Abstract
Back muscles of rabbits were homogenized in Tris-HCl buffer (20 mM, pH 8.0) containing mercaptoethanol (10 mM) and centrifuged at 104rpm for 10 minutes. The activities of phosphofructokinase in the supernatants were variable and had a tendency to be higher upon injection of a muscle relaxant (3-0-toloxy-1, 2-propanediol), to be lower only upon decapitation, in spite of an equal amount of lactic dehydrogenase and aldolase. The precipitates were subsequently homogenized in K2HPO4 (50 mM) solution containing mercaptoethanol (10 mM). The homogenates were centrifuged at 104rpm for 10 minutes. The activities in the supernatants were lower upon injection of a muscle relaxant and higher upon decapitation.
The Tris-extracts following injection of the muscle relaxant, described above, were incubated at 30° and then centrifuged at 104rpm for 10 minutes. The activities in the supernatants showed a decrease in a due course. On the contrary, the activities of phosphate-suspension in the precipitates showed an increase. But the activities in the Tris-extracts containing KF showed a little changes.
These findings indicate that phosphofructokinase may convert from a Tris soluble to a Tris-insoluble form according to either relaxation or contraction of muscle. In addition, these conversions may be presumably mediated by phosphorylation of protein. Based on this speculation, a series of studies were undertaken to determine whether muscle tissue contains the activation system associated with kinase for phosphofructokinase.
Procedure: Assay mixture for phosphofructokinase contains F-6-P, ATP, Mg, NADH2, mercaptoethanol, auxiliary enzymes and an enzyme preparation. ATP and Mg in the above assay system is also adequate as a substrate of kinase. So, the activities may include one of the enzymes which is activated by kinase during the assay. If so, the preincubation effect with ATP and Mg on the activities will be demonstrated.
Preparation: The extract by K2HPO4 solution from the back muscle under the effect of the muscle relaxant were used for purification. A treatment with 0.5% streptomycin, fractionation by 33-65% and then 33-50% of ammonium sulfate resulted in a preparation with a 5 fold higher specific activity and yield of 60%.
Results: Phosphofructokinase activities of the preparation, which was kept at 4°., decreased gradually day by day. On the other side, the preincubation effects with ATP and Mg increased, and the activities after preincubation preserved the initial phosphofructokinase activity. These effects were independent of dilution of the enzyme. Radioactivities of gamma 32P-ATP incorporated to TCA-insoluble fraction increased in parallel with the enzyme activity during preincubation.
