Plant Physiol. (1988) 86, 645-6480032-0889 88/86/0645/04/$01.00/0

Communication

Molecular Comparison of Pyrophosphate- and ATP-DependentFructose 6-Phosphate 1-Phosphotransferases from Potato Tuber'

Received for publication August 31, 1987

NICHOLAS J. KRUGER* AND JOHN B. W. HAMMONDBiochemistry Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station,Harpenden, Hertfordshire AL5 2JQ, United Kingdom

ABSTRACT

The aim of this work was to compare the molecular properties ofpyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) andATP:fructose 6-phosphate 1-phosphotransferase (PFK). Both enzymes werepurified to apparent homogeneity from potato tubers (Solanum tuberosumcv Record). Neither PFP nor PFK preparations contained detectable ac-tivity of the other enzyme. PFP was composed of two polypeptides ofapparent molecular weight 58,000 and 55,700 whereas PFK containedfour polypeptides of apparent molecular weight between 46,300 and 53,300.Chemical cleavage of individual PFP and PFK polypeptides gave a dif-ferent set of fragments for each polypeptide. On Western blots antiseraagainst PFP failed to cross-react with any of the four PFK polypeptides,and antibodies against PFK failed to bind to either of the PFP polypeptides.Antibodies that immunoprecipitate PFP activity had no effect on PFKactivity. Conversely, antibodies against the four PFK polypeptides pre-cipitated the activity of PFK, but not that of PFP. This work shows thatpotato tuber PFP and PFK are composed of distinct, unrelated polypep-tides and indicate that interconversion between PFP and PFK is unlikely.

gestion (6), our knowledge of the kinetic properties of these twoenzymes in plants is far too limited to exclude this possibility.

Previously we have reported that contaminants commonly foundin the commercial assay components used to measure PFK andPFP may confound the results and contribute to the apparentinterconversions (12, 13). Wong et al. (26) have since confirmedthat similar impurities can account for the apparent enhancementof PFP activity in PFK preparations by UDPglucose. However,although such contaminants can explain the original kinetic data(3, 4, 25), they do not eliminate the possibility that PFK andPFP can be reversibly interconverted as initially proposed (4).Recently, PFK has been purified to homogeneity from carrotand contains a single polypeptide of apparent mol wt 60,000 (26).This value is quite different from those of PFK from mammalianand bacterial sources (23), but is similar to that of a PFP poly-peptide from many plant tissues (14). These data support thepossibility that PFP and PFK may be related. To test this sug-gestion further we have compared in detail the molecular prop-erties of the two enzymes involved in the apparent metabolite-mediated interconversion. Here we report that PFK and PFPfrom potato tubers are composed of separate, unrelated poly-peptides. This indicates that direct interconversion between PFKand PFP in higher plants is unlikely.

The conversion of fructose 6-P to fructose 1,6-bisP is an im-portant regulated step of glycolysis. This reaction can be cata-lyzed by two distinct enzyme activities, PFK2 and PFP, for whichthe phosphoryl donors are ATP and PPi, respectively. Manyhigher plants contain significant amounts of both activities (2, 9,16), but their relative importance in glycolysis is unknown (1,24).Balogh et al. (4) have proposed that PFK and PFP can be

reversibly interconverted by appropriate metabolites. The ap-parent conversion of PFK to PFP is mediated by UDPglucose,whereas the reconversion of PFP to PFK is promoted by fructose2,6-bisP. In preliminary reports they suggest that these inter-conversions occur in extracts from a range of tissues, and thatsuch changes contribute to the regulation of glycolysis by varyingthe proportions of PFK and PFP in vivo (3, 25).The evidence for the above interconversions has been criti-

cized, and the kinetic data have been reinterpreted as indicatingthat PFK and PFP are separate, distinct enzymes that are dif-ferentially activated by the effector metabolites (11). Althoughthe known properties of PFK and PFP do not support this sug-

I Supported by Agricultural Genetics Co.2Abbreviations: PFK, ATP:fructose 6-phosphate 1-phosphotransfer-

ase (EC 2.7.1.11); PFP, pyrophosphate:fructose 6-phosphate 1-phos-photransferase (EC 2.7.1.90).

