Skip to main content
SpringerLink
Log in

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana (Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 μmol fructose 1,6-bisphosphate produced (mg protein)−1 min−1. Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (α) and 60-kDa (β) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi (K m values = 32, 9.7, 25, and 410 μM, respectively) in the presence of saturating (5 μM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity (K a=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5–6.0 μmol fructose 1,6-bisphosphate produced (mg protein)−1 min−1. Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP (K m=34 and 21 μM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P (S 0.5=540 and 90 μM, respectively). Allosteric effects of phosphoenolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II (I 50=2.3 and 900 μM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig 1.
Fig. 2.
Fig. 3.

Similar content being viewed by others

Notes

  1. This concentration range was estimated from the data reported by Ball and ap Rees (1988) by assuming that Fru-2,6-P2 is restricted to the cytosol and that the cytosol represents 10% of the cell's volume.

Abbreviations

FPLC:

fast protein liquid chromatography

Fru 6-P:

fructose 6-phosphate

Fru 1,6-P2 :

fructose 1,6-bisphosphate

Fru 2,6-P2 :

fructose 2,6-bisphosphate

PEP:

phosphoenolpyruvate

PEPCase:

phosphoenolpyruvate carboxylase

PFK and PFP:

ATP- and PPi-dependent phosphofructokinase, respectively

PKc :

cytosolic pyruvate kinase

References

  • Ball KL, ap Rees T (1988) Fructose 2,6-bisphosphate and the climacteric in bananas. Eur J Biochem 177:637–641

    CAS  PubMed  Google Scholar 

  • Ball KL, Green JH, ap Rees T (1991) Glycolysis at the climacteric of bananas. Eur J Biochem 197:265–269

    CAS  PubMed  Google Scholar 

  • Beaudry RM, Severson RF, Black CC, Kays SJ (1989) Banana ripening: implications of changes in glycolytic intermediate concentrations, glycolytic and gluconeogenic carbon flux, and fructose 2,6-bisphosphate concentration. Plant Physiol 91:1436–1444

    CAS  Google Scholar 

  • Bollag DM, Rozycki MD, Edelstein SJ (1996) Protein methods. Wiley–Liss, New York, pp 62–70

  • Botha FC, Small JGC (1987) Comparison of the activities and some properties of pyrophosphate and ATP dependent fructose 6-phosphate 1-phosphotransferase of Phaseolus vulgaris seeds. Plant Physiol 83:772–777

    CAS  Google Scholar 

  • Botha FC, Cawood MC, Small JGC (1988) Kinetic properties of the ATP-dependent phosphofructokinase isoenzymes from cucumber seeds. Plant Cell Physiol 29:415–421

    CAS  Google Scholar 

  • Brooks SPG (1992) A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques 13:906–911

    CAS  PubMed  Google Scholar 

  • Brooks SPG, Storey KB (1992) Bound and determined: a computer program for making buffers of defined ion concentrations. Anal Biochem 201:119–126

    CAS  PubMed  Google Scholar 

  • Carnal NW, Black CC (1979) Pyrophosphate dependent 6-phosphofructokinase, a new glycolytic enzyme in pineapple leaves. Biochem Biophys Res Commun 86:20–26

    CAS  PubMed  Google Scholar 

  • Cawood MC, Botha FC, Small JGC (1988) Properties of the phosphofructokinase isoenzymes from germinating cucumber seeds. J Plant Physiol 132:204–209

    CAS  Google Scholar 

  • Dennis DT, Greyson MF (1987) Fructose 6-phosphate metabolism in plants. Physiol Plant 69:395–404

    CAS  Google Scholar 

  • Garland WJ, Dennis DT (1980a) Plastid and cytosolic phosphofructokinase from the developing endosperm of Ricinus communis. Separation, purification and initial characterization of the isoenzymes. Arch Biochem Biophys 204:302–309

    CAS  PubMed  Google Scholar 

  • Garland WJ, Dennis DT (1980b) Plastid and cytosolic phosphofructokinase from the developing endosperm of Ricinus communis. II Comparison of the kinetic and regulatory properties of the isoenzymes. Arch Biochem Biophys 204:310–317

    CAS  PubMed  Google Scholar 

  • Hill SA, ap Rees T (1994) Fluxes of carbohydrate metabolism in ripening bananas. Planta 192:52–60

    CAS  Google Scholar 

  • Iyer MG, Surendranathan KK, Nair PM (1989a) Correlation between increase in 6-phosphofructokinase activity and appearance of three multiple forms in ripening banana. Physiol Biochem 27:99–106

    CAS  Google Scholar 

  • Iyer MG, Surendranathan KK, Nair PM (1989b) Evidence for the formation of lower molecular weight forms of phosphofructokinase in ripening banana by dissociation of oligomeric form. Plant Physiol Biochem 27:483–488

    CAS  Google Scholar 

  • Kelly GJ, Latzko E (1977) Chloroplast phosphofructokinase. II. Partial purification, kinetic and regulatory properties. Plant Physiol 60:295–299

    CAS  Google Scholar 

  • Knowles VL, Greyson MF, Dennis DT (1990) Characterization of ATP-dependent fructose 6-phosphate 1-phosphotransferase isozymes from leaf and endosperm tissues of Ricinus communis. Plant Physiol 92:155–159

    CAS  Google Scholar 

  • Kombrink E, Kruger NJ (1984) Inhibition by metabolic intermediates of pyrophosphate:fructose 6-phosphate phosphotransferase from germinating castor bean endosperm. Z Pflanzenphysiol 114:443–453

    CAS  Google Scholar 

  • Kombrink E, Kruger NJ, Beevers H (1984) Properties of pyrophosphate:fructose 6-phosphate phophotransferase from germinating castor bean endosperm. Plant Physiol 74:395–401

    CAS  Google Scholar 

  • Law RD, Plaxton WC (1995) Purification and characterization of a novel phosphoenolpyruvate carboxylase from banana fruit. Biochem J 307:807–816

    CAS  PubMed  Google Scholar 

  • Law RD, Plaxton WC (1997) Regulatory phosphorylation of banana fruit phosphoenolpyruvate carboxylase by a copurifying phosphoenolpyruvate carboxylase-kinase. Eur J Biochem 247:642–651

    CAS  PubMed  Google Scholar 

  • Mertens E, Marcellin P, Van Schaftingen E, Hers H-G (1987) Effects of ethylene treatment on fructose 2,6-bisphosphate and on the activity of phosphofructokinase-2/fructose 2,6-bisphosphatase in banana. Eur J Biochem 167:579–583

    CAS  PubMed  Google Scholar 

  • Moraes TF, Plaxton WC (2000) Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures. Implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation and the integration of glycolysis with nitrogen assimilation. Eur J Biochem 267:4465–4476

    Article  CAS  PubMed  Google Scholar 

  • Nair PM, Darak BG (1981) Identification of multiple forms of phosphofructokinase in ripening dwarf cavendish banana. Phytochemistry 20:605–609

    Article  CAS  Google Scholar 

  • Nielsen TH (1995) Fructose 1,6-bisphosphate is an allosteric activator of pyrophosphate:fructose 6-phosphate 1-phosphotransferase. Plant Physiol 108:69–73

    CAS  PubMed  Google Scholar 

  • Plaxton WC (1996) The organization and regulation of plant glycolysis. Annu Rev Plant Physiol Plant Mol Biol 47:185–214

    Google Scholar 

  • Podestá FE, Moorhead GBG, Plaxton WC (1994) Potato tuber pyrophosphate-dependent phosphofructokinase: effect of thiols and polyalcohols on its intrinsic fluorescence, oligomeric structure, and activity in dilute solutions. Arch Biochem Biophys 313:50–57

    Article  PubMed  Google Scholar 

  • Sabularse DC, Anderson RL (1981) d-fructose 2,6-bisphosphate: a naturally occurring activator for inorganic pyrophosphate:d-fructose 6-phosphate 1-phosphofructotransferase. Biochem Biophys Res Commun 103:848–855

    CAS  PubMed  Google Scholar 

  • Salminen SO, Young RE (1975) The control properties of phosphofructokinase in relation to the respiratory climacteric in banana fruit. Plant Physiol 55:45–50

    CAS  Google Scholar 

  • Seymour GB (1993) The banana. In: Seymour GB, Taylor JA, Tucker GA (eds) The biochemistry of fruit ripening. Chapman and Hall, London, pp 83–106

  • Stitt M (1989) Product inhibition of potato tuber pyrophosphate-dependent phosphofructokinase by phosphate and pyrophosphate. Plant Physiol 89:628–633

    CAS  Google Scholar 

  • Stitt M (1990) Fructose 2,6-bisphosphate as a regulatory molecule in plants. Annu Rev Plant Physiol Plant Mol Biol 41:153–185

    CAS  Google Scholar 

  • Surendranathan KK, Nair PM (1989) ADP as a phosphoryl donor for banana 6-phosphofructokinase. Plant Sci 59:45–51

    CAS  Google Scholar 

  • Surendranathan KK, Iyer MG, Nair PM (1990) Characterization of a monomeric phosphofructokinase from banana—role of magnesium in its regulation. Plant Sci 72:27–35

    CAS  Google Scholar 

  • Surendranathan KK, Iyer MG, Nair PM (1992) Mechanism of action of a dimeric phosphofructokinase from banana: role of magnesium on its kinetics and regulation. Plant Sci 81:29–36

    CAS  Google Scholar 

  • Theodorou ME, Kruger NJ (2001) Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase. Planta 213:147–157

    Article  CAS  PubMed  Google Scholar 

  • Theodorou ME, Plaxton WC (1996) Purification and characterization of pyrophosphate-dependent phosphofructokinase from phosphate-starved Brassica nigra suspension cells. Plant Physiol 112:343–351

    Article  CAS  PubMed  Google Scholar 

  • Theodorou ME, Cornel FA, Duff SM, Plaxton WC (1992) Phosphate starvation-inducible synthesis of the alpha-subunit of the pyrophosphate-dependent phosphofructokinase in black mustard suspension cells. J Biol Chem 267:21901–21905

    CAS  PubMed  Google Scholar 

  • Thomas S, Mooney PJ, Burrell MM, Fell DA (1997a) Finite change analysis of glycolytic intermediates in tuber tissue of lines of transgenic potato (Solanum tuberosum) overexpressing phosphofructokinase. Biochem J 322:111–117

    CAS  PubMed  Google Scholar 

  • Thomas S, Mooney PJ, Burrell MM, Fell DA (1997b) Metabolic control analysis of glycolysis in tuber tissue of potato (Solanum tuberosum): explanation for the low control coefficient of phosphofructokinase over respiratory flux. Biochem J 322:119–127

    CAS  PubMed  Google Scholar 

  • Turner WL, Plaxton WC (2000) Purification and characterization of cytosolic pyruvate kinase from banana fruit. Biochem J 352:875–882

    Article  CAS  PubMed  Google Scholar 

  • Turner WL, Plaxton WC (2001) Purification and characterization of banana fruit acid phosphatase. Planta 214:243–249

    CAS  PubMed  Google Scholar 

  • Wong JH, Kiss F, Wu M-X, Buchanan BB (1990) Pyrophosphate fructose-6-P 1-phosphotransferase from tomato fruit. Evidence for change during ripening. Plant Physiol 94:499–506

    CAS  Google Scholar 

Download references

Acknowledgement

Financial support was kindly provided by research and equipment grants from the Natural Sciences and Engineering Research Council of Canada (NSERC).

Author information

Authors and Affiliations

  1. Department of Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada

    William L. Turner & William C. Plaxton

  2. Department of Biochemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada

    William C. Plaxton

Authors
  1. William L. Turner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William C. Plaxton
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William C. Plaxton.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Turner, W.L., Plaxton, W.C. Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit. Planta 217, 113–121 (2003). https://doi.org/10.1007/s00425-002-0962-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-002-0962-7

Keywords

Search

Navigation