530

[72]

PURINE METABOLIZING ENZYMES

substituting for phosphate with comparable results. Xanthosine is an extremely poor substrate, and adenosine is not phosphorylyzed at 20fold excess of the homogeneous enzyme.

Inhibitors. Product inhibition is observed with guanine (Ki 1.25 × 10-5 M) and hypoxanthine (2.5 × 10-5 M) for the enzymic phosphorolysis of guanosine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition (Ki 3.61 x 10-4M) with guanosine as the variable substrate. Noncompetitive inhibition was observed with pchloromercuribenzoate (K~ 5.68 x 10-6 M). The inactivated enzyme was completely reactivated upon the addition of an excess of 2-mercaptoethanol. Photooxidation. The enzyme is highly susceptible to photooxidation in the presence of methylene blue. A pH dependence of photoinactivation is observed with near maximal photoinactivation obtained near pH 8.5. Stability. The enzyme shows no significant changes in catalytic property when incubated in phosphate buffer between pH 5.5 and pH 9.0 t'or 10 min at 40 ° and assayed at pH 7.0. The stability of the enzyme at relatively higher temperatures is an asset to its purification. The enzyme is stabilized by 2-mercaptoethanol during the purification. Molecular Weight. Recent studies show a molecular weight value of 30,500 by sodium dodecyl sulfate electrophoresis and 61,000 by Sephadex G-100 gel filtration. Cross-linking studies with dimethyl suberimidate show two bands indicating a dimer.16 leT. Treuman and M. D. Glantz, Fed. Proc., Fed. Am. Soc. Exp. Biol. 873, Abstr. No. 3176 (1977).

[72] P u r i n e N u c l e o s i d e P h o s p h o r y l a s e Erythrocytes

I from Human

By J. D. STOECKLER, R. P. A~ARWAL, K. C. AGARWAL, and R. E. PARKS, JR.

Inosine (deoxyinosine) + P~ ~ hypoxanthine + ribose- 1-phosphate (deoxyribose- 1-phosphate) Guanosine (deoxyguanosine) + Pl -~ guanine + ribose- 1-phosphate (deoxyribose- 1-phosphate) 1 Purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1. METHODS

IN ENZYMOLOGY,

VOL. LI

Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5

[72]

PURINE NUCLEOSIDEPHOSPHORYLASE

531

Purine nucleoside phosphorylase (PNPase) has been crystallized 2 from human erythrocytes where it is present in relatively high concentration (about 13 units/ml packed ceUs), a PNPase is essential for the reutilization of purine ribo- and deoxyribonucleosides in these ceils which lack the de n o v o pathway for nucleotide synthesis. The enzyme has been found deficient in erythrocytes and lymphocytes of individuals who also lack T-lymphocyte functions. 4 These subjects have hyperactive de n o v o purine biosynthesis, hypouricemia, and hypouricosuria but markedly elevated blood and urine concentrations of nucleosides of guanine and hypoxanthine. 5

Assay

Method 6

Principle. Hypoxanthine formed in the reaction is oxidized to uric acid by xanthine oxidase. The uric acid is measured by following the increase in absorbancy at 293 nm. 7 Reagents s

Potassium phosphate buffer, 0.5 M, pH 7.5 Inosine, 5 mM Xanthine oxidase from milk (commercially available ammonium sulfate ppt.), 0.2 units/ml water Solutions containing PNPase activity are diluted in Tris or phosphate buffer, pH 7.5, so that an aliquot gives a 0.005-0.040 absorbancy change per minute. P r o c e d u r e . In a 1.5-ml quartz cuvette with a 1-cm light path, 0.1 ml each of the buffer, inosine, and xanthine oxidase are mixed with water to allow a final volume of 1.0 ml after enzyme addition. The reaction mixture is preincubated for several minutes at 30 ° to permit oxidation of any hypoxanthine or xanthine that might be present as contaminants in inosine. The reaction is initiated by the addition of PNPase, and activity 2 R. P. Agarwal and R. E. Parks, Jr., J. Biol. Chem. 244, 644 (1969). 3 R. E. Parks, Jr. and R. P. Agarwal, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 7, p. 483. Academic Press, New York, 1971. 4 E. R. Giblett, A. J. Amman, R. Sandman, D. W. Wara, and L. K. Diamond, Lancet 1, 1010 (1975). 5 A. Cohen, D. Doyle, D. W. Martin, Jr., and A. J. Amman, N. Engl. J. Med. 295, 1449 (1976). B. K. Kim, S. Cha, and R. E. Parks, Jr., J. Biol. Chem. 243, 1763 (1968). r H. M. Kalckar, J. Biol. Chem. 167, 429 (1947). * Phosphate and inosine stock solutions may be combined and are stable for many months if stored frozen. Diluted xanthine oxidase may be used for 1-2 days if refrigerated.

