PROTEIN
EXPRESSION
AND
PURIFICATION
2, 29-33 (1991)
High-Yield Purification of Potato Tuber Pyrophosphate: Fructose-6-phosphate 1 -Phosphotransferase’ Greg B. G. Moorhead and W illiam C. Plaxton Department
of Biology, Queen$ University,
Received October l&1990,
Kingston
and in revised form February
Ontario,
1,199l
The procedure of Yuan et al. (1988, Biochem. Biophys. Res. Commun. 154, 111-117) for the isolation of potato pyrophosphate:fructose-6-phosphate lphosphotransferase (PFP) has been modified so that a high yield of homogeneous enzyme could be obtained. Modifications included a lower temperature heat step, a lower percentage initial polyethylene glycol fractionation step (0 to 4%, w/v), stepwise elution following an increase from 30 to 50 mM pyrophosphate during affinity chromatography on Whatman Pll phosphocellulose, anion-exchange chromatography using Q-Sephaand gel filtration chromatography rose “Fast Flow,” with Superose 6 “Prep grade.” Our procedure resulted in an overall 4 2 % yield and a final specific activity of 87 pmol fructose 1,6-bisphosphate produced per minute per milligram protein. Rabbit anti-(potato PFP) polyclonal antibodies effectively immunoprecipitated the activity of both the pure enzyme and the enzyme from a crude extract. Western blot analysis demonstrated that the antibodies were monospecific for PFP. A survey of various potato cultivars demonstrated significant differences in PFP activity with respect to fresh weight. This observation should be taken into consideration before any purification of potato PFP is undertaken. 0 1991
Academic
Press,
Canada K7L 3N6
Inc.
Fructose 2,6-bisphosphate (F1u-2,6-P,)~ is a key regulatory metabolite localized to the cytosol of eukaryotic
cells which plays a vital role in the regulation of glycolysis and gluconeogenesis. Fru-2,6-P, was first shown to be a potent activator of mammalian PFK and an inhibitor of FBPase (1). Subsequent studies revealed that Fru-2,6-P, also modulates the activity of several plant enzymes, including the pyrophosphate-dependent PFK (PFP) and FBPase (2). Quantitation of the very low intracellular concentration of Fru-2,6-P,, ranging from 0.02 to 30 nmol/g tissue (3), depends upon an assay method which couples a low detection limit with adequate reproducibility. The most extensively used assay for Fru-2,6-P, relies on the activation of isolated potato tuber PFP (4). Kruger and Dennis (5) utilized a combination of column chromatography and preparative nondenaturing PAGE to perform the first purification of a plant PFP (from potato tuber). They reported that the native enzyme was a heterotetramer composed of immunologically unrelated a- and P-subunits having molecular masses of 65 and 60 kDa, respectively (5). Yuan et al. (6) subsequently purified potato PFP to 95% homogeneity without the use of preparative PAGE. Their procedure, however, resulted in a relatively poor overall yield of 14%. The present paper describes a much improved purification protocol for potato PFP. Our procedure not only reduces the total time required for isolation but also gives a much higher yield of enzyme activity and milligram quantities of homogeneous protein. MATERIALS
’ Supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). * Abbreviations used: PFK, ATP:fructose-6-phosphate l-phosphotransferase; FBPase, fructose-1,6-bisphosphate 1-phosphohydrolase; PFP, pyrophosphate:fructose-6-phosphate 1-phosphotransferase; Fru-2,6-P,, fructose 2,6-bisphosphate; DTT, dithiothreitoh PMSF, phenylmethylsulfonyl fluoride; EDTA, ethylenediaminetetraacetate; FPLC, fast protein liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; PVDF, polyvinylidene difluoride; Bistris propane, 1,3bis[tris(hydroxymethyl)methylamino]propane. 1046-5928/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
AND
METHODS
Chemicals and plant material. Biochemicals, coupling enzymes, and Tween 20 were from Sigma Chemical Co. Tris base and SDS were from Schwarz-Mann Biotech. Bistris propane was from Research Organics, Inc. Protein assay reagent was from Pierce Chemical Co. or Bio-Rad. Phosphocellulose P-11 was obtained from Whatman. Ribi adjuvant (product code R730) was from Ribi Immunochemical Research Co. and Staphyb COCCZLS aureus cells (Pansorbin) were from Calbiochem. 29
Inc. reserved.
