Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose...

Eur. J. Biochem. 171, 599-608 (1988) 0 FEBS 1988

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast Jean FRANCOIS, Emile VAN SCHAFTINGEN and Henri-Gery HERS Laboratoire de Chimie Physiologique, Universiti. Catholique de Louvain and International Institute of Cellular and Molecular Pathology

(Received June 30/August 21, 1987) - EJB 87 0753

Phosphofructokinase 2 from Succharomyces cerevisiue was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [y-32P]ATP- Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit.

A low-K, (0.1 pM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 pM) and stimulated 2 - 3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-K, fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-K, ( z 100 pM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi.

Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate ( K , > 50 pM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.

Fructose 2,6-bisphosphate is a newly discovered [l] regu- lator of carbohydrate metabolism present in most eukaryotic cells (reviewed in [2]). It is formed from Fru6P and ATP by 6-phosphofructo-2-kinase also called phosphofructokinase 2 (PFK 2) and hydrolyzed to Fru6P and Pi by fructose-2,6- bisphosphatase (FBPase 2). In liver [3, 41, muscle [4], and higher plants [5], PFK 2 and FBPase 2 are associated in a bifunctional protein. The bifunctional liver enzyme is a substrate for cyclic-AMP-dependent protein kinase which causes the inactivation of PFK 2 and the activation of FBPase 2. There is also indirect evidence that PFK 2 is phosphorylated by cyclic-AMP-dependent protein kinase in Succharomyces cerevisiae but, in deep contrast to what is known to occur in the liver, this phosphorylation causes the activation of the enzyme [6]. Up to now, FBPase 2 has not been clearly identified in this microorganism [7]. Recently, Purwin et al. [8] purified from yeast an enzyme that converted Fru(2,6)P2 into a mixture of Fru2P, Fru6P and free fructose. Although this enzyme was reported to have a relatively high affinity for

Correspondence to H.-G. Hers, Laboratoire de Chimie Physiologique, UCL-7539, 75 avenue Hippocrate, B-1200 Brussels, Belgium

Abbreviations. Fru(2,6)P2, fructose 2,6-bisphosphate; Fru6P, fructose 6-phosphate; Glc6P, glucose 6-phosphate; C subunit, catalytic subunit of the cyclic-AMP-dependent protein kinase; PFK 2, 6-phosphofructo-2-kinase; FBPase 2, fructose-2,6-bisphosphatase.

Enzymes. 6-Phosphofructo-2-kinase (EC 2.7.1.105); fructose- 2,6-bisphosphatase (EC 3.1.3.46); pyrophosphate: fructose-6-phosphate I-phosphotransferase (EC 2.7.1.90).

Fru(2,6)P2 (Km: 6 pM), its biological significance is obscured by the fact that it copurified up to apparent homogeneity with a p-nitrophenylphosphate phosphatase and that its activity in the crude extract was at least 20-fold higher than that of the activated PFK 2.

The purpose of the present work was to further characterize the enzymes able to form or destroy Fru(2,6)P2 in yeast and, particularly, to establish firmly the phosphorylation of PFK 2 by cyclic-AMP-dependent protein kinase.

MATERIALS AND METHODS

Materials

The anion-exchange Mono Q column, Superose 6B and the gel filtration calibration standards were from Pharmacia (Uppsala, Sweden), blue Trisacryl from LKB (Bromma, Sweden) and P-81 paper from Whatman (Springfield Mills, England). Ultrafilters were from Amicon (Lexington, MA, USA). Histone 2A, bovine heart type 11 cyclic-AMP-dependent protein kinase and ATP-Agarose were from Sigma (St Louis, USA), Dowex AG-1 and SDS-PAGE standards from Bio-Rad (Richmond, CA). Phenylmethylsulfonyl fluoride and dithiothreitol were from Janssen Chimica (Beerse, Belgium), yeast extract and bactopeptone from Difco (Detroit, MI, USA), chemicals from Merck (Darmstadt, FRG) or Fluka (Buchs, Switzerland). Radioactive products were from Amersham International (Amersham, UK), enzymes and other biochemicals from Boehringer (Mannheim, FRG).

600

Pyrophosphate : fructose-6-phosphate 1 -phosphotransferase [9], Fru(2,6)P2 [lo], [2-32P]Fru(2,6)P2 [I I] and [Y-~~PIATP [I 21 were prepared as described. [U-14C]Fru(2,6)P2 was synthesized by incubation of 5 mU rat liver PFK 2 for 5 h in the presence of 0.2 mM [U-14C]Fru6P (30 x lo6 cpm), 1 mM ATP, 5 mM MgCI2 and 20 mM Pi, pH 8.0 in a final volume of 1 ml. The subsequent procedure was as in [Ill. [32P]Glc6P was synthesized by incubation of 1 U yeast hexokinase for 10 rnin in the presence of 5 pCi [Y-~~PIATP, 2 mM glucose, 5 mM MgC12 and 50 mM Tris/acetate pH 7.8 in a final volume of 0.2 ml. The reaction was stopped by heating for 5 rnin at 60°C. The mixture was diluted 10-fold with water and applied onto a Dowex AG-1 column (0.4 x 4 cm). After washing the column with 4 ml water, [32P]Glc6P was eluted with 4 ml 0.2 M NaC1. The pure catalytic subunit of cyclic-AMP- dependent protein kinase type I1 from beef heart was a gift of Dr F. Hofmann (Homburg-Saar, FRG).