532

PURINE METABOLIZING ENZYMES

[72]

is calculated from the increase in absorbancy at 293 nm (eM = 1.25 X

104).

Definition of Unit and Specific Activity. One unit of PNPase phosphorolyzes 1 /~mole of inosine per minute under standard assay conditions. Specific activity is expressed as units per milligram of protein. The protein concentration is determined from absorbancy at 280 nm during purification. 9 Alternate Assay Procedures. (1) If spectrophotometric measurements at 293 nm are inconvenient or insensitive, e.g., in the presence of substances with high absorbancies near 293 nm, inosine may be replaced as the substrate by 6-thioinosine. The formation of 6-thiouric acid may be followed at 348 nm (eM = 2.45 × 104).10 (2) Direct spectrophotometric assay of either phosphorolysis or synthesis is possible with substrates that show significant absorbancy differences between the nucleoside and base (e.g., guanosine ~ guanine, eM = 5.1 × 103 at 252 nm, pH 7.5). 11 (3) A radioisotope assay has been described that is capable of detecting as little as 0.1 nmole product in either direction. 12 (4) PNPase activity may be demonstrated qualitatively in electrophoretic gels 13 and in spots of whole blood on DEAE-cellulose paper 14 by use of a tetrazolium dye. Various assay procedures are discussed in detail elsewhere, s Purification Procedure 15 All steps are carried out at 0-5 °. The procedure described is for 100 ml of packed cells from fresh or stored heparinized or ACD human blood, but it may be scaled up or down if column sizes are adjusted accordingly.

Step 1. Preparation of Hemolysate. This step is described elsewhere in this volume. 16 a O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941); see this series, Vol. III [73]. 10 M. R. Sheen, B. K. Kim, and R. E. Parks, Jr., Mol. Pharmacol. 4, 293 (1968). 11A. F. Ross, K. C. Agarwal, S. H. Chu, and R. E. Parks, Jr., Biochem. Pharmacol. 22, 141 (1973). 12 G. Milman, D. L. Anton, and J. L. Weber, Biochemistry 15, 4967 (1976). 13 y. H. Edwards, D. A. Hopkinson, and H. Harris, Ann. Hum. Genet. 34, 395 (1971). 14 M. Ansay, V. Baldewijns-Rouma, and J. E. Smith, Anita. Blood Grps. Biochem. Genet. 6, 249 (1975). 15 This method is basically similar to that reported earlier. 2"6 The initial purification steps were adapted from K. K. Tsuboi and P. B. Hudson, J. Biol. Chem. 224, 879 (1957) and F. M. Huennekens, E. Nurk, and B. W. Gabrio, ibid. 221, 971 (1956). Also see V. Zannis, D. Doyle, and D. W. Martin, J. Biol. Chem. 253, 504 (1978) for a new affinity chromatographic method. 16 R. P. Agarwal, K. C. Agarwal, and R. E. Parks, Jr., this volume [79].

