抄録
Phosphofructokinase from chicken liver has been purified to yield a homogeneous protein whose homogenity was confirmed by disc electrophoretic and ultracentrifugal techniques. The purification procedure involves activation of the crude extract of chicken liver by heat treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and molecular sieve filtration on Biogel 1.5M. The purified phosphofructokinase has been crystallized in the presence of 1mM fructose-1, 6-diP and its specific activity is 134 units per mg.
In contrast to other phosphofructokinase, this enzyme undergoes a reversible inactivation at a low temperature. The rate of the inactivation increases with decrease in temperature. The cold inactivation depends on a variety of conditions such as protein concentration, pH, kind and concentration of salt. At 2mg/ml or above the enzyme at 0° is completely stable for at least one day, but below 0.3mg/ml the activity is lost very rapidly at this temperature. At 0° the enzyme is most stable at pH 8.3, but the stability decreased considerably above or below this pH. The cold inactivation is enhanced by monovalent anions such as Cl- or Br-, but polyvalent anions such as phosphate or sulfate tend to protect against inactivation. The enzyme is protected from cold inactivation by fructose-6-P, fructose-1, 6-diP, cyclic AMP or AMP, but ATP, ADP or citrate enhances the inactivation.
The cold inactivated enzyme can be activated by rewarming. The conditions which favor or disfavor the cold inactivation inhibit or promote the heat activation, respectively. The phenomena of cold inactivation and heat activation were demonstrated with phosphofructokinase in the crude extract of chicken liver as well as the purified enzyme.
The cold inactivation is accompanied by dissociation of the enzyme (13.9S) to a protomer with a S20,w of 5.4 as shown by sucrose density gradient centrifugation and gel filtration. Reactivation by warming of the cold inactivated enzyme results in reassociation of the protomers to an aggregate with molecular weight of about 400,000, similar to the calculated value for the native enzyme (13.9 S).
The process of dissociationand association of the enzyme induced by temperature is influenced by the kinds and concentrations of salt, pH and allosteric effectors, which suggests that this process is a complex phenomenon and that a variety of bonds seem to be involved in holding together the associated form of the enzyme.