30
MOORHEAD
Q-Sepharose, Superose 6 “Prep grade,” a HR 16150 column, and the FPLC system were from Pharmacia. Pyrophosphate and all other chemicals were from BDH. All buffers used in the present study were degassed and adjusted to their respective pH values at 25°C. Locally grown potatoes were purchased at the market and used the same day. Enzyme assay. PFP was assayed at 25°C according to Yuan et al. (6). Coupling enzymes were desalted before use. One unit of enzyme is defined as the amount that catalyzes the phosphorylation of 1 pmol of fructose g-phosphate (or utilization of 2 wmol NADH) per minute under these conditions. Antibody production. After collection of preimmune serum, purified PFP [400 pg, dialyzed exhaustively against phosphate-buffered saline (40 m M sodium phosphate, pH 7.4, containing 150 mM NaCl)] emulsified in Ribi adjuvant (total volume 1 ml) was injected (160 pg intramuscularly, 240 pg intradermally) into a 2-kg New Zealand rabbit. Booster injections of the same protein (200 pg) were given at 3 and 4 weeks. One week after the final injection, blood was collected by cardiac puncture. After incubation at 4°C overnight, the clotted cells were removed by centrifugation at 1500g for 10 min. The crude immune serum was frozen in liquid nitrogen and stored at -80°C in 0.04% (w/v) NaN,. Immunotitration of PFP actiuity. Immunoprecipitation of enzyme activity was performed as described in (7) with the exception that 0.011 unit of PFP was incubated with various amounts of immune or preimmune serum. Preimmune serum was added to each sample containing immune serum so that the total volume of serum (immune plus preimmune) remained constant. Each PFP assay was corrected for blank NADH oxidase activity by omitting fructose 6-phosphate from the reaction mixture. Electrophoresis and immunoblotting. SDS-PAGE was performed with a Bio-Rad minigel apparatus and the discontinuous system of Doucet and Trifaro (8) as described in (7). The final acrylamide monomer concentration in the separating gel was 12% (w/v). Western blotting was performed as in (7) except that a PVDF membrane was used in place of nitrocellulose. Western blots and dot blots were probed with anti-(potato tuber PFP) crude immune serum diluted lOOO-fold in 50 m M Tris-HCl (pH 7.5), containing 150 m M NaCl, 0.05% (v/v) Nonidet-P40, and 3% (w/v) bovine serum albumin (fraction V). For dot blot analysis, homogeneous PFP was absorbed directly onto a PVDF membrane and immunodetection was accomplished as in Western blotting. Densitometric scanning of gels and blots was performed with a LKB Ultroscan XL enhanced laser densitometer. Densitometric data were analyzed using the LKB Gelscan XL software (version 2.1).
AND
PLAXTON
Other methods. During purification, protein concentration was determined by the method of Bradford (9). When the Pierce bicinchoninic acid reagent was used to determine protein concentration, the method of Hill and Straka (10) was adopted. In all cases bovine y-globulin was used as the standard. Buffers used in PFP purification. Buffer A: 20 mM Hepes-OH (pH 8.2) and 20 m M potassium acetate-OH (pH 8.2) containing 2 m M DTT, 1 m M PMSF, and 1 mM EDTA. Buffer B: 20 m M imidazole-HCl (pH 6.6) containing 1 mM EDTA and 2 mM DTT. Buffer C: 20 m M Tris-HCl (pH 8.2) containing 20 mM KCl, 1 m M EDTA, and 2 m M DTT. Buffer D: 20 m M Tris-HCl (pH 8.2) containing 1 m M EDTA and 2 m M DTT. RESULTS
Purification
of Potato PFP
A survey of four different locally grown potato cultivars demonstrated PFP activities ranging from 0.19 to 0.54 unit/g fresh weight. The potato cultivar (cv. Chiefton) displaying the highest PFP activity was selected as the source of tissue for PFP purification. Unless otherwise stated, all purification steps were carried out at 4°C. Crude extract. Potato tubers (2 kg) were peeled, diced, and homogenized first in a Waring blender and then with a Polytron, in 2 vol of buffer A. The homogenate was filtered through six layers of cheesecloth and the filtrate was designated the crude extract. To the crude extract were added 0.898 g/liter sodium pyrophosphate and 0.5 ml/liter 4 M MgCl,. The pH was adjusted to 8.2 with 10 M NaOH. Heat treatment. The crude extract was divided into three large flasks and heated to 56°C in a 65°C water bath with constant stirring. The flasks were maintained at 56°C for 5 min and then cooled to 4°C in an ice bath with constant stirring. The heating and cooling steps both required approximately 20 min to perform. The pH was then adjusted to 7.1 with 12 M HCl. PEG fractionation. PEG [average molecular mass 8 kDa; 50% (w/v) dissolved in 20 m M Bistris propaneHCl (pH 7.5) containing 1 m M EDTA] was added to the extract to give a final PEG concentration of 4% (w/v). The solution was gently stirred for 15 min and then centrifuged at 7700g for 20 min. The pellets were discarded and the supernatant was adjusted to 18% (w/v) PEG by the slow addition of solid PEG. The mixture was stirred for 20 min and then centrifuged at 9800g for 20 min. The pellets were resuspended in 150 ml buffer B and clarified by centrifugation at 27,200g for 15 min. Phosphocellulose chromatography. The supernatant was adsorbed at 1 ml/min onto a column of phosphocellulose P-11 (2.5 X 9 cm) preequilibrated with buffer B. The column was connected to an FPLC system and
PYROPHOSPHATE:FRUCTOSE-6-PHOSPHATE TABLE
Purification
Step
Volume (ml)
Crude extract 4% PEG supernatant 18% PEG sediment Phosphocellulose’ Q-Sepharose’ Superose 6 “Prep grade”’
4715 4880 152 17.5 0.72 0.69
31
l-PHOSPHOTRANSFERASE 1
of Potato Tuber PFP
Protein (md 13,343” 4618” 844” 44.8” 9.2” 5.60” 5.18b
Activity (units) 1070 786 702 231 457 453
a Protein determined with the Bradford dye-binding assay (9). b Protein determined with the Pierce bicinchoninic acid reagent according ’ Concentrated pooled fractions.
to the method
Specific activity (units/mg)
Purification (fold)
Yield (%)
-
100 73 66 22 43 42
0.081 0.17 0.83 5.1 49.7 80.9 87.5
of Hill and Straka
2.1 10.2 62.9 614 999
(10).
washed with buffer B until the OD,, decreased to 0.1. The enzyme was eluted with a step from 30 to 50 mM sodium pyrophosphate in buffer B (fraction size, 5 ml). Peak activity fractions were concentrated to about 10 ml with an Amicon PM-30 ultrafilter, diluted sevenfold with buffer C, and then reconcentrated to approximately 17.5 ml.
rified to electrophoretic homogeneity. Laser densitometric scanning of the SDS-gel shown in Fig. 1A confirmed that the purified PFP contained equal amounts of the (Yand a-subunits.
