ISSN:
0749-503X
Keywords:
phosphofructokinase
;
protein purification
;
molecular and kinetic properties
;
Kluyveromyces lactis
;
Life and Medical Sciences
;
Genetics
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
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 β-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. © 1997 John Wiley & Sons, Ltd.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
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