PuriJl'cation of phosphofructokinase 2 and of the low-K,,, jructose-2,6-bisphosphatase

The haploid yeast strain Saccharomyces cerevisiae X2180 cultivated on yeast extract/peptone/dextrose medium (2% bactopeptone, 2% glucose, 1% yeast extract) was used for the purification of enzymes. The cells were harvested in the stationary phase of growth approximately 10 h after exhaus- tion of glucose. For the purification of PFK 2,100 - 150 g of cells were homogenized for two 30-s periods in a cell homogenizer MSK (B. Braun, Melsungen, FRG) with 200 g glass beads (0.5 mm diameter) in 50 ml of ice-cold solution containing 20 mM potassium phosphate pH 7.5, I mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA and 0.5 M KCl (buffer A). The beads were removed and washed with 100 ml of this buffer. The washing and the extract were pooled and centrifuged 8 rnin at 35000 xg. The crude extract was obtained by filtration of this supernatant through glass wool and was applied onto a column (5 x 14 cm) of blue Trisacryl. The column was then washed at a rate of 120 ml/h with 1 1 buffer A and developed overnight with a linear gradient of KCl (0.5 - 3 M in 2 x 250 ml of buffer A) at a rate of 28 ml/h. Fractions 54 to 64 (see Fig. 1) were pooled and concentrated to 5 ml by ultrafiltration on Amicon XM- 300 filters. A portion (2.5 ml) of this preparation was applied onto a column (1.4 x 90 cm) of Superose 6B previously washed with 300ml of an ice-cold solution containing 200 mM NaCl, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride and 20 mM potassium phosphate pH 7.5. The active fractions (8 ml) obtained by elution of the column with the same solution (see Fig. 2) were pooled and concentrated to 1.2 ml on Amicon YM-100 filters. A sample (0.5 ml) of this preparation was diluted 10-fold and further purified by chromatography on an ATP-Agarose column (0.5 x 2 cm) equilibrated with 10 ml of a solution containing 1 mM dithiothreitol, 20 mM NaCI, 0.01 O/O Triton X-100 and 20 mM Hepes, pH 7.6 (buffer B). The column was washed successively with 2 ml buffer B, 2ml buffer B containing 5 mM AMP, 2 ml buffer B containing 0.1 mM ATP and finally PFK 2 was eluted with 10 mM MgC12 in buffer B. The recovery of this step was low because only 70% of PFK 2 applied on the column was retained and only 25% of that amount was measured in the eluate. The most active fractions were 9000-fold purified and were used for the phosphorylation experiment described in Fig. 3 . The ability of PFK 2 to be activated by bovine heart cyclic- AMP-dependent protein

kinase was tested as described in [7] at the various steps of its purification.

The first steps of the purification of FBPase 2 were as described above for the purification of PFK 2, except that other fractions of the blue Trisacryl chromatography were pooled and concentrated on Amicon YM-100 filters before being passed through the Superose 6B column, and that phosphate (a competitive inhibitor of FBPase 2) was replaced by 20 mM Hepes pH 7.1 in buffer A. After gel filtration through Superose 6B, the active fractions (8 ml) were pooled and concentrated to 1.2 ml on an Amicon PM-30 filter. This solution was diluted with 5 vol. of a solution containing 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA and 20 mM Tris/Cl, pH 7.4 (buffer C) and poured on the Mono-Q column equilibrated with the same buffer. After washing the column with 8 ml of buffer C, a continuous NaCl gradient (20 ml; 0 - 0.5 M) made in buffer C and programmed in an FPLC system was applied; the active fractions were pooled and kept at - 80 "C in the presence of 10% glycerol. This preparation was stable for at least one week. The crude extract was also filtered through 20 vol. Sephadex G-25 equilibrated with a solution containing 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 20 mM KCI and 20 mM Hepes, pH 7.1.

Separation of phosphatases by chromatography of a crude extract on the Mono (2 column

An extract was prepared from 10 g cells as described above, except that the homogenization and subsequent washing were performed in a solution containing 20 mM NaCI, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol and 20 mM Tris/HCl, pH 8 (buffer D). A 50% solution of poly(ethyleneglyco1) 6000 was added dropwise to this extract to reach 6% and the suspension was allowed to stand for 10 min before being centrifuged for 15 min at 10000 x g. The concentration of poly(ethyleneglyco1) 6000 in the supernatant was raised to 14% and the precipitate was collected by centrifugation, resuspended in 3 ml buffer D and poured on the column, which was then washed with 5 ml buffer D before application of the gradient (see Fig. 10).

Assay of en.zymes

Unless otherwise indicated, all assays were performed at 30 "C and by the following procedures. PFK 2 was measured as described in [6]. For kinetic studies, Fru6P and ATP were used after elimination of contaminating Pi as described in [13].

FBPase 2 was measured by the release of [32P]Pi from [2- 32P]Fru(2,6)P2. The assay mixture contained 1 mg/ml bovine serum albumin, 5 mM MgC12, 2 pM [2-32P]Fru(2,6)P, (50 cpm/pmol) and 50 mM Tris/acetate pH 7.8 in a total volume of 0.1 ml. The assay was stopped by the addition of 0.2 ml of 0.1 M NaOH containing 5 mM P,. [32P]Pi was separated from [2-32P]Fru(2,6)P2 by chromatography on Dowex AG-1 as described previously [Ill. The formation of a [32P]phosph oenzyme was performed at 20°C upon incubation of the sample (30 pl) in the presence of, unless otherwise stated, 2 pM [2-32P]Fru(2,6)Pz (30000 cpm/pmol) and 10 mM MgClz in a total volume of 50 ~ 1 . For quantification of the labelled phosphoprotein formed, the reaction was stopped by adding 20 p1 0.2 M NaOH; 50 pl of the alkaline mixture was spotted on P-81 papers which were then treated and counted as in [14]. Samples of the incubation mixture were also analyzed by SDS-PAGE (see below). A similar

60 1 2.0

- I 0 Y

0

1.5

L

I - 1.0 < - F C a, .-

0.5 p a

0

Fraction number

Fig. 1 . Elution profile of PFK 2 and FBPase 2 from the blue Trisacryl column. A crude extract (1 50 ml) containing 11 2 mU of PFK 2 as well as an unknown quantily of FBPase 2 was applied on the gel. Fractions of 6 ml were collected. FBPase 2 was measured by the release of [3zP]Pi and by the labclling of the enzyme upon incubation for 3 min in the presence of 2 pM [2-3ZP]Fru(2,6)P2

procedure was used in experiments performed with [32P]Glc6P as a substrate. The acid lability of the phosphoenzyme was determined as in [ 141.