[72]

PURINE NUCLEOSIDE PHOSPHORYLASE

533

Step 2. Adsorption on Calcium Phosphate Gel. The enzyme is adsorbed on calcium phosphate gel as described elsewhere in this volume. 16In the procedure presented here, the enzyme is desorbed from the gel by eluting twice with 100 ml of 0.1 M potassium phosphate buffer, pH 7.5.17 Step 3. Ammonium Sulfate Fractionation. The eluate from step 2 is brought to 40% saturation by the gradual addition of solid ammonium sulfate (23.1 g/100 ml of initial volume) and stirred for at least 30 min. The precipitate is removed by centrifugation at 9000 g for 20 min. The supernatant fluid is brought to 60% saturation by slow addition of solid ammonium sulfate (12.3 g/100 ml of supernatant fluid). After about 1 hr, the suspension is centrifuged at 9000 g for 40 min, and the precipitate is dissolved in about 15 ml of 0.1 M Tris-acetate, pH 7.5. The solution is dialyzed in pretreated dialysis casings is against several changes of 4 liters of 0.03 M Tris-acetate, pH 7.5, for 12-16 hr. Step 4. DEAE-Cellulose Column Chromatography. A 2.5 × 25 cm column of DEAE-cellulose (acetate form) TM is equilibrated with 0.03 M Tris-acetate, pH 7.5, containing 1 mM dithiothreitol. 2° The dialyzed enzyme is adsorbed on the column, washed with about 50 ml of the same buffer, and eluted with a linear gradient (0.03-0.35 M; total volume, 600 ml) of Tris-acetate, pH 7.5. About 95% of the enzyme emerges in the range of 0.06-0.2 M Tris-acetate, and the fractions containing enzyme activity are pooled. 17 Eiution with 0.1 M phosphate, in contrast to the 20% ammonium sulfate eluant used by R. P. Agarwal et al. 16 permits the separation of purine nucleoside phosphorylase from many other proteins, e.g., nucleoside diphosphokinase, which desorb at higher phosphate concentrations. The procedure outlined in this chapter is specifically for the purification of purine nucleoside phosphorylase; however, if large quantities of erythrocytes are employed or purification of other enzymes is desired, it may be preferable to use the general method TM where desorption with ammonium sulfate is followed by precipitation at 70% saturation and calcium phosphate gel chromatography. Fraction 5 of the general method, TM which contains purine nucleoside phosphorylase at a specific activity of 2-5 (approximately equivalent to enzyme from step 3 above), may be dialyzed against 0.03 M Tris-acetate, pH 7.5, and purified further starting at step 4 above. 1~ p. McPhie, this series, Vol. 22, p. 25. 19 DEAE-cellulose is washed free of fines by decantation with distilled water and converted to the -OH form by gentle stirring with 0.5 M NaOH for 1 hr followed by adequate rinsing with distilled water. The fluids may be removed by decantation, or more rapidly, by filtration on a Biichner funnel. The fibers are treated with 1 M sodium acetate until the filtrate pH is 7.5-8.0 and then equilibrated with 0.03 M Tris-acetate, pH 7.5. 20 From this step onward, all solutions used in the purification should contain 5-10 mM mercaptoethanol or 1 mM dithiothreitol. It should be noted that the oxidized form of dithiothreitol may interfere with the measurement of absorbancy at 280 nm.

534

PURINE METABOLIZING ENZYMES

[72]

Step 5. Calcium Phosphate Gel--Cellulose Column Chromatography. Calcium phosphate gel-cellulose is prepared as described elsewhere 16 and poured into a column (2.5 × 25 cm). The column is equilibrated with 0.1 M Tris-acetate, pH 7.5. The pooled enzyme from step 4 is loaded and eluted with a linear gradient of potassium phosphate (0-0.1 M in 0.1 M Tris-acetate, pH 7.5; total volume, 500 ml). Ten-milliliter fractions are collected. The enzyme, which follows the colored proteins, emerges at the phosphate concentration range of 0.05-0.07 M. The pooled enzyme (almost colorless) is concentrated by addition of solid ammonium sulfate to 65% saturation (40.7 g/100 ml). The precipitate is sedimented by centrifugation at 9000 g for 40 rain and then dissolved in 1-2 ml of 0.1 M Tris-acetate buffer, pH 7.5. Step 6. Gel Filtration. The enzyme solution from step 5 is applied to a Sephadex G-100 column (1.9 × 50 cm). Either 0.1 M Tris-acetate or 0.05 M potassium phosphate buffer, pH 7.5, is employed to equilibrate the column and filter the enzyme. The pooled fractions containing the enzymic activity are concentrated by precipitation with ammonium sulfate at 80% saturation (39.5 g/100 nil). To achieve a specific activity of 80-95, it may be necessary to repeat steps 5 or 6. Step 7. Crystallization. ~ The enzyme solution from the last chromato-

