Eur. J. Biochem. 77,77-85 (1977)

Purification and Comparative Study of Adenine and Guanine Phosphoribosyltransferases from Schizosaccharomyces pombe Maria NAGY and Anne-Marie RIBET

Laboratoire de GenCtique, Institut National Agronomique, Paris

(Received January 31, 1977)

1. Adenine and guanine phosphoribosyltransferases from the yeast Schizosaccharomyces pombe were purified respectively 1200-fold and 1 000-fold from the crude extracts giving specific activities respectively of 18 and 5.7 pmol purine nucleotide monophosphates mg protein-' min-'. The purified enzymes remain stable for at least a month when conserved at -20 "C in the presence of 6 mM 5-phosphoribosyl-1 -pyrophosphate (P-Rib-P,) and 2 mM MgCI, .

2. Both phosphoribosyltransferases have multiple, apparently buffer-independent, pH optima; both have an absolute requirement for M g + , but, contrary to other organisms studied, the effects of inhibitory concentrations of Mg2+ are non-competitive towards P-Rib-P, binding.

3. The hyperbolic P-Rib-P, versus rate curve of guanine phosphoribosyltransferase (but not that of adenine phosphoribosyltransferase) becomes sigmoidal in the presence of fixed concentrations of the reaction product (GMP).

4. Guanine phosphoribosyltransferase is ten times more sensitive to inhibition by reaction pro- ducts in comparison to adenine phosphoribosyltransferase. The inhibition of the first enzyme by other purine and pyrimidine nucleotides appears much more specific relative to the last enzyme.

5. Both activities are separated in two active forms by filtration through a controlled-pore glass column CPG-10. The estimation of molecular weights of these forms gives 48000 and 42000 for guanine phosphoribosyltransferase and 50 000 and 44000 for adenine phosphoribosyltransferase. In the presence of 5 mM MgCl, the last enzyme, but not the first, dissociates into a single form of apparent molecular weight 30 000.

6. Electrophoresis on polyacrylamide gel of crude as well as of purified extracts reveals two active bands of adenine phosphoribosyltransferase but only a single active band of guanine phosphoribosyl- transferase,

In the yeast Schizosaccharomyces pombe, as in many other organisms described [ l ] two distinct en- zymes were identified for the conversion of purine bases to their respective nucleotide monophosphates in the presence of 5-phosphoribosyl-1 -pyrophosphate (P-Rib-P,). Adenine phosphoribosyltransferase cata- lyzes the conversion of adenine to adenylic acid (AMP). Guanine phosphoribosyltransferase catalyzes the con- version of guanine to guanilic acid (GMP), of hypo- xanthine to inosinic acid (IMP) and of xanthine to xanthylic acid (XMP). The two genes coding for these two enzymes have been isolated and the mutant strains lacking respectively adenine phosphoribosyltransferase

Abbreviations. P-Rib-P, , 5-phosphoribosyl-1 -pyrophosphate. Enzymes. Adenine phosphoribosyltransferase (EC 2.4.2.7);

guanine (hypoxanthine and xanthine) phosphoribosyltransferase (EC 2.4.2.8); phosphoribosyldiphosphate amidotransferase (gluta- mate-amidating) (EC 2.4.2.14).

and guanine phosphoribosyltransferase have been denominated dap and pur [2].

Beside the important function of adenine phos- phoribosyltransferase in the reconversion of free intracellular adenine to the mononucleotide derivative, this enzyme was assigned in bacteria to a role in the translocation process of exogenous adenine by the mechanism of 'group translocation' [3]. However, recent studies of purine uptake in Novikoff cells [4], in Escherichia coli B [5] and in Saccharomyces cerevi- siae [6], deny any direct participation of phosphori- bosyltransferases in the translocation process.

It was interesting to analyze the mechanism of purine transport and the eventual participation of adenine phosphoribosyltransferase and/or guanine phosphoribosyltransferase by the use of mutants im- paired in phosphoribosyltransferase activities. The necessary prerequisites to this study, an exhaustive

78 Adenine and Guanine Phosphoribosyltransferases from Schizosaccharomyces pombe

purification of both enzymes as well as a detailed study of their properties, are the subject of this communi- cation.