Q-Sepharose FPLC. The phosphocellulose eluent, now equilibrated in buffer C, was adsorbed at 0.75 ml/ min onto a column of Q-Sepharose (1.0 X 3.0 cm) that had been preequilibrated with buffer C. The column was washed with buffer C until the OD,,, reached baseline and then eluted with a linear 20 to 400 mM KC1 gradient (80 ml) in buffer C (fraction size, 1.0 ml). Pooled peak activity fractions were concentrated with an Amicon YM-30 ultrafilter to approximately 1 ml, diluted sixfold with buffer D, and then reconcentrated to 0.72 ml.
Rabbit anti-(potato tuber PFP) immune serum effectively immunoprecipitated the activity of the purified enzyme (Fig. 2). Immunoremoval of 50% of the PFP activity required approximately 5 ~1 of immune serum per unit of enzyme. Similarly, the enzyme was immuno-
Superose 6 FPLC. The concentrated Q-Sepharose pooled fractions were clarified by centrifugation at 16,000g for 10 min in an Eppendorf microcentrifuge. The supernatant was applied to a column (1.6 X 50 cm) of Superose 6 Prep grade (plate number, 13,400/m) at 0.25 ml/min using an FPLC system and buffer D (fraction size, 0.5 ml). Peak activity fractions were concentrated to 0.69 ml with an Amicon YM-30 ultrafilter and then adjusted to contain 50% (v/v) glycerol. Aliquots were frozen in liquid nitrogen and stored at -80°C. After 2 months of storage the enzyme retained 87% of its activity. A second peak of PFP activity was not detected in the Superose 6 effluent. As shown in Table 1, PFP was purified lOOO-fold to a final specific activity of 80.9 units/mg and an overall yield of 42%. SDS-PAGE of the final preparation (Fig. 1A) revealed two equal intensity Coomassie blue staining polypeptides of approximately 65 and 60 kDa (corresponding to the (Y- and P-subunits of PFP, respectively) (5). This indicated that the enzyme had been pu-
Immunology
A
B
FIG. 1. SDS-polyacrylamide minigel electrophoresis and Western blot analysis. (A) SDS-PAGE (127 O, w/v, separating gel) of purified potato PFP. Lanes 1 and 2 contain 1 and 10 pg, respectively, of the Superose 6 “Prep grade” fraction (Table 1). The gel was stained with Coomassie blue R-250. (B) Western blot analysis of potato PFP. Samples were subjected to SDS-PAGE and blot-transferred to a PVDF membrane. Western analysis was performed using lOOO-fold diluted rabbit anti-(potato PFP) immune serum. Antigenic peptides were visualized using an alkaline phosphatase-tagged secondary antibody as described in (7); phosphatase staining was for 15 min at 30°C. Lane 1 contains 50 ng of the homogeneous potato PFP and lane 2 contains 13 fig of protein from a potato crude extract. 0, origin; TD, tracker dye.
32
MOORHEAD
20.
AND
immune J
@ 0
25
50
75
100 125 150 pl serum/unit PFT
0 175
*
200
FIG. 2. Effect of rabbit anti-(potato PFP) immune (0) and preimmune (0) serum on the activity of purified potato tuber PFP. Immunoremoval was performed with 0.011 unit of PFP as described in (7).
precipitated from a crude extract (data not shown). Western blot analysis of the pure enzyme and a potato crude extract demonstrated that the antibodies were monospecific for PFP (Fig. 1B). Densitometric quantitation of the Western blot of purified PFP (Fig. lB, lane 1) revealed that the P-subunit was about 1.5fold more immunoreactive than the a-subunit. By contrast, densitometric scanning revealed that the @-subunit was 4.8fold more immunoreactive than the a-subunit on a Western blot of a potato tuber crude extract (Fig. lB, lane 2). Using the dot blot technique as little as 1 ng of homogeneous potato PFP could be detected when probed with lOOO-fold diluted anti-(potato tuber PFP) immune serum. DISCUSSION
Initial attempts to purify potato PFP according to (6) resulted in a much lower yield of enzyme activity than expected. We obtained a much higher recovery of PFP activity when the heat step was decreased to 56°C and the first PEG fractionation step was lowered to O-4% (w/v). Phosphocellulose chromatography as described by Yuan et al. (6) also yielded unsatisfactory results; the enzyme failed to elute when the column was eluted with equilibration buffer supplemented with 20 mM sodium pyrophosphate. This step was modified by substituting Whatman cellulose phosphate P-11 for Bio-Rad CellexP and eluting PFP with a step from 30 to 50 mM sodium pyrophosphate. The apparent large loss in PFP activity that we observed after phosphocellulose chromatography was largely negated following concentration of the pooled peak activity fractions from the Q-Sepharose column (Table 1). Anion-exchange chromatography was modified in two ways: (i) Q-Sepharose Fast Flow replaced DEAE-Bio-Gel as the anion exchange resin, and (ii) the relative amount of protein applied to the column was increased from 0.3 to 18 mg/ml resin bed volume. This greatly reduced the time required to perform this
PLAXTON
