Plant Physiol. (1988) 86, 645-648 0032-0889 88/86/0645/04/$01.00/0

Communication Molecular Comparison of - and ATP-Dependent 6-Phosphate 1- from Potato Tuber' Received for publication August 31, 1987

NICHOLAS J. KRUGER* AND JOHN B. W. HAMMOND Downloaded from https://academic.oup.com/plphys/article/86/3/645/6082991 by guest on 29 September 2021 Biochemistry Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, United Kingdom

ABSTRACT gestion (6), our knowledge of the kinetic properties of these two in plants is far too limited to exclude this possibility. The aim of this work was to compare the molecular properties of Previously we have reported that contaminants commonly found pyrophosphate:fructose 6-phosphate 1- (PFP) and in the commercial assay components used to measure PFK and ATP:fructose 6-phosphate 1-phosphotransferase (PFK). Both enzymes were PFP may confound the results and contribute to the apparent purified to apparent homogeneity from potato tubers (Solanum tuberosum interconversions (12, 13). Wong et al. (26) have since confirmed cv Record). Neither PFP nor PFK preparations contained detectable ac- that similar impurities can account for the apparent enhancement tivity of the other . PFP was composed of two polypeptides of of PFP activity in PFK preparations by UDPglucose. However, apparent molecular weight 58,000 and 55,700 whereas PFK contained although such contaminants can explain the original kinetic data four polypeptides of apparent molecular weight between 46,300 and 53,300. (3, 4, 25), they do not eliminate the possibility that PFK and Chemical cleavage of individual PFP and PFK polypeptides gave a dif- PFP can be reversibly interconverted as initially proposed (4). ferent set of fragments for each polypeptide. On Western blots antisera Recently, PFK has been purified to homogeneity from carrot against PFP failed to cross-react with any of the four PFK polypeptides, and contains a single polypeptide of apparent mol wt 60,000 (26). and antibodies against PFK failed to bind to either ofthe PFP polypeptides. This value is quite different from those of PFK from mammalian Antibodies that immunoprecipitate PFP activity had no effect on PFK and bacterial sources (23), but is similar to that of a PFP poly- activity. Conversely, antibodies against the four PFK polypeptides pre- peptide from many plant tissues (14). These data support the cipitated the activity of PFK, but not that of PFP. This work shows that possibility that PFP and PFK may be related. To test this sug- potato tuber PFP and PFK are composed of distinct, unrelated polypep- gestion further we have compared in detail the molecular prop- tides and indicate that interconversion between PFP and PFK is unlikely. erties of the two enzymes involved in the apparent metabolite- mediated interconversion. Here we report that PFK and PFP from potato tubers are composed of separate, unrelated poly- peptides. This indicates that direct interconversion between PFK and PFP in higher plants is unlikely.