MATERIALS AND METHODS

Strain and Growth Conditions

The wild-type 972h- strain of the yeast Schizo- saccharomyces pombe was cultured overnight at 30 "C in complete yeast extract medium [7] and harvested at the end of the exponential phase of growth.

Chemicals

14C-labeledpurine bases were from C.E.A. (France), nucleoside mono-, di- and triphosphates were from Calbiochem. Their purity was checked by thin-layer chromatography. P-Rib-P, tetrasodium salt was from Kyowa Hakko Kogyo Co. Ltd, Tokyo (Japan). Its purity, checked by the method of J. Flaks [8], was about 50- 60% (w/w).

Standard Enzymes Assay

Enzymes were assayed as described in [9]. For all the kinetic studies the enzyme preparation purified up to step V was used (Tables 1 and 2). 5 pg protein was added to each assay. The linear relationship between the reaction rate and the time of incubation as well as the protein concentration was checked.

Guanine Phosphoribosyltransferase Purijication

All operations were carried out at 4 "C. Step I : Preparation of Crude Extract. This was as

described in [9]. Step 11: MnClz Treatment. 0.05 ml1 M MnC12 was

added dropwise to each ml of the supernatant from step I, the nucleic acids were allowed to precipitate overnight and further eliminated by centrifugation for 15 rnin at 54000 x g.

Step 111: Chromatography through the CPG-10 Column. A column of 200 ml(l .4 x 100 cm) was filled with controlled-pore glass CPG-10, pore size 12 nm (Electro Nucleonic Inc.) and equilibrated with 0.1 M potassium phosphate buffer (pH 7.8) containing 15% (v/v) glycerol and 0.1 % (v/v) thioglycerol. 10 ml super- natant from step I1 was added on the column and the elution performed with the same buffer at a rate of 40 ml/h. 2-ml fractions were collected. The activity was recovered between 108 and 122 ml of the elution volume (as seen in Fig. 7) and the corresponding frac- tions were pooled.

Step IV: Ammonium Sulfate Fractionation. Solid ammonium sulfate was added to the enzyme solution from step I11 to a concentration of 277 mg/ml (45%

saturation) and the precipitate eliminated by centri- fugation for 15 rnin at 54000 x g. More ammonium sulfate was added to the supernatant up to 134 mg/ml (65% saturation). After 20 min of stirring the pre- cipitate was collected as above and dissolved in 0.75 ml of 0.02 M glycylglycine buffer (pH 6.4) for each ml of enzyme solution used in this step.

Step V : Alumina C y Gel Treatment. 25 mg alumina C y aged gel (Sigma) was added to each ml of enzyme solution from step IV and the suspension was stirred for 10 min to allow the adsorption of proteins. The gel was centrifuged at 54000 x g for 10 min, the super- natant discarded and the gel washed with the same volume of 0.015 M sodium phosphate buffer at pH 7.0. The enzyme was desorbed from the gel with half of the volume of 0.06 M sodium phosphate buffer, pH 7.0. Finally the gel was removed by centrifugation as above.

Step VI: Hydroxyapatite Column. A column of 0.9 x 7 cm of hydroxyapatite was equilibrated with 0.02 M sodium phosphate buffer, pH 6.0, containing 1 mM EDTA. 5 ml supernatant from step V was added on the column. Non-adsorbed material was washed out by 20 mlO.04 M sodium phosphate buffer, pH 7.6. The activity was thereafter eluted by 0.08 M sodium phosphate buffer, pH 7.6, containing 1 mM EDTA at a rate of 0.2 ml/min. The bulk of activity appeared between 3.6 ml and 6.5 ml of the elution volume. It was concentrated about 20-fold in solid poly(ethy1ene glycol) and 6 mM P-Rib-P, was added.

Adenine Phosphoribosyltransferase Purijication

Step I. Crude extract was prepared as described in

Step 11: Heat Treatment. AMP was added to the crude extract up to 10 mM and the mixture was heated for 10 rnin in a water bath at 60 "C. After cooling to 4 "C in an ice bath, it was centrifuged for 20 min at 55 000 x g and the precipitate was discarded.