step and resulted in a purification 6.5-fold greater than Yuan et al. (6) achieved with this step. Finally, a Superose 6 Prep grade column (100 ml bed volume) was substituted for a prepacked Superose 6 HR lo/30 column (25 ml bed volume) (6) so that all of the concentrated Q-Sepharose peak activity fractions could be subjected to gel filtration FPLC in a single run with no loss in activity. Overall, our procedure resulted in a relatively high yield of 42% and generated more than 5 mg of homogeneous PFP (Table 1, Fig. 1A). The specific activity of the final preparation (80.9 or 87.5 units/mg using the Bradford and Pierce protein assays, respectively) was at least 2-fold higher than that obtained for any other plant PFP purified to date (6). PFP eluted from the Q-Sepharose column was purified 614-fold and was free from detectable activities of PFK, FBPase, aldolase, Fru-2,6-P,ase, and pyrophosphatase. The enzyme purified to this stage has successfully been utilized for bioassays of Fru-2,6-P, in extracts of soybean root nodules (L. Sung, W. C. Plaxton, and D. B. Layzell, unpublished results). The availability of milligram quantities of homogeneous PFP allowed us to prepare high titer monospecific polyclonal antibodies (Figs. 1B and 2). The ,&subunit appeared to elicit a slightly stronger immunogenic response than the o-subunit (Fig. lB, lane 1). Although the reason for this is unclear, the immunologically unrelated CY-and /3-polypeptides (5) should not necessarily evoke identical immune responses. Interestingly, laser densitometric quantitation of the Western blot of a potato crude extract (Fig. lB, lane 2) suggested that there might be an approximate 1:3 ratio of a$-polypeptides in vivo.3 A variety of recent studies have provided compelling evidence that the (Y- and P-subunits of higher plant PFP represent the regulatory and catalytic subunits, respectively (11, 12). Possibly, the P-subunit is a constitutively expressed protein and PFP becomes functional only following the induced synthesis of the a-subunit. This could provide a rationale for the significant elevation in extractable PFP activity that follows anoxia stress in rice seedlings (13) or nutritional phosphate starvation in black mustard cell suspension cultures (14). The ability to easily purify relatively large quantities of potato PFP should facilitate further biochemical characterization of this key enzyme of plant carbohydrate and energy metabolism. We are currently investigating the potential occurrence of PFP “binding proteins” through the production of a PFP affinity column. REFERENCES 1. Van Schaftingen, E. (1987) Fructose-2,6-bisphosphate. Ada Enzymol. 59,315-396. 2. Stitt, M. (19901 Fructose-2,6-bisphosphate as a regulatory molea This ratio l&fold more the P-subunit on a Western
was calculated on the basis that: (i) the P-subunit was antigenic than the a-subunit (Fig. lB, lane l), and (ii) was 4.8-fold more immunoreactive than the a-subunit blot of a potato tuber crude extract (Fig. lB, lane 2).
PYROPHOSPHATE:FRUCTOSE-6-PHOSPHATE
3.
4.
5.
6.
cule in plants. Annu. Reu. Plant Physiol. Plant Mol. Biol. 41,153185. Bruni, P., Vasta, V., and Farnararo, M. (1989) An endpoint enxymatic assay for fructose-2,6-bisphosphate performed in 96-well plates. Anal. Biochem. 1'78, 324-326. Stitt, M. (1990) Fructose-2,6-bisphosphate, in “Methods in Plant Biochemistry” (Lea, P. J., Ed.), Vol. 3, pp. 87-92, Academic Press, London. Kruger, N. J., and Dennis, D. T. (1987) Molecular properties of pyrophosphate:fructose-6-phosphate phosphotransferase from potato tuber. Arch. Biochem. Biophys. 256,273-279. Yuan, X. H., Kwiatkowska, D., and Kemp, R. G. (1988) Inorganic pyrophosphate fructose-6-phosphate 1-phosphotransferase of the potato tuber is related to the major ATP-dependent phosphofructokinase of E. coli. Biochem. Biophys. Res. Commun. 154, 111-117.
7. Moorhead, G. B. G., and Plaxton, W. C. (1990) Purification characterization of cytosolic aldolase from carrot storage Biochem. J. 269,133-139.
and root.
8. Doucet, J. P., and Trifaro, J. M. (1988) A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution. Anal. Biochem. 168, 265-271.
33
l-PHOSPHOTRANSFERASE
9. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. And. Biochem. 72, 248-254. 10. Hill, H. D., and Straka, J. G. (1988) Protein determination bicinchoninic acid in the presence of sulfhydryl reagents. Biochem. 170,203-208.
using Anal.
11. Cheng, H.-F., and Tao, M. (1990) Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-l-phosphotransferase. J. Biol. Chem. 265,2173-2177. 12. Carlisle, S. M., Blakeley, S. D., Hemmingsen, S. M., Trevanion, S. J., Hiyoshi, T., Kruger, N. J., and Dennis, D. T. (1990) Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the a- and &subunits and with the ATP-dependent phosphofructokinase. J. Biol. Chem. 265, 18,366-18,371. 13. Mertens, E., Larondelle, Y., and Hers, H.-G. (1990) Induction pyrophosphate:fructose 6-phosphate 1-phosphotransferase anoxia in rice seedlings. Plant Physiol. 93, 584-587.
of by
14. Duff, S. M. G., Moorhead, G. B. G., Lefebvre, D. D., and Plaxton, W. C. (1989) Phosphate starvation inducible ‘bypasses’ of adenylate and phosphate dependent glycolytic enzymes in Brassica nigra suspension cells. Plant Physiol. 90, 1275-1278.
