Evidence for the Existence of Two Interconvertible Forms of the Phosphofructokinase Dimer from Escherichia coli K‐12

Abstract

The isolation by affinity chromatography of a low‐molecular‐weight form of phosphofructokinase from Escherichia coli K‐12 has resulted in an improved 2050‐fold purification. The enzyme was readily separable from the higher‐molecular‐weight form by Sepharose 6B fractionation. The enzyme was found to have a molecular weight of 65000 ± 6500 with a subunit molecular weight of 35000 ± 3500. The enzyme exists in either of two interconvertible forms dependent on the presence or absence of the positive of ectors ADP, fructose 6‐phosphate and ATP. A regulatory mechanism is postulated for this enzyme involving hysteric conformational changes; its importance in the mechanism of the Pasteur effect is discussed.

Use of Cibracon Blue-Sepharose 6B to Detect Modifications of the Non-allosteric 6-Phosphofructokinase from Escherichia Coli K-12

“…Cells (20 g wet weight) were suspended in 100 ml of buffer 1. Two aliquot suspensions were homogenized as described earlier (Ewings and Doelle 1976) and clarified by centrifugation at 40000 g for 20 min at 4°C. Cell-free extract (96 ml, 20 mg/ml protein) was subjected to a 40-70 % (w Iv) ammonium sulfate precipitation and the pellet was divided into four equal parts by weight.…”

“…Reports on the partially purified enzyme from the wild-type parent strain list the molecular weights of both isozymes varying from 65000 to 350000 (Doelle 1974(Doelle , 1975Ewings and Doelle 1976;Babul 1978), whereas purified preparations from a mutant strain list a molecular weight of 140000 for the allosteric and 67000 for the non-allosteric form (Kotlarz and Buc 1977). Both isozymes were separable on Cibracon Blue-Sepharose 6B, owing to the affinity of the allosteric form in contrast to the lack of affinity of the non-allosteric form.…”

The occurrence and distribution of two phosphofructokinase proteins in the facultative anaerobic family of enterobacteriaceae

“…In the transitional stage of oxygen limitation, both forms were present. It was demonstrated [ 4] that the lower molecular form existed in two interconvertible forms, one of which was an active form, tile other an inactive form.…”