The disappearance of Fru(2,6)P2 was assayed in the presence of 50 pM [U-14C]Fru(2,6)P2 (40000 cpm), 5 mM MgC12 and 50 mM Mes, pH 6.0 in a total volume of 0.5 ml. The progress of the reaction was followed by measuring either the remaining Fru(2,6)P2 or the [U-'4C]fructose and the [U- 14C]fructose monophosphates formed. For this purpose, a portion (0.1 ml) of the incubation mixture was mixed with 1 vol. ice-cold 0.2 M NaOH, to which 1 ml ice-cold water was then added. A portion of this solution was used for the determination of Fru(2,6)P2. Another portion (1 ml) was passed through a column (0.4 x 4 cm) of Dowex AG-1 which was washed with 3 ml water; [U-'4C]fructose was counted in this first eluate. The monophosphate esters were then eluted with 4 ml 0.15 M NaCl and the residual [U-14C]Fru(2,6)P2 with 4 ml 0.4 M NaC1, and counted. To determine the pro- portion of Fru2P in the 0.15 M NaCl elution, an aliquot (1 ml) was brought to pH 2 with 0.1 M HCl and incubated 30 min at 30°C to hydrolyze Fru2Pinto fructose and Pi. After neutralization with NaOH and dilution to 2 ml with water, 1 ml of this mixture was fractionated by Dowex AG-1 chromatography as described above.

Enzymatic hydrolysis of Glc6P and sn-glycerol-3-P was measured by the formation of [U-'4C]glucose or [U-'"C]- glycerol in an assay mixture containing 50 pM [U-14C]-Glc6P (100000 cpm) or [U-14C]glycerol-3-P (100000 cpm), 5 mM MgCI2 and 50 mM Mes pH 6.0 in a total volume of 0.1 ml. The reaction was stopped by the addition of 1 vol. ice-cold 0.2 M NaOH followed, after 10 min by 1 ml water. The mixture was passed through a Dowex AG-1 column (0.4 x 4 cm) and radioactive glucose or glycerol were counted in the eluate (see above). The enzymic hydrolysis of p-nitrophenylphosphate was measured spectrophotometrically at 405 nm in the presence of 10 mM p-nitrophenylphosphate, 50 mM Tris/ acetate pH 7.8 an 5 mM MgCI2. Protein kinase activity was determined with histone 2A as a substrate according to [15].

One unit is the amount of enzyme that catalyzes the conversion of 1 pmol substrdte/min under the standard con-

LO 50 60 70 80 Fraction number

Fig. 2. Elution profile of PFK 2 and FBPase 2 from the Superose 6B column. A sample containing PFK 2 (42 m u ) and FBPase 2 (0.7 m u ) was applied on the column and fractions of 2 m l were collectcd. The column was calibrated with thyroglobulin (660 kDa), ferritin (440 kDa), aldolase (240 kDa), albumin (66 kDa) and cytochromc c (12.5 kDa) as standards for the determination of molecular mass (inset)

ditions of the assay, or describes an activity of 1 pmol substrate converted/min under variable experimental conditions.

Other methods

Protein was determined according to Bradford [16] with bovine immunoglobulin G as a standard. For SDS-PAGE analysis, the samples were mixed with 0.25 vol. of a solution containing 8% SDS, 37% sucrose, 40 mM dithiothreitol, 0.08% blue bromophenol and 165 mM Tris/Cl pH 6.8. The mixtures were heated in a boiling water bath for 7 min and applied on gels that were prepared as in [17]. The gels were silver-stained according to [18]. For autoradiography the gels were dried in a Bio-Rad 1125B slab gel dryer and exposed at -80°C to Fuji X-ray films with a Cronex intensifying screen.

RESULTS

Purijkution of phosphofructokinuse 2

PFK 2 was purified 8500-fold by a procedure involving blue Trisacryl chromatography (Fig. l), gel filtration on Superose 6B (Fig. 2) and ATP-agarose chromatography (not shown). The difficulty encountered during this purification was to separate PFK 2 from cyclic-AMP-dependent and cyclic-AMP-independent protein kinases. Indeed, procedures of purification involving as a first step fractionation with poly(ethyleneglyco1) or chromatography on a DEAE- Trisacryl or blue Sepharose column resulted in the copurifica- tion of PFK 2 and protein kinases and in the activation of PFK 2 either during the procedure of the purification or in the assay mixture. This problem could be solved by using chromatography on blue Trisacryl as a first purification step. Indeed, as much as 98% of the protein kinases was eluted from the column by 0.5 M KCl together with 90% of the protein, whereas PFK 2 was strongly retained and came out only with 1.1 M KC1. Attempts at eluting specifically PFK 2 with various ligands, e.g. MgC12, Fru6P, ATP, Fru(2,6)P2, etc., as was done for the purification of the liver and the

602

Table 1. Purification of yeast phosphofructokinase 2 Values shown are cdlcuhled by making the assumption that all the enzyme obtained at one step is used for the next step

Step ~ ~ ~ ~~

Protein Totdl activity Specific activity Purification Yield

Extract Blue Trisacryl Superose 6B ATP-agarose

mg mU 6800 112

33.2 85.5 0.83 26.7 0.022 2.42

mU/mg protein -fold 0.013 1 2.56 196

32 2506 110 8500

~

%

100 I7 24 2.2

muscle enzyme [14], were unsuccessful. As is apparent from Table 1, gel filtration on Superose 6B was a very efficient step, since PFK 2 was found to have a molecular mass higher (estimated to 600 kDa, see inset of Fig. 2) than most other proteins. SDS-PAGE analysis of the fractions eluted from the Superose 6B column revealed that the major constituent of the high-molecular-mass fraction was a 53-kDa peptide also present at much higher concentration in the smaller- molecular-mass fractions. Many other minor bands were apparent in the fractions containing P F K 2 activity. When a pool of these fractions was incubated with [Y-~~PIATP, six or seven major 32P-labelled peptides were apparent on the SDS gel. The intensity of only two of these bands (at 53 and 105 kDa) was markedly increased by the presence of the C subunit in the incubation medium, PFK 2 activity was increased fivefold during this procedure (not shown). The 53-kDa phosphorylatable band was, however, found also in fractions devoid of PFK 2 activity and was, therefore, presumably not to be a subunit of this enzyme.

