TY - JOUR
T1 - Affinity chromatography of phosphofructokinase
AU - Ramadoss, Candadai S.
AU - Luby, Lynne J.
AU - Uyeda, Kasaku
N1 - Funding Information:
was supported by Grant AM16194 Public Health Service. N., and Uyeda, K., manuscript
PY - 1976/8
Y1 - 1976/8
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0017111661&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0017111661&partnerID=8YFLogxK
U2 - 10.1016/0003-9861(76)90536-1
DO - 10.1016/0003-9861(76)90536-1
M3 - Article
C2 - 134285
AN - SCOPUS:0017111661
SN - 0003-9861
VL - 175
SP - 487
EP - 494
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -