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. 1983 Mar 1;209(3):669–676. doi: 10.1042/bj2090669

Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger.

A Habison, C P Kubicek, M Röhr
PMCID: PMC1154144  PMID: 6223622

Abstract

Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran--Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis--Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.

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Selected References

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  1. Bañuelos M., Gancedo C., Gancedo J. M. Activation by phosphate of yeast phosphofructokinase. J Biol Chem. 1977 Sep 25;252(18):6394–6398. [PubMed] [Google Scholar]
  2. Boonsaeng V., Sullivan P. A., Shepherd M. G. Phosphofrucktokinase and glucose catabolism of Mucor and Penicillium species. Can J Microbiol. 1977 Sep;23(9):1214–1224. doi: 10.1139/m77-182. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Dennis D. T., Coultate T. P. The regulatory properties of a plant phosphofructokinase during leaf development. Biochim Biophys Acta. 1967 Sep 12;146(1):129–137. doi: 10.1016/0005-2744(67)90079-4. [DOI] [PubMed] [Google Scholar]
  5. Jaenicke L. A rapid micromethod for the determination of nitrogen and phosphate in biological material. Anal Biochem. 1974 Oct;61(2):623–627. doi: 10.1016/0003-2697(74)90429-1. [DOI] [PubMed] [Google Scholar]
  6. Kubicek C. P., Hampel W., Röhr M. Manganese deficiency leads to elevated amino acid pools in citric acid accumulating Aspergillus niger. Arch Microbiol. 1979 Oct;123(1):73–79. doi: 10.1007/BF00403504. [DOI] [PubMed] [Google Scholar]
  7. Kubicek C. P., Röhr M. Regulation of citrate synthase from the citric acid-accumulating fungus, Aspergillus niger. Biochim Biophys Acta. 1980 Oct;615(2):449–457. [PubMed] [Google Scholar]
  8. Lundin A., Thore A. Analytical information obtainable by evaluation of the time course of firefly bioluminescence in the assay of ATP. Anal Biochem. 1975 May 26;66(1):47–63. doi: 10.1016/0003-2697(75)90723-x. [DOI] [PubMed] [Google Scholar]
  9. Mavis R. D., Stellwagen E. The role of cations in yeast phosphofructokinase catalysis. J Biol Chem. 1970 Feb 25;245(4):674–680. [PubMed] [Google Scholar]
  10. Osmani S. A., Marston F. A., Selmes I. P., Chapman A. G., Scrutton M. C. Pyruvate carboxylase from Aspergillus nidulans. Regulatory properties. Eur J Biochem. 1981 Aug;118(2):271–278. doi: 10.1111/j.1432-1033.1981.tb06396.x. [DOI] [PubMed] [Google Scholar]
  11. Paveto C., Passeron S. Some kinetic properties of Mucor rouxii phosphofructokinase. Effect of cyclic adenosine 3', 5'-monophosphate. Arch Biochem Biophys. 1977 Jan 15;178(1):1–7. doi: 10.1016/0003-9861(77)90164-3. [DOI] [PubMed] [Google Scholar]
  12. Reinhart G. D., Lardy H. A. Rat liver phosphofructokinase: kinetic activity under near-physiological conditions. Biochemistry. 1980 Apr 1;19(7):1477–1484. doi: 10.1021/bi00548a034. [DOI] [PubMed] [Google Scholar]
  13. Reuter R., Eschrich K., Schellenberger W., Hofmann E. Kinetic modelling of yeast phosphofructokinase. Acta Biol Med Ger. 1979;38(8):1067–1079. [PubMed] [Google Scholar]
  14. Ryan L. D., Vestling C. S. Rapid purification of lactate dehydrogenase from rat liver and hepatoma: a new approach. Arch Biochem Biophys. 1974 Jan;160(1):279–284. doi: 10.1016/s0003-9861(74)80035-4. [DOI] [PubMed] [Google Scholar]
  15. SALAS M. L., VINUELA E., SALAS M., SOLS A. CITRATE INHIBITION OF PHOSPHOFRUCTOKINASE AND THE PASTEUR EFFECT. Biochem Biophys Res Commun. 1965 Apr 23;19:371–376. doi: 10.1016/0006-291x(65)90471-7. [DOI] [PubMed] [Google Scholar]
  16. SLAYMAN C. W., TATUM E. L. POTASSIUM TRANSPORT IN NEUROSPORA. I. INTRACELLULAR SODIUM AND POTASSIUM CONCENTRATIONS, AND CATION REQUIREMENTS FOR GROWTH. Biochim Biophys Acta. 1964 Nov 29;88:578–592. [PubMed] [Google Scholar]
  17. Smith J. E., Ng W. S. Fluorometric determination of glycolytic intermediates and adenylates during sequential changes in replacement culture of Aspergillus niger. Can J Microbiol. 1972 Nov;18(11):1657–1664. doi: 10.1139/m72-257. [DOI] [PubMed] [Google Scholar]
  18. Streckbein R. G., Wöltge E., Kubicek J. Tierexperimentelle Erprobung von Ca-P-Keramik zum Verschluss von Knochendefekten in der fazialen Kieferhöhlenwand. Dtsch Zahnarztl Z. 1981 Jan;36(1):34–37. [PubMed] [Google Scholar]
  19. TAKETA K., POGELL B. M. ALLOSTERIC INHIBITION OF RAT LIVER FRUCTOSE 1,6-DIPHOSPHATASE BY ADENOSINE 5'-MONOPHOSPHATE. J Biol Chem. 1965 Feb;240:651–662. [PubMed] [Google Scholar]
  20. Tsao M. U., Madley T. I. Kinetic properties of phosphofructokinase of Neurospora crassa. Biochim Biophys Acta. 1972 Jan 20;258(1):99–105. doi: 10.1016/0005-2744(72)90969-2. [DOI] [PubMed] [Google Scholar]
  21. Uyeda K. Phosphofructokinase. Adv Enzymol Relat Areas Mol Biol. 1979;48:193–244. doi: 10.1002/9780470122938.ch4. [DOI] [PubMed] [Google Scholar]

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