Affinity partitioning of erythrocytic phosphofructokinase in aqueous two-phase systems containing poly(ethylene glycol)-bound cibacron blue. Influence of pH, ionic strength and substrates/effectors.
Phosphofructokinase (PFK) from rat erythrocyte haemolysates has a high affinity for Cibacron Blue F3G-A covalently bound to poly(ethylene glycol) (PEG-Cb) and thus the enzyme can be extracted into the top phase of poly(ethylene glycol)-dextran aqueous two-phase systems containing PEG-Cb. The pH, ionic strength and presence of substrates/effectors affect to different extents the affinity of the enzyme for PEG-Cb and the number of PEG-Cb molecules attached per molecule of PFK (the latter probably reflecting, at saturation, the influence on the aggregation state of the enzyme) and thus influence the yield of enzyme recovered in the top phase. Increasing the pH from 6 to 7 and then to 8 leads to a higher yield of PFK in the top phase. A change in pH from 6 to 7 and 8 results in an increased number of PEG-Cb molecules attached per molecule of enzyme while the affinity of PFK for PEG-Cb shows a minimum at pH 7. The ionic strength in the range 0.017-0.164 has less influence on the partitioning of PFK. The presence of substrates or effectors of the enzyme in general reduces the recovery of PFK in the top phase. Fructose 6-phosphate increases the number of PEG-Cb molecules attached but greatly reduces the affinity of PFK for PEG-Cb. In contrast, AMP slightly reduces the number of PEG-Cb molecules attached and the affinity of PFK for PEG-Cb. ATP and ATP-Mg2+ compete with PEG-Cb for the same binding sites in PFK.(ABSTRACT TRUNCATED AT 250 WORDS)
Tejedor
M C
MC
Departamento de Bioquimica y Biologia Molecular, Universidad de Alcala, Madrid, Spain.
Delgado
C
C
Grupeli
M
M
Luque
J
J
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008297:Male; D009994:Osmolar Concentration; D010732:Phosphofructokinase-1 / Q000097:blood*; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D014227:Triazines
D014227:Triazines; D011092:Polyethylene Glycols; C012842:Cibacron Blue F 3GA; D010732:Phosphofructokinase-1