Purification and characterization of phosphofructokinase from the yeast Kluyveromyces lactis.
Phosphofructokinase from Kluyveromyces lactis was purified by 180-fold enrichment, elaborating the following steps: cell disruption, polyethylene glycol precipitation, affinity chromatography, size exclusion chromatography on Sepharose 6B and on Bio-Sil SEC 400 and ion exchange chromatography. The homogeneous enzyme exhibits a molecular mass of 845 +/- 20 kDa as determined by sedimentation equilibrium measurements and a specific activity of 100 units/mg protein. The apparent sedimentation coefficient was found to be s20,c = 20.7 +/- 0.6 S and no significant dependence on the protein concentration was observed in a range from 0.2 to 8 mg protein/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to molecular masses of 119 +/- 5 kDa and 102 +/- 5 kDa, respectively. Thus, the enzyme assembles as octamer composed of two types of subunits. From Western blot analysis applying subunit-specific monoclonal antibodies raised against Saccharomyces cerevisiae phosphofructokinase and from the determination of the N-terminal amino acid sequence, the conclusion was drawn that the 102 kDa-subunit corresponds to the beta-subunit of the S. cerevisiae enzyme. In contrast to bakers' yeast phosphofructokinase, the K. lactis enzyme exhibits no cooperativity with respect to the substrate fructose 6-phosphate. Both activators AMP and fructose 2,6-bisphosphate decrease the Michaelis constant with respect to this substrate. The enzyme from K. lactis is also inhibited by ATP. Fructose 2,6-bisphosphate or AMP diminish the ATP-inhibition. In contrast to the phosphofructokinase from S. cerevisiae, where fructose 2,6-bisphosphate turned out to be more efficient than AMP, both activators exert similar effects on the K. lactis enzyme.
Bär
J
J
Institut für Biochemie, Universitätsklinikum, Universität Leipzig, Germany.
Schellenberger
W
W
Kopperschläger
G
G
D004789:Enzyme Activation; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D010732:Phosphofructokinase-1