FIG. 1. Erythrocytic purine nucleoside phosphorylase.

[72]

535

PURINE NUCLEOSIDE PHOSPHORYLASE

PURIFICATION OF HUMAN ERYTHROCYTICPURINE NUCLEOSIDEPHOSPHORYLASE

Purification steps 1. 2. 3. 4. 5.

Hemolysate Calcium phosphate gel adsorption Ammonium sulfate fractionation DEAE-cellulose chromatography Calcium phosphate gel-cellulose chromatography 6. Gel filtration a Repetition of steps 5 and 6, concentration and lyophilizationb 7. Crystallization Recrystallization

Total activity (units) 14,500 14,820 14,520 10,780

Specific Recovery activity Purification (%) 0.01 0.35 2.23 9.19

1 35 223 919

100 102 100 74

6,670 5,120

48.3 69.6

4833 6962

46 35

2,750

93.1

9310

19

1,340

93.0

9300

9~

If seed crystals are available, crystallization is possible after step 6 with an overall recovery of 15-20%. b The enzyme was concentrated and dialyzed by DIAFLO ultrafiltration (Model 50, Amicon Corp., Lexington, Massachusetts) under 40 psi of Ne pressure; however, other techniques that do not increase the salt concentration may be employed. A Collodion Bag Apparatus (Schleicher and Schuell, Inc., Keene, New Hampshire) is preferable for smaller volumes.

graphic step is concentrated in a Collodion Bag Apparatus (Schleicher & Schuell, Inc., Keene, New Hampshire) to a protein concentration of about 10 mg/ml. Alternatively, the enzyme may be concentrated by precipitation at 70% ammonium sulfate saturation. The precipitate is dissolved in 30 mM Tris-acetate buffer, pH 7.5, to give a protein concentration of about 10 mg/ml. The enzyme solution is adjusted very slowly and with continuous mixing to about 35% saturation with saturated ammonium sulfate (recrystaUized) solution. The turbid mixture is centrifuged at 9000 g for 30 min to remove the amorphous precipitate. Saturated ammonium sulfate is added to the supernatant fluid until slight turbidity develops (at about 40% saturation). Upon overnight storage at 4 °, about 50% of the enzyme crystallizes as fine needles and bundles of needles (Fig. 1). The crystals are harvested by centrifugation at 9000 g for 10 rain. A few crystals are reserved for seeding, and the remainder are dissolved in 30 mM Tris-acetate, pH 7.5, and brought to 40% saturation with saturated ammonium sulfate. Seed crystals are added, and 95% of the enzyme crystallizes within 12 hr. The table summarizes the purification procedure from approximately 2 liters of packed erythrocytes.

536

PURINE METABOLIZING ENZYMES

[72]

