Purification and properties of phosphofructokinase from Dictyostelium discoideum.
Phosphofructokinase (PFruK) from the slime mold Dictyostelium discoideum has been purified to homogeneity over 15,000-fold with a 29% yield. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the final preparation revealed a single band of 95 kDa. The native molecular mass was determined by gel filtration to be 382 kDa, indicating that the enzyme is a homotetramer. An antibody raised in rabbits against the 95-kDa band immunoprecipitated PFruK activity while it did not react with the enzyme from yeast and mammalian cells. The apparent pI was 6.8 and the pH optimum was 7.6. The enzyme had an activation energy (Ea) of 29.1 kJ/mol. The amino acid composition was distinctive in having high Ser, Gly and Glx and low Ala, Val and Tyr compared with other eukaryotic PFruKs. Enzyme activity did not have a sigmoidal saturation curve for fructose 6-phosphate, was only mildly inhibited by MgATP at acidic pH values, was not affected by enzyme concentration and was insensitive to any of the typical allosteric effectors of PFruKs from other sources. However, the enzyme binds fructose 2,6-bisphosphate as indicated by protection against thermal denaturation. Treatment with cAMP-dependent protein kinase led to phosphorylation of the enzyme without change in activity. The metabolic significance of these properties and their relationship to structure/function are discussed.
Martínez-Costa
O H
OH
Departamento de Bioquímica de la UAM, Facultad de Medicina de la Universidad Autónoma, Madrid, Spain.
Estévez
A M
AM
Sánchez
V
V
Aragón
J J
JJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017421:Sequence Analysis; D013816:Thermodynamics
D000596:Amino Acids; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D010732:Phosphofructokinase-1