MATERIALS AND METHODS

Materials. Mature potato tubers (Solanum tuberosum L. cvRecord) were supplied by Walkers Crisps Ltd. and were storedat 9 to 15°C. Biochemicals, auxiliary enzymes, and Reactive Red12-Agarose (type 3000-CL) were purchased from Sigma. Seph-adex G-25, DEAE-Sephacel, and ATP-Agarose (type 2) werefrom Pharmacia.Enzyme Purification. PFK was purified over 15,000-fold to a

specific activity of about 200 ,umol-min-'1mg- 1 protein using acombination of ion-exchange, dye-ligand, and affinity chroma-tography as described elsewhere (8).PFP was purified to apparent homogeneity as described pre-

viously (14).Enzyme Assays. PFK and PFP were measured spectrophoto-

metrically as described previously (16). All assays were carriedout at 25°C in a total volume of 1 ml. Ammonium sulfate wasremoved from auxiliary enzymes before use. The PFK assaycontained 100 mm Tris-HC1 (pH 8.0), 5 mM MgCl2, 5 mM fruc-tose 6-P, 1 mm ATP, 0.1 mm NADH, 1 IU aldolase, 10 IUtriosephosphate isomerase, 1.2 IU glycerol 3-P dehydrogenase.Assay conditions for PFP were the same as those for PFK exceptthat 1 mM MgCl2 was present,- 0.2 mm PPi replaced ATP andthe assay contained 1 ,u/M fructose 2,6-bisP. The reactions werestarted with ATP and PPi, respectively.

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KRUGER AND HAMMOND

Electrophoresis. Polypeptides were separated by SDS-PAGEin the presence of 4 M urea using the system described by Lae-mmli (17). Proteins were stained with Coomassie brilliant blue.Chemical Cleavage of Polypeptides. Polypeptides were excised

individually from a polyacrylamide gel and cleaved in situ withcyanogen bromide (21), N-chlorosuccinimide (19), or hydrox-ylamine (18). After cleavage, the peptide fragments were sep-arated by SDS-PAGE and visualized by silver staining (20).Immunochemical Techniques. Antibodies against PFK and PFP

were raised in New Zealand white rabbits as described previously(8, 14). Immunoglobulin G was purified by affinity chromatog-raphy on protein A-Sepharose (15).

Electroblotting and Immunodetection of Proteins. Polypeptideswere separated by SDS-PAGE and electroblotted onto nitro-cellulose (50 V for 3 h, Bio-Rad Trans-Blot cell) as describedby Burnette (7). The resulting blot was probed with up to 0.2ml antiserum in 50 ml PBS (150 mm NaCl, 10 mM NaH2PO4adjusted to pH 7.2 using NaOH) containing 8% (w/v) BSA for12 h, and then washed thoroughly with PBS. Bound antibodieswere detected by incubating the nitrocellulose with 5 ,g proteinA-alkaline phosphatase conjugate in 50 ml PBS containing 4%(w/v) BSA for 2 h. The nitrocellulose was washed exhaustivelywith 1% (v/v) Triton X-100 in phosphate buffered saline andthen rinsed with 100 mm diethanolamine-HCl buffer (pH 9.8).Alkaline phosphatase activity was detected using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (5).

Immunoprecipitation of Enzyme Activity. Potato tuber washomogenized in 1 volume of 100 mm Tris-HCl (pH 8.0), 2 mMMgCl2, 1 mm EDTA, 28 mm 2-mercaptoethanol, 1 mm phen-ylmethylsulfonylfluoride, 2% (w/v) insoluble PVP. The extractwas filtered through four layers of cheesecloth and centrifugedat 18,000g for 30 min. A 2.5 ml sample of the resulting super-natant was passed through a Sephadex G-25 column (1.5 x 5cm) equilibrated with extraction buffer from which phenylme-thylsulfonylfluoride and PVP were omitted. The activities of PFKand PFP in the desalted extract were about 70 and 350nmol-min-1lml- , respectively. A 20 ,ul aliquot of this extractwas added to 80 ul PBS containing up to 40 ,ug purified IgG.The mixture was incubated at room temperature for 60 min.Antibody-antigen complex was precipitated by adding 20 Al 200mg-ml-' insoluble protein A suspended in PBS. The mixturewas incubated for a further 30 min and then centrifuged (Ep-pendorf microfuge, 5 min). PFK and PFP activity remaining inthe supernatant were measured.