Step 111: MnC1, Treatment was identical to that described above for guanine phosphoribosyltrans- ferase.

Step IV: Ammonium Sulfate Precipitation. Solid ammonium sulfate was added to the clear supernatant of fraction I11 to a concentration of 45% saturation (277 mgiml). After 20 rnin of stirring the precipitate was collected by centrifugation at 54000 x g for 10 rnin and was dissolved in half of the initial volume of 0.02 M glycylglycine buffer at pH 6.4.

Step V : Alumina C y Gel Treatment. 25 mg alumina C y aged gel was added to each ml fraction IV, the suspension was stirred for 10 min and the enzyme ad- sorbed on the gel was collected by centrifugation at 54000 x g for 10 min. The supernatant was discarded and the gel washed by resuspension in the same volume of 4 mM sodium phosphate buffer at pH 7.0 contain-

[91.

M. Nagy and A,-M. Ribet 79

Table 1. Summary ofpurijication of guanine phosphoribosyltransferase

Steps Total Yield Specific Purifi- activity activity cation

Table 2. Summary ofpurlfieation of adeninephosphoribosyltransferase

Steps Total Yield Specific Purifi- activity activity cation

nmol % GMP/ min

I. Crude extract 700 100 11. MnC1, treatment 700 100

111. CPG-10 column 581 83 IV. (NH,),SO, precipitation 427 61 V. Alumina gel adsorption 217 31

VI. Hydroxyapatite column 175 25

nmol -fold GMP min-' mg protein-'

5.3 1.0 5.8 1.1

19.1 3.6 79.5 15.0

374 71 .O 5746 1084

nmol AMP/ min

I. Crude extract 4310 11. Heat treatment 3875

111. MnC1, treatment 3792 IV. (NH,),SO, precipitation 2155 V. Alumina gel adsorption 1939

VI. Affinity chromatography 1336

% nmol -fold min - ' mg protein-'

14.8 1.0 90 35.5 2.4 88 51.8 3.5

251.6 17 50 45 1080 73 31 18041 1219

100

ing 1 mM EDTA. The gel was settled again by centri- fugation, the washing buffer discarded and the enzyme desorbed in half of the volume of 0.03 M sodium phosphate buffer, pH 7.3, containing 1 mM EDTA. The gel was then eliminated by centrifugation as above.

Step VI: Chromatography on AMP-Sepharose 4B. A 3-ml aliquot of fraction V was added to the AMP- Sepharose 4B column (15 x 110 mm) equilibrated with 0.05 M sodium phosphate buffer, pH 7.0. No attached material was washed out by about 20 ml of the same buffer at a rate of 2 ml in 10 min. The enzyme was eluted selectively with the equilibrating buffer con- taining 6 mM P-Rib-P, and 2 mM MgC1,. Fractions containing the enzyme were pooled (about 10 ml) in a dialysis bag and concentrated to 0.5 ml in solid poly(ethy1ene glycol) 6000.

Both enzymes purified until stage VI can be stored at -20 "C without any loss of activity for at least 1 month when concentrated in the presence of P-Rib-P, as described.

Analytical Polyacrylamide Disc Gel Electrophoresis

50 - 100 p1 adenine phosphoribosyltransferase or guanine phosphoribosyltransferase enzyme sample purified up to stage VI or 10 p1 of crude extracts was electrophoresed on 7.5% polyacrylamide gel in a Tris/glycine buffer system (pH 8.9). The gels were made up in glass tubes (0.5 x 7.5 cm) and a current of 3 mA/gel was maintained for about 3 h. One pair of the gels was stained with Coomassie blue for proteins and the other was frozen to an appropriate consistency for slicing. 1.5-mm slices were obtained, transferred into tubes containing the assay mixture, and the activity was determined after 2 h of incubation at 30 "C.

RESULTS AND DISCUSSION

A. de Groodt et al. [lo] described in 1971 a method for the partial purification of guanine phosphoribosyl-

transferase and adenine phosphoribosyltransferase enzymes from the 972h- strain of Schizosaccharomyces pombe. The method allowed complete separation of both proteins leading to preparations with specific activities of 75 and 80 nmol nucleotides formed min-' mg proteins-' respectively. This activity, as well as the stability of the enzymes, was too low for a further study of their properties.

