Use of Cibacron Blue--Sepharose 6B to detect modifications of the non-allosteric 6-phosphofructokinase from Escherichia coli K-12.
In crude cell-free extracts of aerobically grown E. coli K-12, the non-allosteric form of 6-phosphofructokinase has a tetrameric molecular weight 140 000 with a low affinity (less than 5%) for the blue dextran chromophore--Cibacron Blue. The allosteric form has the same tetrameric molecular weight, but possesses a strong affinity for the blue dextran chromophore. Under conditions of prolonged storage, purification procedures of mild heat treatment (50 degrees C), the non-allosteric form converts to an active dimer (mol. wt 67 000), which binds to Cibacron Blue (less than 90%). Acid precipitation plus heat treatment prevents the conversion to the dimeric form and retains low Cibacron Blue affinity. These results are consistent with the isolation of a low molecular weight form and suggest that the inherent lability of this enzyme might be due to both non-specific proteolytic modification and a weak quaternary structure.
D000873:Anthracenes; D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D007527:Isoenzymes / Q000302:isolation & purification*; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000302:isolation & purification*; D011134:Polysaccharides; D012685:Sepharose / Q000031:analogs & derivatives
D000873:Anthracenes; D004396:Coloring Agents; D007527:Isoenzymes; D011134:Polysaccharides; C017842:cibacron blue-sepharose; D012685:Sepharose; D010732:Phosphofructokinase-1