A further fourfold purification of PFK 2 could be performed by adsorption on ATP-agarose and elution by MgC12. However, PFK 2 purified by this method was very unstable and the total recovery of this step did not exceed 25%. In addition, the purified fraction was very diluted and no peptide band was detected after silver staining of the gel. As shown in Fig. 3, the activity of this fraction could be increased fivefold upon incubation with [Y-~~PIATP and the C subunit. The specific activity of this activated enzyme reached then 550 mU/mg, a value similar to that of the purified spinach leaf PFK 2 (700 mU/mg [5 ] ) and severalfold greater than that of the animal enzyme (50 - 100 mU/mg [2,3,14]). Three peptides were labelled during this activation and could be detected by their radioactivity after SDS-PAGE. The major one was again the 53-kDa peptide, which was not likely to be PFK 2, since its phosphorylation was not entirely C-subunit- dependent and was maximal already after 10 min whereas full activation took more than 30 min. The second one in intensity had a molecular mass of 105 kDa and its phosphorylation roughly paralleled the activation of PFK 2. A third faint band had a molecular mass of 162 kDa. Both the latter peptides were detectably phosphorylated only in the presence of the C subunit.

Kinetic properties of purified phosphofructokinase 2

As previously reported [13], yeast PFK 2 is completely dependent upon the presence of Pi (Fig. 4) or of arsenate (not shown) for its activity. The new fact which is apparent in Fig. 4 is that Pi, at concentrations above 1 mM, is also an inhibitor of the non-activated form of the enzyme, resulting in a greater effect of the activation at high than at low Pi concentration. Under all conditions, the Pi effect was greatly

0.5 L A

~

O O 10 20 30 LO 50 60 Time at 3OoC (m id

Fig. 3. Phosphorylution and simultaneous activation of purified PFK 2 by the catalyticsuhunit of cyclic-AMP-dependentprotein kinase. PFK 2 (0.3 mu) , obtained by elution of the ATP-agarose column, was incubated at 20°C with 2 pg of the catalytic subunit/ml, 20 pM [y-3zP]ATP (20000 cpm/pmol), 5 mM MgC12, 10 mM potassium phosphate, pH 7.5. The change in activity (A) and the incorporation of 32P into peptide subunits separated by SDS-PAGE (B) were recorded as a function of time

to increase I/ with only a minimal decrease (20% if any) of the K, for Fru6P.

When measured at 5 mM Pi, V of the 3000-fold-purified PFK 2 was increased fivefold and K , for Fru6P was decreased from 1.5 mM to 0.4 mM after phosphorylation by the catalytic subunit of bovine heart cyclic-AMP-dependent protein

603

3 0.01 0.1 1 10 [Pi] (mM)

Fig. 4. Phosphate dependency qf the native and of the phosphorylated form of PFK 2. The enzyme purified by blue Trisacryl chromatography was used either as such or after activation as described in Fig. 3 , except that ATP was 2 mM. Both preparations were filtered through Sephadex (3-25 to remove Pi and the reaction was run in the presence of 5 mM Fru6P (without GIc-6-P), 5 mM ATP and the indicated concentrations of Pi

kinase; the K, for ATP was not affected by this treatment. Since, under similar conditions, the enzyme present in a crude yeast extract could be activated tenfold [6], we cannot discard the possibility that a partial activation occurred during the early steps of the purification. The two forms of enzyme were non-competitively inhibited by P-enolpyruvate (Ki = 2 mM) and citrate (Ki = 6 mM). As previously reported, sn-glycerol- 3-P was also an inhibitor, and the phosphorylation of the enzyme increased Ki from 0.7 mM to 3 mM when the assay was performed at 0.5 mM Fru6P.

Identification of a low-K,J~uctose-2,6-bisphosphutuse and its separation f rom phosphofructokinuse 2

The conversion of Fru(2,6)P2 by a crude extract (not gel- filtered) of yeast into Fru6P and Pi at pH 7.8, as measured by the liberation of [32P]Pi from [2-32P]Fru(2,6)P2, was a first- order reaction up to a substrate concentration of 50 pM (not shown). At 10 pM, the rate of reaction was approximately 0.4 nmol min-' (g wet cells)-'. The presence of a low-K,,, FBPase 2 could be demonstrated in this preparation only by its ability to form a [32P]phosphoprotein when incubated in the presence of [2-32P]Fru(2,6)P2. A control reaction was also run with [32P]Glc6P and, as shown in Fig. 5a, a half-maximal incorporation of phosphate was obtained with 5 pM Fru(2,6)P2 and at least 100 FM Glc6P. However, eight times more phosphoprotein could be formed with Glc6P than with Fru(2,6)P2. That the two substrates were utilized by different enzymes was also demonstrated by the different molecular mass of the labelled subunits, which was 56 kDa for FBPase 2 and 60 kDa for the enzyme acting on Glc6P (Fig. 5).

In the various steps of the purification of PFK 2 (see above) FBPase 2 was measured by the two methods described above, which were in close agreement. The enzyme was clearly distinct from PFK 2 from which it was separated in the course of both blue Trisacryl chromatography (Fig. 1) and gel filtration on Superose 6B (Fig. 2). The latter method revealed

Fig. 5. Formation of [32P]phosphoproteins upon a 3 min incubation of a crude extract of yeast in the presence of labelled phosphate esters. The yeast extract was filtered through Sephadex (3-25 before use. The reaction product obtained at (a) 10 pM [32P]Glc6P or (b) 1 pM [2-3'P] Fru(2,6)P2 was submitted to SDS-PAGE and the gel was analyzed by autoradiography. In another similar experiment, FBPase 2 was half- saturated by 1 pM [2-32P]Fru(2,6)Pz, probably because of a more complete elimination of Fru6P by gel filtration

an apparent molecular mass of 110 kDa indicating that FBPase 2 is a dimer. The 56-kDa 32P-labelled subunit was detected by SDS-PAGE performed at all stages of purification (not shown). During chromatography on Mono Q, the two activities coeluted at 0.22 M NaCl, immediately after the main peak of protein and the enzyme was then purified 300-fold (Table 2). A minor fraction of phosphatase active on Fru(2,6)P2 was eluted at 0.4 M NaCI, but it did not catalyze the formation of an intermediary phosphoprotein. It most likely corresponded to the high-K, FBPase 2 present in peak V of the experiment shown in Fig. 10b (see below).