The procedures summarized in Tables 1 and 2 lead to specific activities of 5746 nmol GMP and 18041 nmol AMP formed min-' mg proteins-'. The ex- haustively purified enzymes were stable for at least a month in the presence of 6 mM P-Rib-P, when con- centrated about 20-fold by solid poly(ethy1ene glycol).

Resistance to Heat

Although de Groodt et al. [lo] described a lower resistance to heat for adenine phosphoribosyltrans- ferase relative to guanine phosphoribosyltransferase, this property is reversed when the enzymes are pro- tected by their respective reaction products AMP and GMP. In the presence of saturating (10 mM) AMP, 90 - 100% adenine phosphoribosyltransferase activity is recovered after 10 min of heating at 60 "C.

In the same conditions guanine phosphoribosyl- transferase in the presence of saturating (1.5 mM) concentrations of GMP is completely inactivated. For this reason, the heat step was not included in the gua- nine phosphoribosyltransferase purification procedure. On the other hand, since the heat step in adenine phosphoribosyltransferase purification could be re- sponsible for the absence of allosteric properties ob- served during the kinetic study of the enzyme, an alternative purification procedure was developed where the heat step was replaced by a CPG-10 chromato- graphy under the same conditions as described for guanine phosphoribosyltransferase. This method, al- though more time consuming, leads to a similar degree of purification to that described in Table 2, but with

80 Adenine and Guanine Phosphoribosyltransferases from Schizosaccharomyces pombe

1500 A I B

I I I I I I

4 5 6 7 8 9 10 PH

Fig. 3 . Effect o f p H on guanine and adenine phosphoribosyltransferase activities. Assays were performed as outlined in Methods except for the buffers, which were at pH indicated: 0.05 M Tris glycine histidine (0-0); 0.1 M Tris maleate (O- - -0); 0.1 M glycine NaOH ( A - ~ -A). (A) Guanine phosphoribosyltransferase. (B) Adenine phosphoribosyltransferase

a lower yield. The kinetic properties of the enzyme purified by this last method appeared identical to those obtained with the enzyme purified by the pro- cedure which included the heat step.

Effect o f p H on Guanine and Adenine Phosphoribosyltransferase Activities

The relationship between the pH and the initial rate of activity of both phosphoribosyltransferases is obviously complex.

For guanine phosphoribosyltransferase, two zones of pH optima were repeatedly found, one between pH 7.6-8.0 and another between pH 9.2-9.5. As seen in Fig. lA, very similar results were obtained with either the Tris/glycine/histidine buffer used from pH 5.2 - 10.2 or the Tris/maleate buffer used from pH 5.2 - 8.4, followed by the glycine/NaOH buffer from

Adenine phosphoribosyltransferase activity has also (independently of the buffer used) two zones of pH optima at pH 7.5 and 8.5 with two additional minor peaks at pH 6.5 and pH 9.25 (Fig. 1B). For both enzymes, rigorously identical curves of pH de- pendency were found with the crude extracts, with extracts purified to stage V or with the two forms of enzymes separated on the CPG-10 column; so it does not seem to be linked with different molecular species. A rather analogous pH dependency has been described for adenine phosphoribosyltransferase from blood platelets [Ill.

pH 8.6 - 9.8.

EfSect o fMgZt Concentration on Guanine and Adenine Phosphoribosyltransferase Activities

Both enzymes have an absolute requirement for Mg2+.

Guanine phosphoribosyltransferase enzyme reach- es maximum activity at 0.5 - 1 mM MgC1, , i.e. 1 - 2 times the P-Rib-P, concentration used (Fig. 2A).

Adenine phosphoribosyltransferase enzyme shows maximum activity at twice the MgCl, concentrations, 1-2 mM (Fig. 2B).

In contradiction to the adenine phosphoribosyl- transferase from monkey liver [12] and the guanine phosphoribosyltransferase of human erythrocytes [13], the inhibitory effect of high MgCl, concentrations is non-competitive towards P-Rib-P, binding for both adenine and guanine phosphoribosyltransferases from Schizosaccharomyces pombe (figure not shown).

