Purification and Properties of 1-Phosphofructokinase from Clostridium pasteurianum

1-Phosphofructokinase from Clostridium pasteurianum has been purified by chromatography on DEAE-Sephadex and hydroxyapatite and by gel filtration. The procedure employed resulted in a 712-fold purification with a yield of 41%. The use of Tris-HCl buffer in purification and electrophoresis of the enzyme led to partial denaturation and loss of activity; in phosphate buffer the enzyme was stable. The molecular weight of 1-phosphofructokinase was estimated to be 63000. Treatment with sodium dodecylsulfate and mercaptoethanol dissociated the enzyme into two subunits with molecular weights of approximately 40000 and 28500, respectively. The reaction rates for adenosine triphosphate and fructose 1-phosphate followed Michaelis-Menten kinetics (Km values: 0.14 mM for ATP and 0.62 mM for fructose 1-phosphate). ITP could replace ATP, other nucleoside triphosphates were not effective. Product inhibition studies indicated a mechanism of the ordered Bi Bi type for 1-phosphofructokinase.