Kinetic properties of the low-K, fructose-2,6-bisphosphatase and its stimulation by benzoate

Purified yeast FBPase 2 had several similarities with the animal or plant enzymes namely a K , of the order of 0.1 pM, an absence of requirement for Mg2+ and a strong inhibition by Fru6P (Fig. 6A). This inhibition was of the mixed type with a Ki of 5 pM for the competitive aspect and of 25 pM for the non-competitive part. Pi, glycerol-2-P and glycerol- 3-P, at a concentration of 5 mM, increased K, threefold without affecting V . Pi could also partially antagonize the inhibition by Fru6P (not shown). Nucleoside triphosphates (GTP and ATP), which are activators of liver FBPase 2 [ll], were without effect on the activity of the yeast enzyme. The enzymic activity was also little affected by variation of pH between 6 an 8.5. Remarkably, benzoate and salicylate, which cause a rapid disappearance of Fru(2,6)P2 in yeast cells [7], increased FBPase 2 activity 2.5 - 3-fold at pH 6 (Fig. 6B) but only 2-fold at pH 7.8 (not shown). Benzoate also slightly increased K, from 0.1 pM to 0.35 pM and did not significantly relieve the inhibition by Fru6P. All attempts made at modulating the activity of yeast FBPase 2 by incubation in the presence of cyclic-AMP-dependent protein kinase, cyclic AMP and ATP- Mg gave negative results.

604

Table 2. Purification of the low-K, fructose-2,6-bisphosphatase The activity was measured either by the release of [32P]Pi from [2-32P]Fru(2,6)P2 (A) or by the formation of a [32P]phosphoenzyme (B). Values shown are calculated by making the assumption that all the enzyme obtained at one step is used for the next step

Step Protein A B Purification Yield

total specific total [E - PI activity activity amount

O f E - P

A B A B

mg clu pU/mg protein pmol pmol/mg protein -fold Yo

Extract 5117 3172 0.62 Blue Trisacryl 95.5 2691 27.1 Superose 6B 19.2 922 48 Mono Q 3.84 636 166

2252 0.44 754 7.14 857 44.6 645 168

1 1 100 100 45 18 85 33 71.5 102 29 38

267 382 20 28

0.2

- c ar .- c g 0.1

? \ 3 E I

I x o

z 0.L

.- > .- +

N

0.3 a m LL 0.2

0.1

0

lFru6Pl (pM) A

B +salicylate 20mM

0:2 0:L 0:6 d.8 ;'y [Fructose 2.6-bisphosphatel (pM)

Fig. 6. Effect of Fru6P ( A ) , benzoate andsalicylate ( B ) on the activity ofthe low-K,= FBPase 2 studied at different concentrations of substrate. The purified enzyme was used. The effects of benzoate and salicylate were studied at pH 6 in the presence of a 50 mM Mes buffer

We show in Fig. 7 that when a partially purified preparation of FBPase 2 was incubated in the presence of 1 pM [2-32P]Fru(2,6)P2, the formation of the radioactive phosphoprotein was at least 60-fold faster than its hydrolysis. The addition of 0.1 mM Fru6P, 5 mM Pi or 5 mM glycerol-2-P markedly decreased both the velocity of formation of the phosphoprotein and the quantity of phosphoenzyme formed. These effects are compatible with the property of these agents to be competitive inhibitors of the enzyme. Benzoate increased about twofold the rate of hydrolysis of the phosphoenzyme (not shown). When incubated at 48°C the phosphoprotein was stable at pH 12 but was destroyed at pH 2.8 at a first- order rate with a half-life of 63 min.

In search of other phosphatases acting on fructose 2,6-bisphosphate

Purwin et al. [8] have recently reported the purification of an enzyme able to convert Fru(2,6)P2 into a mixture of

20" LO 1 3 10 Time at 2OoC (rninl

Fig. 7. Effect of Fru6P, glycerol-2-P and Pi on the labelling of yeast FBPase 2 upon incubation with [2-32P]Fru(2,6)P2. Fraction 58 of the Superose 6B column was incubated in the presence of 1 pM [2- 32P]Fru(2,6)P2 with or without 0.1 mM Fru6P, 5 mM glycerol-2-P or 5 mM Pi

approximately 80% Fru2P and 20% Fru6P, ultimately hydrolyzed into free fructose. This enzyme was most active at pH 6 and copurified with an alkaline phenylphosphate phosphatase which was about 100-fold more active. It was called a fructose-2,6-bisphosphate 6-phosphohydrolase.

In partial agreement with these observations, Fig. 8 shows that Fru(2,6)P2, when incubated at pH 6 in the presence of a gel-filtered yeast extract and at a concentration of 50 pM, was converted into almost equimolar amounts of Fru2P and Fru6P; free fructose was formed after a short delay, more rapidly from Fru6P than from Fru2P. The disappearance of Fru(2,6)P2 under these conditions was close to first order (as it was also, even more strictly, with a purified enzyme [S]), indicating that the enzyme(s) responsible for this conversion had a K, above 50 pM. Its initial rate was about 50 nmol min-' (g wet cells)-'. However, this rate was as low as 0.3 nmol min- (g wet cells)-' (the value reported in [7]) when measured at the more physiological concentration of 10 pM Fru(2,6)P2 and in the presence of 10 mM Pi, 2 mM Glc6P and 0.5 mM Fru6P.

As shown in Fig. 9, changing the pH from 5.5 to 8 greatly reduced the rate of disappearance of Fru(2,6)P2 when present

Fru6P

- _ m aJ 0

\ 3

- -

Cn 50 -

E ; 10 - V C

3 0 - aJ CL Q 20-

~

TI

a_" 10- a. N 3 I

I I _ I; 0 '

& "0 10 20 LO 60 80 100 12

Fig. 8. Action of a yeast extract on 50 pM Fru(2,6) P2. The incubation mixture contained 50 pM [U-'4C]Fru(2,6)P2, 5 mM MgCIZ, 50 mM Mes buffer pH 6 and 0.2 ml of a crude Sephadex filtrate in a total volume of 0.5 ml