Effect of Substrate Concentrations

Because of the complex pH dependence of both phosphoribosyltransferases, the effect of substrate concentration was studied at several pH values, 7.1, 8.0 and 9.25, using either the Tris/HCl buffer, or the Tris/glycine/histidine buffer. At all pH values the double-reciprocal plots of reaction velocity against the increasing concentrations of the substrates studied were linear and gave the same apparent K, values as those taken from the plots in Fig. 3-5, which were performed at pH 8. Adenine phosphoribosyltrans-

M. Nagy and A.-M. Ribet 81

40K

300C

E 2 2mc

."

. v) I c

-

lo0C

0

Fig. 2. Effect of Mgz i- concentration on phosphoribosyltransferase activities. Assays were as outlined in Methods, except for the MgC1, concentrations, which were as indicated. (A) Guanine phospho- ribosyltransferase (0-0). (B) Adenine phosphoribosyltrans- ferase (0-0)

0 1 2 3 1/[S] (mM-')

Fig. 3 . Lineweaver and Burk plots of the effect of the variation of adenine and guanine concentrations on the initial rate of adenine phosphoribosyltransferase activity (.I) and guanine phos- phoribosyltransferase activity (o--o)

ferase has an apparent K, for P-Rib-P, of 32 pM and for adenine of 69 pM. For guanine phosphoribosyl- transferase, the apparent K , for P-Rib-P, is 100 pM and for guanine it is 28 pM. The affinity of both enzymes for their substrates was not modified by the purification procedure.

40

30

I

c - E - - g 2c c I - . .--

1c

1/ [ P- Rib- Pz] (rnM-')

Fig. 4. Product inhibition of guanine phosphoribosyltransferase. (Lineweaver and Burk plot) (o--o) 0 GMP; (A-A) 0.1 mM GMP; (o--n) 0.2 mM GMP

5

I I I I 0 10 20 30 40 50

1 / [ P-Rib- P2] ( r n M - l )

Fig. 5 . Product inhibition of adenine phosphoribosyltransferase activity. (Lineweaver and Burk plot) (0-0) 0 AMP; (A-A) 1 mM AMP; (0-0) 2 mM AMP

82 Adenine and Guanine Phosphoribosyltransferases from Schizosaccharomyces pombe

-E 1000

;; aoo E

5 600 0,

400

5 2 0 0 -

L

0

Table 3. Effects of various nucleotides on guanine and adenine phos- phoribosyltransferase activities In order to exclude the effect of Mg complexation by nucleotide triphosphates this series of experiments was performed at 5 mM MgCl, concentration. NaPPi = sodium pyrophosphate

C -

- - -

Expt no. Inhibitor Concn Inhibition

guanine adenine

ribosyl trans- ribosyl trans- ferase ferase

phospho- phospho-

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

AMP AMP ADP ATP ATP dAMP dATP GMP GMP GDP GTP IMP IDP ITP XMP UMP UDP UTP CMP CDP CTP OMP NaPP, NaPP,

mM

2 5 5 2 5 5 5 0.1 0.5 or 5 0.5 or 5 0.5 or 5 0.5 or 5 0.5 0.5 5 5 5 5 5 5 5 5 2

20

% 0

23 0

0 0

50 90-100 52-100

~

-

30-100 32-100 41 14 50 0

10 0

20

31 43

-

50 77 79 30 50 73 74 0 0-10 0-18 0-51 0-0 -

-

0 0

15

0 0

65 0 0

80

-

Regulation of Phosphoribosyltran$e rase Activities by Reaction Products and Other Nucleotides

While a very close similarity was observed in the properties of both phosphoribosyltransferases de- scribed above, the enzymes studied differ in their sensitivity towards inhibitors, as well as in the mecha- nism of product binding.

Product inhibition is much more efficient for the guanine phosphoribosyltransferase activity relative to adenine phosphoribosyltransferase ; as shown in Table 3,90 % inhibition of the guanine phosphoribosyltrans- ferase activity is reached in the presence of 0.5 mM GMP, whereas adenine phosphoribosyltransferase activity is inhibited by 77% by 5 mM AMP.