The conversion of fructose 6-P to fructose 1,6-bisP is an im- MATERIALS AND METHODS portant regulated step of . This reaction can be cata- lyzed by two distinct enzyme activities, PFK2 and PFP, for which Materials. Mature potato tubers (Solanum tuberosum L. cv the phosphoryl donors are ATP and PPi, respectively. Many Record) were supplied by Walkers Crisps Ltd. and were stored higher plants contain significant amounts of both activities (2, 9, at 9 to 15°C. Biochemicals, auxiliary enzymes, and Reactive Red 16), but their relative importance in glycolysis is unknown (1, 12-Agarose (type 3000-CL) were purchased from Sigma. Seph- 24). adex G-25, DEAE-Sephacel, and ATP-Agarose (type 2) were Balogh et al. (4) have proposed that PFK and PFP can be from Pharmacia. reversibly interconverted by appropriate metabolites. The ap- Enzyme Purification. PFK was purified over 15,000-fold to a parent conversion of PFK to PFP is mediated by UDPglucose, specific activity of about 200 ,umol-min-'1mg- 1 protein using a whereas the reconversion of PFP to PFK is promoted by fructose combination of ion-exchange, dye-ligand, and affinity chroma- 2,6-bisP. In preliminary reports they suggest that these inter- tography as described elsewhere (8). conversions occur in extracts from a range of tissues, and that PFP was purified to apparent homogeneity as described pre- such changes contribute to the regulation of glycolysis by varying viously (14). the proportions of PFK and PFP in vivo (3, 25). Enzyme Assays. PFK and PFP were measured spectrophoto- The evidence for the above interconversions has been criti- metrically as described previously (16). All assays were carried cized, and the kinetic data have been reinterpreted as indicating out at 25°C in a total volume of 1 ml. Ammonium sulfate was that PFK and PFP are separate, distinct enzymes that are dif- removed from auxiliary enzymes before use. The PFK assay ferentially activated by the effector metabolites (11). Although contained 100 mm Tris-HC1 (pH 8.0), 5 mM MgCl2, 5 mM fruc- the known properties of PFK and PFP do not support this sug- tose 6-P, 1 mm ATP, 0.1 mm NADH, 1 IU aldolase, 10 IU triosephosphate , 1.2 IU glycerol 3-P dehydrogenase. I Supported by Agricultural Genetics Co. Assay conditions for PFP were the same as those for PFK except 2Abbreviations: PFK, ATP:fructose 6-phosphate 1-phosphotransfer- that 1 mM MgCl2 was present,-0.2 mm PPi replaced ATP and ase (EC 2.7.1.11); PFP, pyrophosphate:fructose 6-phosphate 1-phos- the assay contained 1 ,u/M fructose 2,6-bisP. The reactions were photransferase (EC 2.7.1.90). started with ATP and PPi, respectively. 645 646 KRUGER AND HAMMOND Plant Physiol. Vol. 86, 1988 Electrophoresis. Polypeptides were separated by SDS-PAGE in 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 with cyanogen bromide (21), N-chlorosuccinimide (19), or hydrox- ~ ~ ~ ~ w 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. Polypeptides were separated by SDS-PAGE and electroblotted onto nitro- Downloaded from https://academic.oup.com/plphys/article/86/3/645/6082991 by guest on 29 September 2021 cellulose (50 V for 3 h, Bio-Rad Trans-Blot ) as described by Burnette (7). The resulting blot was probed with up to 0.2 ml antiserum in 50 ml PBS (150 mm NaCl, 10 mM NaH2PO4 adjusted to pH 7.2 using NaOH) containing 8% (w/v) BSA for 12 h, and then washed thoroughly with PBS. Bound antibodies were detected by incubating the nitrocellulose with 5 ,g protein A-alkaline phosphatase conjugate in 50 ml PBS containing 4% (w/v) BSA for 2 h. The nitrocellulose was washed exhaustively with 1% (v/v) Triton X-100 in phosphate buffered saline and then 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 was FIG. 1. Molecular comparison of potato tuber PFK and PFP poly- homogenized in 1 volume of 100 mm Tris-HCl (pH 8.0), 2 mM peptides. Purified PFK and PFP were subject to SDS-PAGE in the MgCl2, 1 mm EDTA, 28 mm 2-mercaptoethanol, 1 mm phen- presence of 4 M urea and then stained with Coomassie brilliant blue. ylmethylsulfonylfluoride, 2% (w/v) insoluble PVP. The extract The apparent Mr of each polypeptide was determined by comparison was filtered through four layers of cheesecloth and centrifuged with the migration