Affinity chromatography of phosphofructokinase

Candadai S. Ramadoss, Lynne J. Luby, Kasaku Uyeda

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

The behavior of mammalian phosphofructokinase on immobilized adenine nucleotides was investigated. Three different insolubilized ligands were compared using a pure rabbit muscle phosphofructokinase. N6-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose bound at least 90 times more enzyme than either N6-(6-aminohexyl)-AMP-agarose or ATP-adipic acid hydrazide-Sepharose. The elution of phosphofructokinase from the ATP-Sepharose with various metabolites and combinations of metabolites was investigated. The enzyme is eluted specifically from N6-[(6-aminohexyl)-carbamoyl]-ATP-Sepharose with a mixture of 25 μm each of fructose 6-phosphate and ADP (±Mg2+). The enzyme is not eluted either with ATP (25 μm), fructose 1,6-diphosphate (1 mm), ADP (25 μm), fructose 6-phosphate (1 mm) alone, or with a mixture of fructose 1,6-diphosphate (25 μm) and ATP (25 μm). The recovery of bound enzyme was usually greater than 90%. A mixture of glucose 6-phosphate and ADP or a mixture of IDP and fructose 6-phosphate also elutes the enzyme, but the recovery with these eluants was only about 40%. It was concluded that the "dead-end" complex is the most effective in the elution. Using this method, phosphofructokinase has been prepared in an essentially homogeneous form from muscle and brain of rabbit and rat. The overall isolation procedure involves a high speed centrifugation of crude extracts which sediments phosphofructokinase as a pellet, followed with adsorption on N6-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose and specific elution with the mixture of fructose 6-phosphate and ADP.

Original languageEnglish (US)
Pages (from-to)487-494
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume175
Issue number2
DOIs
StatePublished - 1976

Fingerprint

Affinity chromatography
Phosphofructokinases
Affinity Chromatography
Adenosine Diphosphate
Enzymes
Adenosine Triphosphate
Metabolites
Muscle
Inosine Diphosphate
Rabbits
Recovery
Muscles
Glucose-6-Phosphate
Adenine Nucleotides
Centrifugation
Complex Mixtures
Sepharose
Adsorption
Rats
Brain

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Affinity chromatography of phosphofructokinase. / Ramadoss, Candadai S.; Luby, Lynne J.; Uyeda, Kasaku.

In: Archives of Biochemistry and Biophysics, Vol. 175, No. 2, 1976, p. 487-494.

Research output: Contribution to journalArticle

Ramadoss, Candadai S. ; Luby, Lynne J. ; Uyeda, Kasaku. / Affinity chromatography of phosphofructokinase. In: Archives of Biochemistry and Biophysics. 1976 ; Vol. 175, No. 2. pp. 487-494.
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