Similarly, the common reaction product, pyro- phosphate, at 2 mM concentration lowers the guanine phosphoribosyltransferase activity by 40 % but is without effect on the adenine phosphoribosyltrans- ferase reaction rate.

The same difference between AMP and GMP in their efficiency of inhibition of their own biosynthesis

5000 I A c E - 2 3000 40001 .

1000 1

0 1 ao 90 100 110 120 130 140 Elution vol . (ml)

Fig. 6. Chromatography of adenine phosphoribosyltransferase on controlled-pore glass CPG-10 column. Conditions as described in Methods (step I11 of guanine phosphoribosyltransferase purifi- cation). (A) Elution profile of the activity from the crude extract clarified by MnCl, precipitation. (B) Rechromatography of the hatched area of the first peak from (A). (C) Rechromatography of the hatched area of the second peak from (A)

was found for the first enzyme of the common de novo pathway in Schizosaccharomyces pombe : the P-Rib-P, amidotransferase is 10 times more sensitive to GMP than to AMP [14]. This is in agreement with the ratio of the intracellular concentrations of GMP and AMP which is 1 :10 [15].

The mechanism of the inhibition by products (as far as it was studied) seems also different for the two enzymes. The Lineweaver-Burk plot of guanine phos- phoribosyltransferase saturation by P-Rib-P, (Fig. 4), which is linear in the absence of added GMP, becomes non-linear in the presence of fixed concentrations of GMP. Thus, GMP converts the polymeric enzyme into a form having a lower affinity for P-Rib-P, and the kinetics of saturation by this last substrate exhibit cooperative homotropic effects. On the other hand, the Michaelis-Menten saturation kinetics of adenine phosphoribosyltransferase by P-Rib-P, is not modi- fied in the presence of AMP, which acts as a non- competitive inhibitor with respect to P-Rib-P, (Fig. 5).

M. Nagy and A,-M. Ribet

5 - - f E . 2? 4 - 5 s 'f 3 -

? 0

- V

2 -

1 -

83

0

A

, , pJ;Ld I ?hl., '.* * r'

6 -

5 - I

5 $ 4 - c c 3 s v 3 - 'f

? 0 2 -

1

1 -

0

1- 1.01.1 1.2 1.3 1.4 1.5 1.6 1.7

v / v,

B

I I

Elution volume (mi)

Fig. 7. ( A ) Chromatography of adenine (ap@) and guanine (@- - - @) phosphoribosyltransferases on controlled-pore glass CPG-10. ( B ) Elution profile of adenine and guanine phosphoribosyltransferases from the CPG-10 column equilibrated by the Tris HCI buffer added by 5 m M MgCI,. (A) Conditions as in Fig. 6 except for the column-equilibrating buffer, which was 0.1 M Tris HCI pH 8.2. In the insert: estimation of molecular weights of adenine (@) and guanine (A) phosphoribosyltransferases. Standard proteins used for the calibration of the column were: (1) albumin ( M , = 67000), (2) ovalbumin ( M , = 45 000) and ( 3 ) trypsin ( M , = 23 800). The void volume (V,,), determined by the elution of blue dextran 2 x lo6, was 87.3 ml.

Evidence f o r Heterogeneity among Active Adenine Phosphoribosyltransferase and Guanine Phosphoribosyltransferase Molecules

Molecular Sieve Chromatography. Two close peaks of enzymatic activity for both adenine and guanine phosphoribosyltransferases were separated by the CPG-10 column. A typical elution profile is shown in Fig. 6A for the adenine phosphoribosyltransferase activity, the Vi V, values being 1.25 and 1.32. Guanine phosphoribosyltransferase activity (not shown) gave a very similar pattern, with Vi V, values of 1.23 and 1.33. When the fractions eluted in the first peak (hatched area on Fig. 6A) were pooled and rechroma- tographed in identical conditions, the same two peaks were again found (Fig. 6B). On the other hand, rechromatography of the pooled fractions correspond- ing to the hatched area of the second peak of Fig. 6A,