RESULTS AND DISCUSSIONBoth PFK and PFP from potato tubers have been purified to

apparent homogeneity and are compared by urea/SDS-PAGE(Fig. 1). The PFK preparation contains four polypeptides (PFKad) of apparent mol wt 46,300, 49,500, 50,000, and 53,300. Evi-dence that all four of these polypeptides are associated with PFKactivity will be presented in a separate publication. In contrast,PFP is composed of two polypeptides (a and f3) of apparent molwt 58,800 and 55,700 (14). Neither of these preparations con-tained detectable activity of the opposite enzyme. We stress thatboth preparations were obtained from the same batch of tuberswhich was grown and stored under uniform conditions. Otherpreparations of PFK and PFP have yielded essentially identicalresults. The mol wt of PFPa, and PFP, we have determined inthe presence of urea are lower than those reported previouslyfor this enzyme using conventional SDS-PAGE (14). Such dif-ferences in the relative migration of polypeptides in the presenceand absence of urea are well established (22) and have also beenobserved with potato tuber PFK (8).

Since PFK and PFP were purified using separate procedures,we investigated whether differential proteolysis in the two pro-tocols could account for the mol wt differences between the two

~ ~ ~ ~ w

FIG. 1. Molecular comparison of potato tuber PFK and PFP poly-peptides. Purified PFK and PFP were subject to SDS-PAGE in thepresence of 4 M urea and then stained with Coomassie brilliant blue.The apparent Mr of each polypeptide was determined by comparisonwith the migration of the following Mr standards: a2-macroglobulin, 170kD; phosphorylase b, 97.4 kD; glutamate dehydrogenase, 55.4 kD; lac-tate dehydrogenase, 36.5 kD; trypsin inhibitor, 20.1 kD. The positionsof PFPa,, PFP,, and PFKa-d are indicated by arrows and the resolutionof the polypeptides is illustrated in the inset.

enzymes. Partial chemical cleavage of each polypeptide usingcyanogen bromide and chlorosuccinimide produced unique setsof peptide fragments (Fig. 2). Under the conditions used, thesechemicals selectively cleave Met-X and Trp-X bonds, respec-tively (19, 21). Similarly, treatment of the polypeptides withhydroxylamine, which hydrolyzes Asn-Gly bonds (18), differ-entially cleaved PFP, and PFP,, but failed to cut any of the PFKpolypeptides. Specific amino- or carboxyl-terminal proteolysis ofthe peptides during enzyme purification could not readily accountfor the differences in the patterns of cleavage fragments derivedfrom PFK and PFP.

Despite differences in the distribution of specific amino acids,PFK and PFP polypeptides could contain extensive regions ofhomology. To test this we have investigated the immunochemicalrelationship between the two enzymes. The polypeptides in pur-ified PFK and PFP were resolved by SDS-PAGE, electroblottedonto nitrocellulose, and then probed with a mixture of antibodiesraised against PFKa-C. In combination these antisera recognizeall four PFK polypeptides (8). Two replicate nitrocellulose filterswere challenged separately with antibodies against PFPG andPFP,. The antisera each reacted strongly with their correspond-ing immunogens, but failed to recognise polypeptides present inthe alternative enzyme (Fig. 3).