Properties

Stability. The crude enzyme is stable for several days, even at room temperature, in concentrated solutions, e.g. 1% in 0.02% sodium azide. Purified enzyme is stable as an ammonium sulfate precipitate at 4 ° or frozen for long periods of time. 21 Enzyme preparations of high specific activity (70-96) can decrease in activity rapidly in the absence of sulfhydryl compounds, e.g., dithiothreitol, 1 mM. Inactivation by the sulfhydryl reagents, PCMB and 5,5'-dithio(2-nitrobenzoic acid), can be reversed by dithiothreitol, s Freezing the enzyme in the presence of dithiothreitol results in greater loss of activity than in the absence of a sulfhydryl reagent. 21 Homogeneous enzyme loses about 50% of its activity upon incubation at 57 ° for 15 min and is also rapidly inactivated at pH values below 6.2 and above 10.0. 2 Molecular Properties. Multiple peaks of enzymic activity with pI values from 5.85-6.25 are detectable after column isoelectric focusing of the crystalline enzyme (Fig. 2). 22 Starch gel electrophoresis of hemolysates of washed erythrocytes also reveals multiple bands of enzymic activity, and the presence of the more acidic variants is related to senescence of the cells. 23 Molecular weights have been estimated at 80,000-92,000, based on gel filtration and sedimentation equilibrium analysis. 2"~4"~5Other physical parameters are: Stokes radius, 38 /~ (gel filtration); Sz0,w, 5.4 and 5.5 (from sedimentation velocity and sucrose density gradient centrifugation, respectively); D20.w, 5.7 × 10 - r c m 2 s e c - 1 ; frictional ratio, 1.29; partial specific volume, 0.73 cm 3 g-l.z5 Three moles of hypoxanthine bind per mole of enzyme, 2 and a single protein band of molecular weight 30,000 __+ 500 is observed on SDS gel electrophoresis. ~5 Therefore the enzyme appears to be a homologous trimer. The CD spectrum of the enzyme indicates approximately 65% random coil structure and a very low a-helix content. Tryptophan, cysteine, methionine, histidine, and isoleucine are present in lowest concentration. ~1% = 9.6 at 280 nm. 25 ~lcm Catalytic Properties. The enzyme catalyzes the reversible phospho21 Our unpublished observations. 22 K. C. Agarwal, R. P. Agarwal, J. D. Stoeckler, and R. E. Parks, Jr., Biochemistry 14, 79 (1975). ~s B. M. Turner, R. A. Fisher, and H. Harris, Fur. J. Biochern. 24, 288 (1971). ,4 y. H. Edwards, P. A. Edwards, and D. A. Hopkinson, FEBS Lett. 32, 235 (1973). 25 j. D. Stoeckler, R. P. Agarwal, K. C. Agarwal, K. Schmid, and R. E. Parks, Jr., Biochemistry 17, 278 (1978).

[72]

537

PURINE NUCLEOSIDE PHOSPHORYLASE

I

"5

2.C ~;

5.97'

6.5

1.6

6o I

1.2

'~

0,8

)N UJ

0.4

55

5.0

32

40

48

56

64

77'

80

88

96

104

117'

120

17'8

t~6

FRACTION NUMBER ( 0 . 9 M L )

FIG. 2.

rolysis o f the ribo- a n d d e o x y r i b o n u c l e o s i d e s o f g u a n i n e , h y p o x a n t h i n e , and xanthine. T h e Km values o f s o m e substrates are: h y p o x a n t h i n e , 1.9 x 10 -5 M ; 26 inosine, 4.8 x 10 -5 M ; 21 d e o x y i n o s i n e , 6.6 × l0 -5 M ; 6 g u a n o s i n e , 4.7 x 10 -5 M ; 21 Pi, 3.2 × 10 -5 M . 6 C o m p a r e d to h y p o x a n thine, adenine is a v e r y p o o r substrate (Vmax, 0.6%; Kin, 4.1 X l0 -4 M). 26 F o r m y c i n B inhibits the e n z y m e with a Ki value o f l x l0 -4 M.l° M a n y o t h e r analogs h a v e b e e n t e s t e d for substrate or inhibitory activity. 27 T h e r e a c t i o n follows an o r d e r e d bi-bi m e c h a n i s m with n u c l e o s i d e being the first s u b s t r a t e to a d d and b a s e the last p r o d u c t to leave. 6 N o c o f a c t o r s o r metal r e q u i r e m e n t s are k n o w n . N o e v i d e n c e has b e e n f o u n d o f a r i b o s y l a t e d o r p h o s p h o r y l a t e d intermediate, and the existence o f a ribosyl t r a n s f e r r e a c t i o n f r o m n u c l e o s i d e to base in the a b s e n c e o f Pi r e m a i n s doubtful. 2"6 26 T. P. Zimmerman, N. Gersten, A. F. Ross, and R. P. Miech, Can. J. Biochem. 49, 1050