Time (rninl

PH

Fig. 9. Effect o f p H on the hydrolysis ofhigh (50 p M ) and low (5 p M ) concentrations of Fru(2,6)P2 by a Sephadexfiltrate. Same conditions as in Fig. 8 except that a Mes/Hepes buffer was used

at 50 pM, but much less when present at 5 pM. Since this indicates the presence of several enzymes with different affin- ities for Fru(2,6)Pz, we applied on a Mono Q column the fraction obtained by precipitation of the extract between 6% and 14% poly(ethyleneglyco1). As shown in Fig. 10 and in Table 3 , five peaks acting on phosphate esters could be separated. Peak I (fraction 10) and peak I11 (fraction 15) were, at pH 6, the most active in causing the disappearance of Fru(2,6)P2 which they converted into a mixture of Fru2P and Fru6P and only for a small part into free fructose. These two peaks were inactive at pH 7.8 (Fig. 10B) and displayed only 20% of their activity at pH 7 (not shown). Their K, for Fru(2,6)P2 was above 50 pM and their action was completely (peak I) or for 50% (peak 111) inhibited by 2 mM Glc6P. Peak I1 (fraction 12) contained the alkaline phenylphosphate phosphatase. It was incompletely separated from peak I and I11 and its action on Fru(2,6)Pz was similar to that of peak I. Its activities on Fru(2,6)P2 at pH 6 and 7.8 were less than 1 YO and 0.01 YO, respectively, of that on phenylphosphate and were 90% inhibited by 2 mM Glc6P or Fru6P. At pH 6 but not at pH 7.8, this fraction was nearly as active on sn-glycerol-3-P

3

2

1 - - E

E \ 3 I

c x o .- .z 0.15 Q

0.10

0.05

C

P D I

A. pH 6.0 \ m 7

Fru-P formation

pNpPase (xIO-') h B. p t i 7.8

605

3 3 E \

j E L :

C .-

L a 2

3

0.1

- 0.3

0.2 = - 0 0

0.1

0 0 10 20 30

Froction number Fig. 10. Separation of several phosphatases by Mono Q chromatography of the 6 - 14% poly(ethyleneglyco1) fraction. Fractions of 1 ml were collected. In A, the disappearance of Fru(2,6)P2 was obtained by direct measurement of the remaining substrate. The formation of Fru-P was measured by the radioactivity of the fraction eluted from Dowex columns at 0.1 5 M NaCl when [U-'4C]Fru(2,6)P2 was used as the substrate. Similar results were obtained when [2-3ZP]Fru(2,6)P2 was used as the substrate (not shown), since Pi and monophosphate esters are eluted simultaneously. In B, FBPase 2 was measured at both 2 pM (V) and 50 pM (V) [2-32P]Fru(2,6)P2. pNpPase = p- nitrophenylphosphatase

than on p-nitrophenylphosphate. In another experiment, and in agreement with [8], this peak was superimposed on peak I (not shown).

Peak IV (fraction 19) was the low-K, FBPase 2, easily recognizable by its ability to form a radioactive phosphoprotein upon incubation in the presence of 1 yM [2-32P]- Fru(2,6)P2 (not shown). Peak V was an apparently specific, high-K, fructose-2,6-bisphosphatase. Indeed, it was inactive on Glc6P and on sn-glycerol-3-P and its activity on Fru(2,6)Pz was not affected by the presence of 2 mM Glc6P, 0.5 mM Fru6P or 1 mM Fru(1,6)Pz; it was also unaffected by 10 mM Pi or by 50 mM benzoate. Its K, for Fru(2,6)PZ was around 100 pM and its activity was little modified by pH changes between 6 and 7. Like the low-K, FBPase 2, the high-K, enzyme converted Fru(2,6)Pz into equimolar amounts of Fru6P and Pi, and neither free fructose nor Fru2P could be detected among the reaction products. In the absence of inhibitors, its activity measured at 10 pM Fru(2,6)Pz was similar to that of peak IV (the low-K, FBPase 2). When the hydrolysis of Fru(2,6)P2 was measured at 10 pM Fru(2,6)Pz and pH 7.1, and in the presence of 2 mM Glc6P, 0.5 mM Fru6P and 10 mM Pi, peak V was the only one to display an important activity.

None of the enzymes present in peaks I-V formed a [32P]phosphoprotein upon incubation with [32P]Glc6P as was shown in Fig. 5. This activity was in great part recovered in the supernatant of the centrifugation performed in the presence of 14% poly(ethyleneglyco1).

606

Table 3. Hydrolysis of Fru(2,6)P2 by fractions from Mono Q chromatography

Parameter Value for peak

I (fraction 10)

I1 111 IV V (fraction 12) (fraction 15) (fraction 19) (fraction 26)

K m [ F ~ ( ~ , ~ ) P Z I > 50 pM

Optimum pH 6.0

Produiformed within- fruczse 7 ' 7 7 -

15 min from Fru2P 60% 50 pM Fru(2,6)PZ Fru6P 33%

Inhibition 100% by 2 mM Glc6P or Fru6P

80% by 10 mM glycerol-2-P 50% by 10 mM Pi

n.d."

6.0

fruiose W!& Fru2 P 60 YO Fru6P 20%

2 mM Glc6P or 1 mM Fru6P or 10 mM Pi

90% by

> 50 pM

5.5

fructose 8% Fru2P 30% Fru6P 62%

2 mM Glu6P or 1 mM Fru6P 80% by 10 mM Pi

50% by

0.2 1M = 100 pM

6.0 - 8.0 6.0-7.0

Fru6P Fru6P

none by 2 mM Glc6P or Fru6P 1 mM Fru(1,6)P2 or 10 mM Pi

inhibited by Fru6 P

a n.d. = not determined

DISCUSSION PURIFICATION AND PROPERTIES OF YEAST PHOSPHOFRUCTOKINASE 2

Structure and phosphorylution

Previous work from this laboratory has revealed the presence in a crude yeast extract, of a PFK 2 that could be activated 5 - 10-fold upon incubation in the presence of ATP-Mg and cyclic AMP. This enzyme had also been purified 150-fold and could then be activated fivefold upon incubation with ATP-Mg and the pure catalytic subunit of beef heart cyclic- AMP-dependent protein kinase [6]. We have now purified yeast PFK 2 8500-fold and could demonstrate that its activation by the C subunit, when performed in the presence of [pJ2P]ATP, was accompanied by the incorporation of 32P into the protein. However, despite its very high specific activity, the purified enzyme was not homogeneous and three radioactive bands were detected by SDS-PAGE. The phosphorylatable 53-kDa peptide was also found in fractions with no PFK 2 activity and, furthermore, its phosphorylation was not entirely dependent on the presence of C subunit; it is therefore most likely not a subunit of PFK 2. In contrast, the labelling of the other two subunits (105 kDa and 162 kDa) was completely cyclic-AMP-dependent; of these two bands, the 105-kDa peptide was the most abundant and since its phosphorylation paralleled the activation, it is most likely the PFK 2 subunit. Since the apparent molecular mass of PFK 2, as measured by gel filtration, is close to 600 kDa, the enzyme could be an hexameric protein. By contrast, Yamoshoji and Hess [19] reported a molecular mass close to 250 kDa for yeast PFK 2, a discrepancy which presumably results from the fact that the sizing gel (Ultragel Aca 34) used by these authors is not expected to separate proteins with a molecular mass above 300 kDa and also that the separation was performed in the absence of salt.