EXPRESSION
AND
PURIFICATION
2, 29-33 (1991)
High-Yield Purification of Potato Tuber Pyrophosphate: Fructose-6-phosphate 1 -Phosphotransferase’ Greg B. G. Moorhead and W illiam C. Plaxton Department
of Biology, Queen$ University,
Received October l&1990,
Kingston
and in revised form February
Ontario,
1,199l
The procedure of Yuan et al. (1988, Biochem. Biophys. Res. Commun. 154, 111-117) for the isolation of potato pyrophosphate:fructose-6-phosphate lphosphotransferase (PFP) has been modified so that a high yield of homogeneous enzyme could be obtained. Modifications included a lower temperature heat step, a lower percentage initial polyethylene glycol fractionation step (0 to 4%, w/v), stepwise elution following an increase from 30 to 50 mM pyrophosphate during affinity chromatography on Whatman Pll phosphocellulose, anion-exchange chromatography using Q-Sephaand gel filtration chromatography rose “Fast Flow,” with Superose 6 “Prep grade.” Our procedure resulted in an overall 4 2 % yield and a final specific activity of 87 pmol fructose 1,6-bisphosphate produced per minute per milligram protein. Rabbit anti-(potato PFP) polyclonal antibodies effectively immunoprecipitated the activity of both the pure enzyme and the enzyme from a crude extract. Western blot analysis demonstrated that the antibodies were monospecific for PFP. A survey of various potato cultivars demonstrated significant differences in PFP activity with respect to fresh weight. This observation should be taken into consideration before any purification of potato PFP is undertaken. 0 1991
Academic
Press,
Canada K7L 3N6
Inc.
Fructose 2,6-bisphosphate (F1u-2,6-P,)~ is a key regulatory metabolite localized to the cytosol of eukaryotic
cells which plays a vital role in the regulation of glycolysis and gluconeogenesis. Fru-2,6-P, was first shown to be a potent activator of mammalian PFK and an inhibitor of FBPase (1). Subsequent studies revealed that Fru-2,6-P, also modulates the activity of several plant enzymes, including the pyrophosphate-dependent PFK (PFP) and FBPase (2). Quantitation of the very low intracellular concentration of Fru-2,6-P,, ranging from 0.02 to 30 nmol/g tissue (3), depends upon an assay method which couples a low detection limit with adequate reproducibility. The most extensively used assay for Fru-2,6-P, relies on the activation of isolated potato tuber PFP (4). Kruger and Dennis (5) utilized a combination of column chromatography and preparative nondenaturing PAGE to perform the first purification of a plant PFP (from potato tuber). They reported that the native enzyme was a heterotetramer composed of immunologically unrelated a- and P-subunits having molecular masses of 65 and 60 kDa, respectively (5). Yuan et al. (6) subsequently purified potato PFP to 95% homogeneity without the use of preparative PAGE. Their procedure, however, resulted in a relatively poor overall yield of 14%. The present paper describes a much improved purification protocol for potato PFP. Our procedure not only reduces the total time required for isolation but also gives a much higher yield of enzyme activity and milligram quantities of homogeneous protein. MATERIALS
’ Supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). * Abbreviations used: PFK, ATP:fructose-6-phosphate l-phosphotransferase; FBPase, fructose-1,6-bisphosphate 1-phosphohydrolase; PFP, pyrophosphate:fructose-6-phosphate 1-phosphotransferase; Fru-2,6-P,, fructose 2,6-bisphosphate; DTT, dithiothreitoh PMSF, phenylmethylsulfonyl fluoride; EDTA, ethylenediaminetetraacetate; FPLC, fast protein liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; PVDF, polyvinylidene difluoride; Bistris propane, 1,3bis[tris(hydroxymethyl)methylamino]propane. 1046-5928/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
AND
METHODS
Chemicals and plant material. Biochemicals, coupling enzymes, and Tween 20 were from Sigma Chemical Co. Tris base and SDS were from Schwarz-Mann Biotech. Bistris propane was from Research Organics, Inc. Protein assay reagent was from Pierce Chemical Co. or Bio-Rad. Phosphocellulose P-11 was obtained from Whatman. Ribi adjuvant (product code R730) was from Ribi Immunochemical Research Co. and Staphyb COCCZLS aureus cells (Pansorbin) were from Calbiochem. 29
Inc. reserved.