I-esulted in elution of only one peak of activity (Fig. t5C) corresponding to the position of the second peak c>f Fig. 6A. An analogous behaviour has been de- $scribed for E. coli adenine phosphoribosyltransferase and was found to be due to desaggregation of the en- zyme. Desaggregation of the bacterial enzyme was prevented by Mg2+ ions [3]. In order to check whether o r not Mg ions have the same effect on the yeast en- zymes, crude extracts were prepared in Tris/HCl buf- f'ers containing 0 or 5 mM MgCl, (low solubility of Inagnesium phosphate prevented the use of the phos- phate buffer). These extracts, freed of nucleic acids, were applied onto the CPG-10 column equilibrated with the Tris/HCl buffers with or without MgCl,. As shown in Fig. 7A in the absence of MgCl,, the elution profile of both activities was essentially the same as that found with phosphate buffer. However, in the Iiresence of 5 mM MgCl, , adenine phosphoribosyl-

84 Adenine and Guanine Phosphoribosyltransferases from Schizosaccharomyces pombe

- 0 10 20 30 40 50 60 0 10 20 30 40 50 60

D

r 0 10 20 30 40 50 60 0 10 20 30 40 50 60

Distance from origin (mm)

Fig. 8. Polyacrylamide gel electropherograms of adenine phosphoribosyltransferase purified to stage VI ( A ) and to stage I ( B ) and of guanine phosphoribosyltransferase puriJied to stage VI ( C ) and II (0). Conditions as described in Methods

transferase activity appeared much later in a single peak (at V/Vo=1.55), while the elution pattern of guanine phosphoribosyltransferase was essentially un- changed (V/V, = 1.31 and 1.40) (Fig. 7B). The estima- tion of molecular weights of the active proteins eluted in the above conditions was made from the plot in the insert of Fig. 7A accordingly to Andrews [16]. For guanine phosphoribosyltransferase, molecular weights of 48 000 and 42 000 were found in the absence as well as in the presence of MgCl, . The estimated molecular weights of the two peaks of adenine phosphoribosyl- transferase obtained in the absence of MgCl, were 50000 and 44000 and that found in the presence of 5 mM MgCl, was 30000. These two forms of adenine as well as guanine phosphoribosyltransferases have the same affinity for P-Rib-P, and the same pH de- pendence. Hence the loss of a molecule of apparent molecular weight about 6000 does not seem to affect the catalytic properties of the smaller protein compar- ed to the big one. This loss is apparently not due to the

action of a protease, since the elution profile appeared independent of the time of conservation of the extracts at 4 "C (which varied from 1 to 7 days), and remained also the same in the presence of the protease inhibitor phenylmethylsulfonyl fluoride.

Electrophoresis on Acrylamide Gels. As shown in Fig. 8, the heterogeneity of the adenine phosphori- bosyltransferase was evidenced also by electrophoresis on acrylamide gel. The crude, as well as the exhaustively purified enzymes, separate into two active bands. In the same conditions, guanine phosphoribosyltrans- ferase migrates in a single band. However, by electro- phoresis on sodium dodecyl sulfate/acrylamide gel by the method of K. Weber et al. [17] (experimental details not given) of the adenine phosphoribosyltrans- ferase purified to stage VI, only one protein band with a mobility corresponding to a molecular weight of 40000 was found.

Similar multiplicities of molecular forms with very close molecular weights have been described for phos-

M. Nagy and A.-M. Ribet 85

phoribosyltransferases from other sources, namely for guanine phosphoribosyltransferase from human ery- throcytes [18] and adenine phosphoribosyltransferase from E. coli [3]. The nature and the significance of this heterogeneity are not clear.

Preliminary studies on the cellular localisation of phosphoribosyltransferases in Schizosaccharomyces pombe seem to indicate that a significant amount of these activities are membrane bound [19]. If these results are confirmed, the multiple forms described in this report could be interpreted by association of the enzymes molecules with some membrane material.

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M. Nagy*, Laboratoire d’Enzymologie du C.N.R.S., F-91190 Gif-sur-Yvette, France

A.-M. Ribet, Laboratoire de GCnktique, Institut National Agronomique, 16 Rue Claude-Bernard, F-75005 Paris, France

* To whom correspondence should be addressed.