n d e b t e d with Mr. G.L. Dall'Olio, B E C A of P r u n a r o di Budrio, for the kind supply of b o v i n e lenses a n d to Dr. T. Mazza ( B E C A ) for his cooperation.
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7 Wiginton, D.A., Coleman, M.S. and Hutton, J.J. (1980) J. Biol. Chem. 255, 6663-6669. 8 Mora, M. and Bozal, J. (1985) Comp. Biochem. Physiol. 82B, 805-813. 90sman, A.M., Del Corso, A., Mohamed A.S., Ipata, P.L. and Mura, U. (1990) Comp. Biochem. Physiol. 97B, 177-182. 10 Engelbrecht, A.L. and Sadoff, H.L. (1969) J. Biol. Chem. 244, 6228-6232. 11 Jensen, K.F. and Nygaard, P. (1975) Eur. J. Biochem. 51,253-265. 12 Jensen, K.F. (1976) Eur. J. Biochem. 61, 377-386. 13 Koszalka, G.W., Vanhooke, J., Short, S.A. and Hall, W.W. (1988) J. Bacteriol. 170, 3493-3498. 14 Manzanero, J.C., Mora, M. and Bozal, J. (1986) Rev. Esp. Fisiol. 42, 29-36. 15 Mora, M., Manzanero, J.C. and Bozal, J. (1987) Comp. Biochem. Physiol. 88B, 1143-1149. 16 Lewis, A.S. and Lowy, B.A. (1979) J. Biol. Chem. 254, 9927-9932. 17 Savage, B. and Spencer, N. (1977) Biochem. J. 167, 703-710. 18 Kim, B.K., Cha, S. and Parks, R.E., Jr. (1968) J. Biol. Chem. 243, 1771-1776. 19 Moyer, T.P. and Fischer, A.G. (1976) Arch. Biochem. Biophys. 174, 622-629. 20 Savage, B. and Spencer, N. (1979) Biochem. J. 179, 21-27. 21 Ropp, P.A. and Traut, T.W. (1991) Arch. Biochem. Biophys. 288, 614-620. 22 Turner, B.M., Fisher, R.A. and Harris, H. (1971) Eur. J. Biochem. 24, 288-295. 23 Zannis, V., Doyle, D. and Martin, D.W., Jr. (1978) J. Biol. Chem. 253, 504-510. 24 Mora, M., Manzanero, J.C. and Bozal, J. (1987) Comp. Biochem. Physiol. 87B, 403-409. 25 Cappiello, M., Barsacchi, D., Del Corso, A., Tozzi, M.G., Camici, M., Mura, U. and Ipata, P.L. (1992) Curr. Eye Res. 11, 435-444. 26 Kalckar, H.M. (1947) J. Biol. Chem. 167, 423-443. 27 Mura, U., Sgarrella, F., Felicioli, R.A., Senesi, S. and Ipata, P.L. (1976) Bull. Mol. Biol. Med. 1, 129-139. 28 Stoeckler, J.D., Cambor, C. and Parks, R.E., Jr. (1980) Biochemistry 19, 102-107. 29 Laemmli, U.K. (1970) Nature 227, 680-685. 30 Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70-77. 31 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 32 Ropp, P.A. and Traut, T.W. (1991) J. Biol. Chem. 266, 7682-7687. 33 Rudolph, F.B. and Fromm, H.J. (1979) Methods Enzymol. 63, 138-159. 34 Rudolph, F.B. (1979) Methods Enzymol. 63, 411-436. 35 Lueck, J.D., Ellison, W.R. and Fromm, H.J. (1973) FEBS Lett. 30, 321-324. 36 Carlson, J.D. and Fischer, A.G. (1979) Biochim. Biophys. Acta 571, 21-34. 37 Well. L. (1965) Arch. Biochem. Biophys. 110, 57-68. 38 Dixon, M. and Webb, E.C. (1979) in Enzymes, 3rd Edn., pp. 109-110, Longman, London. 39 Yamada, E.H. (1961) J. Biol. Chem. 236, 3043-3046. 40 De Verdier, C.-H. and Gould, B.J. (1963) Biochim. Biophys. Acta 68, 333-341.