Kinetics

As previously reported [13], yeast PFK 2 requires Pi for its activity, the effect of which is mainly to increase V. The new fact reported in this paper (Fig. 4) is that the effect of Pi is dependent on the activation state of PFK 2. Indeed, the non-activated form of the enzyme was already maximally active at 1 mM Pi and was inhibited by higher concentrations,

whereas the activated form was only saturated at 5 mM Pi and was not inhibited by an excess of this anion. The conse- quence of this difference in sensitivity to Pi is that the activity ratio of activatedlnon-activated PFK 2 increases with the concentration of Pi. When the enzyme was measured in the presence of 5 mM Pi, its phosphorylation resulted in a fivefold increase in V and threefold decrease in K, for Fru6P, without change in K, for ATP. The relatively high value of K , for Fru6P (0.5 mM) can explain why, under some circumstances [6], the rate of Fru(2,6)P2 formation can be limited by the availability of this substrate. Similarly to other PFK 2 enzymes, both forms of yeast PFK 2 were inhibited by P- enolpyruvate, citrate and glycerol-3-P, but these effectors have 'most likely no physiological significance since their Ki is 2 - 5-fold higher than their intracellular concentration.

IDENTIFICATION AND PROPERTIES OF A LOW-K, AND A HIGH-K, FRUCTOSE-2,6-BISPHOSPHATASE AND OF OTHER PHOSPHATASES

In previous work, we have been able to detect only a minimal ability (0.2 nmol min-' g yeast-') of a yeast extract to hydrolyze 10 pM Fru(2,6)P2 into Pi and Fru6P. We have now reconsidered this problem using two different ap- proaches. First, a low-K, FBPase 2, presenting many similarities with the animal [I31 and plant [4] enzymes, was identified thanks to its ability to form a labelled phosphoprotein when incubated in the presence of low concentrations of [2- 32P]Fru(2,6)P2. Second, several other phosphatases, includ- ing an apparently specific high-K, FBPase 2, could be identified after separation by anion-exchange chromatography.

The low-K, FBPase 2 and its separation from PFK 2

The ability of rat liver FBPase 2 to form an acid-labile intermediary phosphoenzyme when incubated with its substrate was first reported by Pilkis et al. [20] and confirmed for the FBPase 2 from chicken liver [14], pigeon muscle [14] and spinach leaves [5]. This property was extensively used to identify the 32F'-labelled subunits of spinach leaf FBPase 2 by autoradiography of the gel obtained by SDS-PAGE [5]. It has now been a key for the identification of FBPase 2 in S . cerevisiue. The labelling method allowed us to identify, already in the crude extract, an enzyme with a K, of 5 pM (Fig. 5), a value presumably overestimated because of the presence of

607

Fru6P in the preparation. This activity was also measured in the eluate of the various chromatographic procedures where it coincided with the release of [32P]Pi from 2 pM [2- 32P]Fru(2,6)Pz and was eventually purified 270-fold. We have also verified the acid lability of the intermediary phosphoenzyme. Its half-life at 48°C and pH 2.8 was longer (63 min) than that reported for other FBPases 2 [5, 14, 201.

Remarkably, the low-K, FBPase 2 clearly separated from PFK 2 in the course of each chromatographic procedure indicating that, in contrast to what is known of the liver, muscle [4] and spinach leaf [5] enzymes, but similarly to the situation in Trypanosoma brucei [21], PFK 2 and FBPase 2 in yeast are not associated in a single bifunctional protein.

It is unlikely that this separation of the two enzymes results from proteolysis since PFK 2, which in animals [2] and plants [5] is much more sensitive to proteolysis than FBPase 2, was, in its native form, 20-50-fold more active than the latter enzyme. The apparent molecular mass of yeast FBPase 2 was close to 110 kDa and the labelling technique allowed us to recognize a subunit of 56 kDa, indicating that the enzyme is a dimer.

The yeast low-K, FBPase 2 displays some kinetic properties similar to those of enzymes found in animals and plants, i.e. a K, lower than micromolar and an inhibition by Fru6P. However, the potent inhibitory action of Fru6P (Ki = 5 pM) raises the question of whether FBPase 2 can be active in vivo at the prevailing concentration of Fru6P (0.2-0.5 mM) present in yeast incubated with glucose. The fact that benzoate and salicylate stimulate by threefold the activity of FBPase 2 is in agreement with the effect of these agents to cause a rapid decrease in the concentration of Fru(2,6)P2 [7].

A high-K, FBPuse 2

The presence of this new enzyme in a crude extract of yeast and in fractions from blue Trisacryl and Superose chromatography was not apparent because the assay was performed at low (2 pM) substrate concentration. It was also masked by the activity of other phosphatases (see next paragraph), from which it was only separated by anion-exchange chromatography. Since it converts Fru(2,6)P2 into equimolar amounts of Fru6P and Pi and does not form Fru2P or fructose, this enzyme appears to be specific to the hexosyl phosphate linkage of Fru(2,6)P2, and can be called a FBPase 2 . As expected, it is inactive on Glc6P and other phosphate esters and is not inhibited by them or by Pi. Furthermore, it is also unaffected by Fru6P, a potent mixed inhibitor of the low-K, FBPase 2. It is therefore qualified to act in yeast cells, in the presence of the high physiological concentrations of Pi and hexose 6-phosphates observed when Fru(2,6)Pz is also present. In contrast to the low-K, FBPase 2, the high-K, enzyme does not form a detectable phosphoenzyme intermediate when incubated in the presence of its substrate. This might be due to the fact that the concentration of Fru(2,6)P2 used in these experiments was far from saturating for the high-K, enzyme.