30
MOORHEAD
Q-Sepharose, Superose 6 “Prep grade,” a HR 16150 column, and the FPLC system were from Pharmacia. Pyrophosphate and all other chemicals were from BDH. All buffers used in the present study were degassed and adjusted to their respective pH values at 25°C. Locally grown potatoes were purchased at the market and used the same day. Enzyme assay. PFP was assayed at 25°C according to Yuan et al. (6). Coupling enzymes were desalted before use. One unit of enzyme is defined as the amount that catalyzes the phosphorylation of 1 pmol of fructose g-phosphate (or utilization of 2 wmol NADH) per minute under these conditions. Antibody production. After collection of preimmune serum, purified PFP [400 pg, dialyzed exhaustively against phosphate-buffered saline (40 m M sodium phosphate, pH 7.4, containing 150 mM NaCl)] emulsified in Ribi adjuvant (total volume 1 ml) was injected (160 pg intramuscularly, 240 pg intradermally) into a 2-kg New Zealand rabbit. Booster injections of the same protein (200 pg) were given at 3 and 4 weeks. One week after the final injection, blood was collected by cardiac puncture. After incubation at 4°C overnight, the clotted cells were removed by centrifugation at 1500g for 10 min. The crude immune serum was frozen in liquid nitrogen and stored at -80°C in 0.04% (w/v) NaN,. Immunotitration of PFP actiuity. Immunoprecipitation of enzyme activity was performed as described in (7) with the exception that 0.011 unit of PFP was incubated with various amounts of immune or preimmune serum. Preimmune serum was added to each sample containing immune serum so that the total volume of serum (immune plus preimmune) remained constant. Each PFP assay was corrected for blank NADH oxidase activity by omitting fructose 6-phosphate from the reaction mixture. Electrophoresis and immunoblotting. SDS-PAGE was performed with a Bio-Rad minigel apparatus and the discontinuous system of Doucet and Trifaro (8) as described in (7). The final acrylamide monomer concentration in the separating gel was 12% (w/v). Western blotting was performed as in (7) except that a PVDF membrane was used in place of nitrocellulose. Western blots and dot blots were probed with anti-(potato tuber PFP) crude immune serum diluted lOOO-fold in 50 m M Tris-HCl (pH 7.5), containing 150 m M NaCl, 0.05% (v/v) Nonidet-P40, and 3% (w/v) bovine serum albumin (fraction V). For dot blot analysis, homogeneous PFP was absorbed directly onto a PVDF membrane and immunodetection was accomplished as in Western blotting. Densitometric scanning of gels and blots was performed with a LKB Ultroscan XL enhanced laser densitometer. Densitometric data were analyzed using the LKB Gelscan XL software (version 2.1).
AND
PLAXTON
Other methods. During purification, protein concentration was determined by the method of Bradford (9). When the Pierce bicinchoninic acid reagent was used to determine protein concentration, the method of Hill and Straka (10) was adopted. In all cases bovine y-globulin was used as the standard. Buffers used in PFP purification. Buffer A: 20 mM Hepes-OH (pH 8.2) and 20 m M potassium acetate-OH (pH 8.2) containing 2 m M DTT, 1 m M PMSF, and 1 mM EDTA. Buffer B: 20 m M imidazole-HCl (pH 6.6) containing 1 mM EDTA and 2 mM DTT. Buffer C: 20 m M Tris-HCl (pH 8.2) containing 20 mM KCl, 1 m M EDTA, and 2 m M DTT. Buffer D: 20 m M Tris-HCl (pH 8.2) containing 1 m M EDTA and 2 m M DTT. RESULTS
Purification
of Potato PFP
A survey of four different locally grown potato cultivars demonstrated PFP activities ranging from 0.19 to 0.54 unit/g fresh weight. The potato cultivar (cv. Chiefton) displaying the highest PFP activity was selected as the source of tissue for PFP purification. Unless otherwise stated, all purification steps were carried out at 4°C. Crude extract. Potato tubers (2 kg) were peeled, diced, and homogenized first in a Waring blender and then with a Polytron, in 2 vol of buffer A. The homogenate was filtered through six layers of cheesecloth and the filtrate was designated the crude extract. To the crude extract were added 0.898 g/liter sodium pyrophosphate and 0.5 ml/liter 4 M MgCl,. The pH was adjusted to 8.2 with 10 M NaOH. Heat treatment. The crude extract was divided into three large flasks and heated to 56°C in a 65°C water bath with constant stirring. The flasks were maintained at 56°C for 5 min and then cooled to 4°C in an ice bath with constant stirring. The heating and cooling steps both required approximately 20 min to perform. The pH was then adjusted to 7.1 with 12 M HCl. PEG fractionation. PEG [average molecular mass 8 kDa; 50% (w/v) dissolved in 20 m M Bistris propaneHCl (pH 7.5) containing 1 m M EDTA] was added to the extract to give a final PEG concentration of 4% (w/v). The solution was gently stirred for 15 min and then centrifuged at 7700g for 20 min. The pellets were discarded and the supernatant was adjusted to 18% (w/v) PEG by the slow addition of solid PEG. The mixture was stirred for 20 min and then centrifuged at 9800g for 20 min. The pellets were resuspended in 150 ml buffer B and clarified by centrifugation at 27,200g for 15 min. Phosphocellulose chromatography. The supernatant was adsorbed at 1 ml/min onto a column of phosphocellulose P-11 (2.5 X 9 cm) preequilibrated with buffer B. The column was connected to an FPLC system and
PYROPHOSPHATE:FRUCTOSE-6-PHOSPHATE TABLE
Purification
Step
Volume (ml)
Crude extract 4% PEG supernatant 18% PEG sediment Phosphocellulose’ Q-Sepharose’ Superose 6 “Prep grade”’
4715 4880 152 17.5 0.72 0.69
31
l-PHOSPHOTRANSFERASE 1
of Potato Tuber PFP
Protein (md 13,343” 4618” 844” 44.8” 9.2” 5.60” 5.18b
Activity (units) 1070 786 702 231 457 453
a Protein determined with the Bradford dye-binding assay (9). b Protein determined with the Pierce bicinchoninic acid reagent according ’ Concentrated pooled fractions.
to the method
Specific activity (units/mg)
Purification (fold)
Yield (%)
-
100 73 66 22 43 42
0.081 0.17 0.83 5.1 49.7 80.9 87.5
of Hill and Straka
2.1 10.2 62.9 614 999
(10).
washed with buffer B until the OD,, decreased to 0.1. The enzyme was eluted with a step from 30 to 50 mM sodium pyrophosphate in buffer B (fraction size, 5 ml). Peak activity fractions were concentrated to about 10 ml with an Amicon PM-30 ultrafilter, diluted sevenfold with buffer C, and then reconcentrated to approximately 17.5 ml.
rified to electrophoretic homogeneity. Laser densitometric scanning of the SDS-gel shown in Fig. 1A confirmed that the purified PFP contained equal amounts of the (Yand a-subunits.