In complementary experiments we investigated the relation-ship between PFK and PFP in their native conformation by test-ing the ability of antibodies to precipitate enzyme activity in thepresence of insoluble protein A. A combination of antibodiesagainst PFKaC are known to precipitate all four forms of PFKidentified in potato tuber (8). Such a mixture removed substantialPFK activity from a crude extract of potato tubers, but had no

646 Plant Physiol. Vol. 86, 1988

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PPi AND ATP DEPENDENT PHOSPHOFRUCTOKINASE FROM POTATO TUBER

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FIG. 3. Immunochemical comparison of PFK and PFP polypeptides.The individual polypeptides of PFK and PFP were resolved by SDS-PAGE, electroblotted onto nitrocellulose, and probed with antibodiesraised against PFK, PFP,t, and PFP,, as indicated. Bound antibodieswere detected using alkaline phosphatase conjugated protein A.

effect on PFP activity (Fig. 4). Correspondingly, anti-PFPa andanti-PFP,, precipitated the activity of PFP, but not that of PFK.As observed previously in a partially purified preparation (14),anti-PFP, was more effective than anti-PFPa at removing PFPactivity from the crude potato extract.

Despite the similarity of the reactions catalyzed by PFK andPFP, the above results indicate that the two enzymes are com-

posed of separate, distinct polypeptides. The PFK and PFP poly-peptides clearly differ in mol wt and each yields a different pat-

tern of peptide fragments after chemical cleavage. In addition,these polypeptides are immunologically unrelated both in theirnative conformation and when denatured. Limited data suggestthat PFK and PFP from endosperm of developing castor bean,the only other tissue to have been investigated, are also probablyunrelated (10). Antibodies raised against castor bean leucoplastPFK can immunoprecipitate both cytoplasm and plastid formsof PFK, but do not affect PFP. Conversely, antibodies againstpotato tuber PFP can precipitate endosperm PFP activity, buthave no effect on the corresponding PFK.These results provide compelling evidence that PFK and PFP

in higher plants are independent enzymes and are not intercon-vertible. In combination with earlier reports (11-13), the presentwork suggests that the apparent interconversion of PFK and PFPis an artifact arising from contaminants in the assays of the twoenzymes.

Acknowledgments-We thank Walkers Crisps Ltd. for supplying the potatotubers used in these experiments.

LITERATURE CITED

1. AP REES T 1985 The organization of glycolysis and the oxidative pentosephosphate pathway in plants. In R Douce, D Day, eds, Encyclopedia ofPlant Physiology NS, Vol 18, Springer-Verlag, Berlin, pp 391-417

2. AP REES T, JH GREEN, PM WILSON 1985 Pyrophosphate:fructose 6-phosphate1-phosphotransferase and glycolysis in non-photosynthetic tissues of higherplants. Biochem J 227: 199-204

3. BALOGH A, JH WONG, BB BUCHANAN 1984 Metabolite-mediated intercon-version of PFP/PFK: a regulatory mechanism to direct cytosolic carbon flux.Plant Physiol 75: S-53

4. BALOGH A, JH WONG, C W6TZEL, J SOLL. C CSgKE, BB BUCHANAN 1984Metabolite-mediated catalyst conversion of PFK and PFP: a mechanism ofenzyme regulation in green plants. FEBS Lett 169: 287-292

647

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KRUGER AND HAMMOND

75[

50[

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PFP

PFK

- anti-PFK

if*. * I*/-

PFK

- PFP

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anti-PFP

25\PFP0 5 10 15 20 40

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FIG. 4. Immunoprecipitation of potato tuber PFK and PFP. A crudepotato tuber extract was incubated with antibodies raised against PFK,PFP,,, and PFP,, as indicated. Insoluble protein A was added to themixture which was then centrifuged. The activity of PFK (0) and PFP

(M) remaining in the supernatant was measured and is expressed as a

percentage of the activity prior to the addition of antibodies. Each value

is the mean of three separate samples for which the SE was less than 5%.

Equivalent amounts of preimmune IgG had no detectable effect on the

enzymes. The initial activities of PFK and PFP were 70 and 350nmol min- 'ml- 1, respectively.