of the following Mr standards: a2-macroglobulin, 170 at 18,000g for 30 min. A 2.5 ml sample of the resulting super- kD; b, 97.4 kD; glutamate dehydrogenase, 55.4 kD; lac- natant was passed through a Sephadex G-25 column (1.5 x 5 tate dehydrogenase, 36.5 kD; trypsin inhibitor, 20.1 kD. The positions cm) equilibrated with extraction buffer from which phenylme- of PFPa,, PFP,, and PFKa-d are indicated by arrows and the resolution thylsulfonylfluoride and PVP were omitted. The activities of PFK of the polypeptides is illustrated in the inset. and PFP in the desalted extract were about 70 and 350 nmol-min-1lml- , respectively. A 20 ,ul aliquot of this extract was added to 80 ul PBS containing up to 40 ,ug purified IgG. The mixture was incubated at room temperature for 60 min. enzymes. Partial chemical cleavage of each polypeptide using Antibody-antigen complex was precipitated by adding 20 Al 200 cyanogen bromide and chlorosuccinimide produced unique sets mg-ml-' insoluble protein A suspended in PBS. The mixture of peptide fragments (Fig. 2). Under the conditions used, these was incubated for a further 30 min and then centrifuged (Ep- chemicals selectively cleave Met-X and Trp-X bonds, respec- pendorf microfuge, 5 min). PFK and PFP activity remaining in tively (19, 21). Similarly, treatment of the polypeptides with the supernatant were measured. hydroxylamine, which hydrolyzes Asn-Gly bonds (18), differ- entially cleaved PFP, and PFP,, but failed to cut any of the PFK RESULTS AND DISCUSSION polypeptides. Specific amino- or carboxyl-terminal proteolysis of the peptides during enzyme purification could not readily account Both PFK and PFP from potato tubers have been purified to for the differences in the patterns of cleavage fragments derived apparent homogeneity and are compared by urea/SDS-PAGE from PFK and PFP. (Fig. 1). The PFK preparation contains four polypeptides (PFKa Despite differences in the distribution of specific amino acids, d) of apparent mol wt 46,300, 49,500, 50,000, and 53,300. Evi- PFK and PFP polypeptides could contain extensive regions of dence that all four of these polypeptides are associated with PFK homology. To test this we have investigated the immunochemical activity will be presented in a separate publication. In contrast, relationship between the two enzymes. The polypeptides in pur- PFP is composed of two polypeptides (a and f3) of apparent mol ified PFK and PFP were resolved by SDS-PAGE, electroblotted wt 58,800 and 55,700 (14). Neither of these preparations con- onto nitrocellulose, and then probed with a mixture of antibodies tained detectable activity of the opposite enzyme. We stress that raised against PFKa-C. In combination these antisera recognize both preparations were obtained from the same batch of tubers all four PFK polypeptides (8). Two replicate nitrocellulose filters which was grown and stored under uniform conditions. Other were challenged separately with antibodies against PFPG and preparations of PFK and PFP have yielded essentially identical PFP,. The antisera each reacted strongly with their correspond- results. The mol wt of PFPa, and PFP, we have determined in ing immunogens, but failed to recognise polypeptides present in the presence of urea are lower than those reported previously the alternative enzyme (Fig. 3). for this enzyme using conventional SDS-PAGE (14). Such dif- In complementary experiments we investigated the relation- ferences in the relative migration of polypeptides in the presence ship between PFK and PFP in their native conformation by test- and absence of urea are well established (22) and have also been ing the ability of antibodies to precipitate enzyme activity in the observed with potato tuber PFK (8). presence of insoluble protein A. A combination of antibodies Since PFK and PFP were purified using separate procedures, against PFKaC are known to precipitate all four forms of PFK we investigated whether differential proteolysis in the two pro- identified in potato tuber (8). Such a mixture removed substantial tocols could account for the mol wt differences between the two PFK activity from a crude extract of potato tubers, but had no PPi AND ATP DEPENDENT FROM POTATO TUBER 647