Q-Sepharose FPLC. The phosphocellulose eluent, now equilibrated in buffer C, was adsorbed at 0.75 ml/ min onto a column of Q-Sepharose (1.0 X 3.0 cm) that had been preequilibrated with buffer C. The column was washed with buffer C until the OD,,, reached baseline and then eluted with a linear 20 to 400 mM KC1 gradient (80 ml) in buffer C (fraction size, 1.0 ml). Pooled peak activity fractions were concentrated with an Amicon YM-30 ultrafilter to approximately 1 ml, diluted sixfold with buffer D, and then reconcentrated to 0.72 ml.
Rabbit anti-(potato tuber PFP) immune serum effectively immunoprecipitated the activity of the purified enzyme (Fig. 2). Immunoremoval of 50% of the PFP activity required approximately 5 ~1 of immune serum per unit of enzyme. Similarly, the enzyme was immuno-
Superose 6 FPLC. The concentrated Q-Sepharose pooled fractions were clarified by centrifugation at 16,000g for 10 min in an Eppendorf microcentrifuge. The supernatant was applied to a column (1.6 X 50 cm) of Superose 6 Prep grade (plate number, 13,400/m) at 0.25 ml/min using an FPLC system and buffer D (fraction size, 0.5 ml). Peak activity fractions were concentrated to 0.69 ml with an Amicon YM-30 ultrafilter and then adjusted to contain 50% (v/v) glycerol. Aliquots were frozen in liquid nitrogen and stored at -80°C. After 2 months of storage the enzyme retained 87% of its activity. A second peak of PFP activity was not detected in the Superose 6 effluent. As shown in Table 1, PFP was purified lOOO-fold to a final specific activity of 80.9 units/mg and an overall yield of 42%. SDS-PAGE of the final preparation (Fig. 1A) revealed two equal intensity Coomassie blue staining polypeptides of approximately 65 and 60 kDa (corresponding to the (Y- and P-subunits of PFP, respectively) (5). This indicated that the enzyme had been pu-
Immunology
A
B
FIG. 1. SDS-polyacrylamide minigel electrophoresis and Western blot analysis. (A) SDS-PAGE (127 O, w/v, separating gel) of purified potato PFP. Lanes 1 and 2 contain 1 and 10 pg, respectively, of the Superose 6 “Prep grade” fraction (Table 1). The gel was stained with Coomassie blue R-250. (B) Western blot analysis of potato PFP. Samples were subjected to SDS-PAGE and blot-transferred to a PVDF membrane. Western analysis was performed using lOOO-fold diluted rabbit anti-(potato PFP) immune serum. Antigenic peptides were visualized using an alkaline phosphatase-tagged secondary antibody as described in (7); phosphatase staining was for 15 min at 30°C. Lane 1 contains 50 ng of the homogeneous potato PFP and lane 2 contains 13 fig of protein from a potato crude extract. 0, origin; TD, tracker dye.
32
MOORHEAD
20.
AND
immune J
@ 0
25
50
75
100 125 150 pl serum/unit PFT
0 175
*
200
FIG. 2. Effect of rabbit anti-(potato PFP) immune (0) and preimmune (0) serum on the activity of purified potato tuber PFP. Immunoremoval was performed with 0.011 unit of PFP as described in (7).
precipitated from a crude extract (data not shown). Western blot analysis of the pure enzyme and a potato crude extract demonstrated that the antibodies were monospecific for PFP (Fig. 1B). Densitometric quantitation of the Western blot of purified PFP (Fig. lB, lane 1) revealed that the P-subunit was about 1.5fold more immunoreactive than the a-subunit. By contrast, densitometric scanning revealed that the @-subunit was 4.8fold more immunoreactive than the a-subunit on a Western blot of a potato tuber crude extract (Fig. lB, lane 2). Using the dot blot technique as little as 1 ng of homogeneous potato PFP could be detected when probed with lOOO-fold diluted anti-(potato tuber PFP) immune serum. DISCUSSION
Initial attempts to purify potato PFP according to (6) resulted in a much lower yield of enzyme activity than expected. We obtained a much higher recovery of PFP activity when the heat step was decreased to 56°C and the first PEG fractionation step was lowered to O-4% (w/v). Phosphocellulose chromatography as described by Yuan et al. (6) also yielded unsatisfactory results; the enzyme failed to elute when the column was eluted with equilibration buffer supplemented with 20 mM sodium pyrophosphate. This step was modified by substituting Whatman cellulose phosphate P-11 for Bio-Rad CellexP and eluting PFP with a step from 30 to 50 mM sodium pyrophosphate. The apparent large loss in PFP activity that we observed after phosphocellulose chromatography was largely negated following concentration of the pooled peak activity fractions from the Q-Sepharose column (Table 1). Anion-exchange chromatography was modified in two ways: (i) Q-Sepharose Fast Flow replaced DEAE-Bio-Gel as the anion exchange resin, and (ii) the relative amount of protein applied to the column was increased from 0.3 to 18 mg/ml resin bed volume. This greatly reduced the time required to perform this
PLAXTON