Plant Physiol. Vol. 86, 1988

5. BLAKE MS, KH JOHNSTON, GJ RuSSELL-JONES. EC GOTSCHLICH 1984 Arapid, sensitive method for detection of alkaline phosphatase-conjugatedanti-antibody on Western blots. Anal Biochem 136: 175-179

6. BUCHANAN BB 1985 Response to paper by Juana M Gancedo. FEBS Lett180: 65

7. BURNETTE WN 1981 "Western blotting": electrophoretic transfer of proteinsfrom sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellu-lose and radiographic detection with antibody and radioiodinated proteinA. Anal Biochem 112: 195-203

8. BURRELL MM, JBW HAMMOND, NJ KRUGER 1987 Characterization of mul-tiple forms of potato tuber phosphofructokinase. XIV International Botan-ical Congress Abstracts, p 56

9. CARNAL NW, CC BLACK 1983 Phosphofructokinase activities in photosyntheticorganisms. The occurrence of pyrophosphate-dependent-6-phosphofructo-kinase in plants and algae. Plant Physiol 71: 150-155

10. DENNIS DT, WE HEKMAN, A THOMSON, RJ IRELAND, FC BOTHA, NJ KRUGER1985 Compartmentation of glycolytic enzymes in plant cells. /it RL Heath,J Preiss, eds, Regulation of Carbon Partitioning in Photosynthetic tissue.American Society of Plant Physiologists, Rockville, MD, pp 127-146

11. GANCEDO JM 1984 Metabolite-mediated catalyst conversion of PFK and PFP:can PFK really be converted to PFP? FEBS Lett 175: 369-370

12. KRUGER NJ, DT DENNIS 1985 A source of apparent pyrophosphate:fructose6-phosphate phosphotransferase activity in rabbit muscle phosphofructoki-nase. Biochem Biophys Res Commun 126: 320-326

13. KRUGER NJ, DT DENNIS 1985 Reassessment of an apparent hyperactive formof phosphofructokinase from plants. Plant Physiol 78: 645-648

14. KRUGER NJ, DT DENNIS 1987 Molecular properties of pyrophosphate:fructose6-phosphate phosphotransferase from potato tuber. Arch Biochem Biophys256: 273-279

15. KRUGER NJ, JBW HAMMOND 1988 Purification of immunoglobulins usingprotein A-Sepharose. In JM Walker, ed, Methods in Molecular Biology,Vol 3. Humana Press, Clifton, NJ. In press

16. KRUGER NJ, E KOMBRINK, H BEEVERS 1983 Pyrophosphate:fructose 6-phos-phate phosphotransferase in germinating castor bean endosperm. FEBS Lett153: 409-412

17. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

18. LAM KS, CB KASPER 1980 Sequence homology analysis of a heterogenousprotein population by chemical and enzymic digestion using a two-dimen-sional sodium dodecyl sulfate-polyacrylamide gel system. Anal Biochem 108:220-226

19. LISCHWE MA, D OCHS 1982 A new method for partial peptide mapping usingN-chlorosuccinimide/urea and peptide silver staining in sodium dodecyl sul-fate-polyacrylamide gels. Anal Biochem 127: 453-457

20. MERRIL CR, D GOLDMAN, ML VAN KEUREN 1983 Silver staining methodsfor polyacrylamide gel electrophoresis. Methods Enzymol 96: 230-239

21. NIKODEM V, JR FRESCO 1979 Protein fingerprinting by SDS-gel electrophoresisafter partial fragmentation with CNBr. Anal Biochem 97: 283-286

22. PICCIONI R, G BELLEMARE, N-H CHUA 1982 Methods of polyacrylamide gelelectrophoresis in the analysis and preparation of plant polypeptides. In MEdelman, RB Hallick. N-H Chua, eds, Methods in Chloroplast MolecularBiology. Elsevier Biomedical, Amsterdam, pp 985-1014

23. UYEDA K 1979 Phosphofructokinase. Adv Enzymol Rel Areas Mol Biol 48:193-244

24. VAN SCHAFTINGEN E 1987 Fructose 2,6-bisphosphate. Adv Enzymol Rel AreasMol Biol 59: 315-395

25. WONG JH, A BOLOGH, C WOTZEL, J SOLL, BB BUCHANAN 1984 Metabolite-mediated catalyst conversion in C3 and C4 plants. Plant Physiol 75: S-53

26. WONG JH, BC YEE, BB BUCHANAN 1987 A novel type of phosphofructokinasefrom plants. J Biol Chem 262: 3185-3191

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