u m L Q m L 0. 0L LL LL LL. U. LL LL L- a0 0. II. Y. CL CL 0Q 0.

"W*,*. 1, 44"t", W= qw''o Imik-L.

I Downloaded from https://academic.oup.com/plphys/article/86/3/645/6082991 by guest on 29 September 2021 609i ml.. I R?N:o9. fighm6l.

......

CNBr ._m: *

NCS FIG. 2. Chemical cleavage of PFK and PFP polypeptides. PFK and PFP polypeptides were separated by SDS-PAGE. About 2,g of each polypeptide was treated in situ with either cyanogen bromide (CNBr) or N-chlorosuccinimide (NCS). Each polypeptide was digested separately except for PFKb and PFKC which could not be completely resolved and were treated together. The cleavage products of each reaction were separated by SDS-PAGE and silver stained.

ant i- ant i-PFP tern of peptide fragments after chemical cleavage. In addition, PFK these polypeptides are immunologically unrelated both in their a X native conformation and when denatured. Limited data suggest that PFK and PFP from endosperm of developing castor bean, U. U. Y. X LL LL 0. 0. 0L the only other tissue to have been investigated, are also probably unrelated (10). Antibodies raised against castor bean leucoplast PFK can immunoprecipitate both cytoplasm and plastid forms of PFK, but do not affect PFP. Conversely, antibodies against potato tuber PFP can precipitate endosperm PFP activity, but have 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 present work suggests that the apparent interconversion of PFK and PFP is an artifact arising from contaminants in the assays of the two enzymes. FIG. 3. Immunochemical comparison of PFK and PFP polypeptides. Acknowledgments-We thank Walkers Crisps Ltd. for supplying the potato The individual polypeptides of PFK and PFP were resolved by SDS- tubers used in these experiments. PAGE, electroblotted onto nitrocellulose, and probed with antibodies raised against PFK, PFP,t, and PFP,, as indicated. Bound antibodies LITERATURE CITED were detected using alkaline phosphatase conjugated protein A. 1. AP REES T 1985 The organization of glycolysis and the oxidative pentose effect on PFP activity (Fig. 4). Correspondingly, anti-PFPa and phosphate pathway in plants. In R Douce, D Day, eds, Encyclopedia of precipitated the activity of PFP, but not that of PFK. Plant Physiology NS, Vol 18, Springer-Verlag, Berlin, pp 391-417 anti-PFP,, 2. AP REES T, JH GREEN, PM WILSON 1985 Pyrophosphate:fructose 6-phosphate As observed previously in a partially purified preparation (14), 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher anti-PFP, was more effective than anti-PFPa at removing PFP plants. Biochem J 227: 199-204 activity from the crude potato extract. 3. BALOGH A, JH WONG, BB BUCHANAN 1984 Metabolite-mediated intercon- Despite the similarity of the reactions catalyzed by PFK and version of PFP/PFK: a regulatory mechanism to direct cytosolic carbon flux. Plant two enzymes are com- Physiol 75: S-53 PFP, the above results indicate that the 4. BALOGH A, JH WONG, C W6TZEL, J SOLL. C CSgKE, BB BUCHANAN 1984 posed of separate, distinct polypeptides. The PFK and PFP poly- Metabolite-mediated catalyst conversion of PFK and PFP: a mechanism of peptides clearly differ in mol wt and each yields a different pat- enzyme regulation in green plants. FEBS Lett 169: 287-292 648 KRUGER AND HAMMOND Plant Physiol. Vol. 86, 1988

