Natural Activators for Liver Fructose 1,6-Diphosphatase and the Reversal of Adenosine 5′-Monophosphate Inhibition by Muscle Phosphofructokinase

Abstract

1. The addition of a chelating agent was necessary in order to obtain maximal activity of rabbit liver fructose 1,6-diphosphatase at neutral pH. All attempts to demonstrate that this 10-fold or greater activation was merely due to the removal of trace heavy metal contaminants have been negative. 2. A naturally occurring material has been found in rabbit liver supernatant which produces a similar increase in enzymatic activity. Properties of the activator may be summarized as follows: (a) it is nondialyzable and destroyed by crystalline Bacillus subtilis proteolytic enzyme, and therefore it is probably a protein; (b) it can be precipitated at pH 6 and redissolved at pH 7.8; (c) it is unaffected by either p-mercuribenzoate or N-ethylmaleimide; (d) it is readily converted to aggregates upon purification. A very high concentration of activator was found in extracts of Novikoff hepatoma. 3. Purification of the muscle activator of liver fructose 1,6-diphosphatase paralleled phosphofructokinase activity. Twice recrystallized rabbit muscle phosphofructokinase activated rabbit liver fructose 1,6-diphosphatase 4-fold and both prevented and reversed the specific inhibition of the enzyme by adenosine 5′-monophosphate. Both phenomena were very specific for phosphofructokinase. The reversal of inhibition appears to be due to protein-protein interaction. Addition of adenosine 5′-triphosphate to this system counteracted the inhibition reversal. Similar results have been found with rabbit muscle fructose 1,6-diphosphatase. 4. Dissociation of muscle phosphofructokinase into active subunits of molecular weights 235,000 and 125,000 has been observed upon sucrose density gradient centrifugation in the presence of sulfate ions and liver supernatant (pH 6). It is suggested that these may be the active dimer and monomer, respectively, of phosphofructokinase. 5. The molecular and physiological significance of these observations is discussed.