step and resulted in a purification 6.5-fold greater than Yuan et al. (6) achieved with this step. Finally, a Superose 6 Prep grade column (100 ml bed volume) was substituted for a prepacked Superose 6 HR lo/30 column (25 ml bed volume) (6) so that all of the concentrated Q-Sepharose peak activity fractions could be subjected to gel filtration FPLC in a single run with no loss in activity. Overall, our procedure resulted in a relatively high yield of 42% and generated more than 5 mg of homogeneous PFP (Table 1, Fig. 1A). The specific activity of the final preparation (80.9 or 87.5 units/mg using the Bradford and Pierce protein assays, respectively) was at least 2-fold higher than that obtained for any other plant PFP purified to date (6). PFP eluted from the Q-Sepharose column was purified 614-fold and was free from detectable activities of PFK, FBPase, aldolase, Fru-2,6-P,ase, and pyrophosphatase. The enzyme purified to this stage has successfully been utilized for bioassays of Fru-2,6-P, in extracts of soybean root nodules (L. Sung, W. C. Plaxton, and D. B. Layzell, unpublished results). The availability of milligram quantities of homogeneous PFP allowed us to prepare high titer monospecific polyclonal antibodies (Figs. 1B and 2). The ,&subunit appeared to elicit a slightly stronger immunogenic response than the o-subunit (Fig. lB, lane 1). Although the reason for this is unclear, the immunologically unrelated CY-and /3-polypeptides (5) should not necessarily evoke identical immune responses. Interestingly, laser densitometric quantitation of the Western blot of a potato crude extract (Fig. lB, lane 2) suggested that there might be an approximate 1:3 ratio of a$-polypeptides in vivo.3 A variety of recent studies have provided compelling evidence that the (Y- and P-subunits of higher plant PFP represent the regulatory and catalytic subunits, respectively (11, 12). Possibly, the P-subunit is a constitutively expressed protein and PFP becomes functional only following the induced synthesis of the a-subunit. This could provide a rationale for the significant elevation in extractable PFP activity that follows anoxia stress in rice seedlings (13) or nutritional phosphate starvation in black mustard cell suspension cultures (14). The ability to easily purify relatively large quantities of potato PFP should facilitate further biochemical characterization of this key enzyme of plant carbohydrate and energy metabolism. We are currently investigating the potential occurrence of PFP “binding proteins” through the production of a PFP affinity column. REFERENCES 1. Van Schaftingen, E. (1987) Fructose-2,6-bisphosphate. Ada Enzymol. 59,315-396. 2. Stitt, M. (19901 Fructose-2,6-bisphosphate as a regulatory molea This ratio l&fold more the P-subunit on a Western
was calculated on the basis that: (i) the P-subunit was antigenic than the a-subunit (Fig. lB, lane l), and (ii) was 4.8-fold more immunoreactive than the a-subunit blot of a potato tuber crude extract (Fig. lB, lane 2).
PYROPHOSPHATE:FRUCTOSE-6-PHOSPHATE
3.
4.
5.
6.
cule in plants. Annu. Reu. Plant Physiol. Plant Mol. Biol. 41,153185. Bruni, P., Vasta, V., and Farnararo, M. (1989) An endpoint enxymatic assay for fructose-2,6-bisphosphate performed in 96-well plates. Anal. Biochem. 1'78, 324-326. Stitt, M. (1990) Fructose-2,6-bisphosphate, in “Methods in Plant Biochemistry” (Lea, P. J., Ed.), Vol. 3, pp. 87-92, Academic Press, London. Kruger, N. J., and Dennis, D. T. (1987) Molecular properties of pyrophosphate:fructose-6-phosphate phosphotransferase from potato tuber. Arch. Biochem. Biophys. 256,273-279. Yuan, X. H., Kwiatkowska, D., and Kemp, R. G. (1988) Inorganic pyrophosphate fructose-6-phosphate 1-phosphotransferase of the potato tuber is related to the major ATP-dependent phosphofructokinase of E. coli. Biochem. Biophys. Res. Commun. 154, 111-117.
7. Moorhead, G. B. G., and Plaxton, W. C. (1990) Purification characterization of cytosolic aldolase from carrot storage Biochem. J. 269,133-139.
and root.
8. Doucet, J. P., and Trifaro, J. M. (1988) A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution. Anal. Biochem. 168, 265-271.
33
l-PHOSPHOTRANSFERASE
9. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. And. Biochem. 72, 248-254. 10. Hill, H. D., and Straka, J. G. (1988) Protein determination bicinchoninic acid in the presence of sulfhydryl reagents. Biochem. 170,203-208.
using Anal.
11. Cheng, H.-F., and Tao, M. (1990) Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-l-phosphotransferase. J. Biol. Chem. 265,2173-2177. 12. Carlisle, S. M., Blakeley, S. D., Hemmingsen, S. M., Trevanion, S. J., Hiyoshi, T., Kruger, N. J., and Dennis, D. T. (1990) Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the a- and &subunits and with the ATP-dependent phosphofructokinase. J. Biol. Chem. 265, 18,366-18,371. 13. Mertens, E., Larondelle, Y., and Hers, H.-G. (1990) Induction pyrophosphate:fructose 6-phosphate 1-phosphotransferase anoxia in rice seedlings. Plant Physiol. 93, 584-587.
of by
14. Duff, S. M. G., Moorhead, G. B. G., Lefebvre, D. D., and Plaxton, W. C. (1989) Phosphate starvation inducible ‘bypasses’ of adenylate and phosphate dependent glycolytic enzymes in Brassica nigra suspension cells. Plant Physiol. 90, 1275-1278.