Other phosphatases

We were able to confirm the finding by Purwin et al. [8] that yeast contains one or several phosphatases able to convert Fru(2,6)P2, at a relatively rapid rate, into Fru2P, Fru6P and eventually fructose. However, the rate of disappearance of 250 pM Fru(2,6)P2 in the presence of the purified enzyme

reported in [8] was first order as indicated by a strictly linear semi-log plot of the data (not shown). This kinetics is not compatible with a K, of 6 pM for Fru(2,6)P2 as reported in [8] but is in agreement with the K, values reported by us in Table3, all above 50pM. Among the various peaks with phosphatase activity identified in Fig. 10, peak I probably corresponds, at least in part, to that purified in [8] since it is most active at pH 6 and forms Fru2P, Fru6P and fructose. Since it was partially separated from p-nitrophenylphosphate phosphatase, at least in the experiment shown in Fig. 10, it does not appear to be identical to that enzyme. The ability of this and other phosphatases to hydrolyze Fru(2,6)P2 is questioned by their low affinity for this substrate relative to its physiological concentration. Furthermore, their activity on Fru(2,6)Pz in vivo would be competitively inhibited by the presence of much higher concentrations of other substrates and would also be greatly reduced by Pi.

The mechanism of Fru(2,6)P~ disappearance in the presence of benzoate

The high-K, FBPase 2 could account for the basal activity of 0.3 nmol min-' (g wet cells)-' observed in the crude, unfiltered extract. As discussed in [7], this activity is too low to explain the rapid disappearance of Fru( 2,6) Pz occurring after the addition of benzoate to yeast incubated with glucose. The action of stimulating 2-3-fold the activity of the low-K, FBPase 2 by benzoate is of interest in this respect, although the high concentration of Fru6P in the cell under these conditions would inhibit the enzyme considerably. The acidification of the intracellular medium by benzoic acid [22] would also greatly increase the activity of the non-specific phosphatases present in peaks I, I1 and 111, with, however, the restriction mentioned above. We must therefore confess that we have no clear explanation for the action of benzoate in causing the disappearance of Fru(2,6)P2 from yeast cells at a rate of 1 nmol min-' (g wet cells)-'.

This work was supported by the Fonds de la Recherche Scientifique Medicale and by the US Public Health Service (grant DK09235). Jean Franqois is Chargd de Recherches and Emile Van Schaftingen is Chercheur qualijid of the Belgian Fonds National de la Recherche Scien t iji'que .

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2.

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4.

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6.

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10.

Van Schaftingen, E., Hue, L. & Hers, H.-G. (1980) Biochem. J .

Van Schaftingen, E. (1987) Adv. Enzymol. Relat. Areas Mol. Biol.

Claus, T. H., El-Maghrabi, M. R., Regen, D. M., Stewart, H.- B., McGrane, M., Kountz, P. D., Nyfeler, F., Pilkis, J. & Pilkis, S. J. (1984) Curr. Top. Cell. Re&. 23, 57-86.

Van Schaftingen, E., Coulie, P. G., Van Snick, J. & Hers, H.-G. (1986) Eur. J . Biochem. 159,367-373.

Larondelle, Y., Mertens, E., Van Schaftingen, E. & Hers, H.-G. (1986) Eur. J . Biochem. 161, 351-351.

FranCois, J., Van Schaftingen, E. & Hers, H.-G. (1984) Eur. J . Biochem. 145, 187- 193.

Franqois, J., Van Schaftingen, E. & Hers, H.-G. (1986) Eur. J. Biochem. 154, 141 - 145.

Purwin, C., Laux, M. & Holzer, H. (1987) Eur. J . Biochem. 164,

Van Schaftingen, E., Lederer, B., Bartrons, R. & Hers, H.-G.

Van Schaftingen, E. & Hers, H.-G. (1981) Eur. J. Biochem. 117,

192, 897-901.

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(1982) Eur. J. Biochem. 129,191 - 195.

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11. Van Schaftingen, E., Davies, D. R. & Hers, H.-G. (1982) Eur. J.

12. Walseth, T. F. & Johnson, R. A. (1979) Biochim. Biophys. Acta

13. Laloux, M., Van Schaftingen, E., Franqois, J. & Hers, H.-G.

14. Van Schaftingen, E. & Hers, H.-G. (1986) Eur. J . Biochem. 159,

15. Roskoski, R. Jr (1983) Methods Enzymol. 99, 3-6. 16. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. 17. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685.

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Note added in proof (received November 9, 1987). After the present manuscript had been sent for publication, Kretchmer et al. (Bio- chem. J . 246, 755-759, 1987) described the purification of PFK 2 and of the low-K,,, FBPase 2 from baker’s yeast and found, as we did, that the two enzymes can be separated from each other. In contrast to our results, however, the activity of their low-K,,, FBPase 2 was almost equal to that of PFK 2 and could be decreased 2.5-fold upon incubation in the presence of ATP-Mg and the C subunit of the bovine heart cyclic AMP-dependent protein kinase. Using commercial baker’s yeast (Levure Royale, Brussels) as a source of enzyme, we have been unable to repeat the observation by Kretchmer et al., but instead obtained results very similar to those reported in this paper. We have no explanation to offer for this discrepancy.

18. Wray, W., Boulikas, T., Wray, V. P. & Hancock, R. (1981) Anal.

19. Yamashoji, S . & Hess, B. (1984) FEBS Lett. 172, 51 -54. 20. Pilkis, S. J., Walderhaug, M., Murray. K., Beth, A,, Venkataramu,

S. D., Pilkis, J. & El-Maghrabi, M. R. (1983) J. Biol. Chem.

21. Van Scbaftingen, E., Opperdoes, F. R. & Hers, H.-G. (1987) Eur.

22. Krebs, H. A., Wiggins, D., Stubbs, M., Sols, A. & Bedoya, F.

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(1983) Biochem. J . 214,651-663.

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Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose...

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