5. BLAKE MS, KH JOHNSTON, GJ RuSSELL-JONES. EC GOTSCHLICH 1984 A rapid, sensitive method for detection of alkaline phosphatase-conjugated -m 100l ---Z . .~~// anti-antibody on Western blots. Anal Biochem 136: 175-179 6. BUCHANAN BB 1985 Response to paper by Juana M Gancedo. FEBS Lett PFP 180: 65 751 7. BURNETTE WN 1981 "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellu- lose and radiographic detection with antibody and radioiodinated protein 50[ A. Anal Biochem 112: 195-203 PFK 8. BURRELL MM, JBW HAMMOND, NJ KRUGER 1987 Characterization of mul- tiple forms of potato tuber phosphofructokinase. XIV International Botan- 25 - anti-PFK ical Congress Abstracts, p 56 9. CARNAL NW, CC BLACK 1983 Phosphofructokinase activities in photosynthetic organisms. The occurrence of pyrophosphate-dependent-6-phosphofructo- 0 n,v 0 in plants and algae. Plant Physiol 71: 150-155 if 10. DENNIS DT, WE HEKMAN, A THOMSON, RJ IRELAND, FC BOTHA, NJ KRUGER Downloaded from https://academic.oup.com/plphys/article/86/3/645/6082991 by guest on 29 September 2021 L- 100 *. * I*/- 1985 Compartmentation of glycolytic enzymes in plant cells. /it RL Heath, 4- PFK J Preiss, eds, Regulation of Carbon Partitioning in Photosynthetic tissue. c American Society of Plant Physiologists, Rockville, MD, pp 127-146 75[ 11. GANCEDO JM 1984 Metabolite-mediated catalyst conversion of PFK and PFP: can PFK really be converted to PFP? FEBS Lett 175: 369-370 0 12. KRUGER NJ, DT DENNIS 1985 A source of apparent pyrophosphate:fructose in muscle 0 50[ 6-phosphate phosphotransferase activity rabbit phosphofructoki- 0 nase. Biochem Biophys Res Commun 126: 320-326 13. KRUGER NJ, DT DENNIS 1985 Reassessment of an apparent hyperactive form E 25 - PFP of phosphofructokinase from plants. Plant Physiol 78: 645-648 14. KRUGER NJ, DT DENNIS 1987 Molecular properties of pyrophosphate:fructose N 6-phosphate phosphotransferase from potato tuber. Arch Biochem Biophys w -i aniPP. 256: 273-279 15. KRUGER NJ, JBW HAMMOND 1988 Purification of immunoglobulins using 100 . * // protein A-Sepharose. In JM Walker, ed, Methods in Molecular Biology, PFK Vol 3. Humana Press, Clifton, NJ. In press 16. KRUGER NJ, E KOMBRINK, H BEEVERS 1983 Pyrophosphate:fructose 6-phos- 75 phate phosphotransferase in germinating castor bean endosperm. FEBS Lett 153: 409-412 anti-PFP 17. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the 50 head of bacteriophage T4. Nature 227: 680-685 18. LAM KS, CB KASPER 1980 Sequence homology analysis of a heterogenous protein population by chemical and enzymic digestion using a two-dimen- 25 sional sodium dodecyl sulfate-polyacrylamide gel system. Anal Biochem 108: 220-226 19. D A - PFP LISCHWE MA, OCHS 1982 new method for partial peptide mapping using 00%1 25lm \ 0 5 10 15 20 40 N-chlorosuccinimide/urea and peptide silver staining in sodium dodecyl sul- fate-polyacrylamide gels. Anal Biochem 127: 453-457 Antiserum (pg IgG) 20. MERRIL CR, D GOLDMAN, ML VAN KEUREN 1983 Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymol 96: 230-239 21. NIKODEM V, JR FRESCO 1979 Protein fingerprinting by SDS-gel electrophoresis after partial fragmentation with CNBr. Anal Biochem 97: 283-286 FIG. 4. Immunoprecipitation of potato tuber PFK and PFP. A crude 22. PICCIONI R, G BELLEMARE, N-H CHUA 1982 Methods of polyacrylamide gel potato tuber extract was incubated with antibodies raised against PFK, electrophoresis in the analysis and preparation of plant polypeptides. In M Edelman, RB Hallick. N-H Chua, eds, Methods in Chloroplast Molecular PFP,,, and PFP,, as indicated. Insoluble protein A was added to the Biology. Elsevier Biomedical, Amsterdam, pp 985-1014 mixture which was then centrifuged. The activity of PFK (0) and PFP 23. UYEDA K 1979 Phosphofructokinase. Adv Enzymol Rel Areas Mol Biol 48: (M) remaining in the supernatant was measured and is expressed as a 193-244 percentage of the activity prior to the addition of antibodies. Each value 24. VAN SCHAFTINGEN E 1987 Fructose 2,6-bisphosphate. Adv Enzymol Rel Areas Mol Biol 59: 315-395 is the mean of three separate samples for which the SE was less than 5%. 25. WONG JH, A BOLOGH, C WOTZEL, J SOLL, BB BUCHANAN 1984 Metabolite- Equivalent amounts of preimmune IgG had no detectable effect on the mediated catalyst conversion in C3 and C4 plants. Plant Physiol 75: S-53 enzymes. The initial activities of PFK and PFP were 70 and 350 26. WONG JH, BC YEE, BB BUCHANAN 1987 A novel type of phosphofructokinase nmol min- 'ml- 1, respectively. from plants. J Biol Chem 262: 3185-3191