(1971). ~7 6-Mercaptopurine ribonucleoside, 6-selenoguanosine, 6-thioguanosine, and 8-azaguano-

sine are readily phosphorolyzed, l°,n Allopurinol is ribosylated [T. A. Krenitsky, G. B. Eiion, R. A. Strelitz, and G. H. Hitchings, J. Biol. Chem. 242, 2675 (1967)]. Arabinosylhypoxanthine, 3'-aminoinosine, N~-methylguanosine, and 3-deazaguanosine have low substrate activities. 21 7-Deazainosine and 7-deazathioinosine are competitive inhibitors [M. R. Sheen, H. F. Martin, and R. E. Parks, Jr., Mol. Pharmacol. 6, 255 (1970)] as are also 2,6-diaminopurine and numerous methyl and methoxy purine derivatives [T. A. Krenitsky, G. B. Elion, A. M. Henderson, and G. H. Hitchings, J. Biol. Chem. 243, 2876 (1968)]. Arsenate is a competitive inhibitor of phosphate6; contrary to an earlier report [F. M. Huennekens, E. Nurk, and B. W. Gabrio, J. Biol. Chem. 221, 971 (1956)], pyrophosphate has no effect on the reaction, z~

538

[73]

PURINE M E T A B O L I Z I N G ENZYMES

Activation of enzymic activity is observed at high concentrations of nucleoside substrates. After electrophoretic fractionation of the enzyme, it is seen that activation is more pronounced in the acidic variants than in the basic variants, m'za Treatment with 5,5'-dithiobis(2-nitrobenzoic acid) results in a 60% decrease in activity and loss of substrate activation. Both the activity and the phenomenon of substrate activation are fully recovered after dithiothreitol treatment.2s Sulfhydryl groups play a key role in the enzymic behavior. With labeled PCMB it is possible to titrate 12 -SH groups per mole of enzyme. Activity is lost after the first 3-4 -SH are reacted but may be fully restored with dithiothreitol. As noted above, 5,5'-dithiobis(2-nitrobenzoic acid) reacts with only 2-4 sulfhydryl groups causing partial loss of activity; however, in the presence of SDS, approximately 12 -SH groups are titratable, zs The effect of pH on kinetic parameters suggests an essential role for cysteine and histidine in catalysis. 2 At pH values below 6 and above 8, substrate activation and inhibition, respectively, are observed with inorganic phosphate, z The synthetic reaction is favored with a Keq value of 54 for both inosine and deoxyinosine at pH 7.4, 24°.29'a° 2sR. P. Agarwaland R. E. Parks, Jr., J. Biol. Chem. 246, 3763 (1971). 2~H. M. Kalckar,J. Biol. Chem. 167, 477 (1947). 3 0 M. Friedkin,J. Biol. Chem. 184, 449 (1950).

[73] C h i n e s e

Hamster

Purine Nucleoside

Phosphorylase 1

B y GREGORY MILMAN ',synthesis"

(Hypoxanthineor guanine) + ribose-l-P .

• (inosineor guanosine) + Pi

"breakdown"

Assay Method Principle. Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) in eukaryotes catalyzes the reversible conversion between the purine bases, hypoxanthine and guanine, and their corresponding nucleosides, inosine and guanosine. NUCLEOSIDE SYNTHESIS ASSAY. The formation of inosine or guanosine is measured in a radioisotope assay in which 14C-labeled purine base

1Abbreviations used are: P and Pi, phosphate and inorganic phosphate, respectively; Tricine, N-Tris(hydroxmethyl)methylglycine. METHODS IN ENZYMOLOGY,VOL. LI

Copyright © 1978 by Academic Press, Inc.

Allrightsofreproducfoninanyformreserved. ISBN (k12-181951-5