Phosphofructokinase was treated with reagents which are known to react with amino groups of proteins, namely, maleic anhydride, succinic anhydride, and pyridoxal 5′-phosphate. All the reagents inactivated the enzyme. Fructose 6-phosphate, fructose 1,6-diphosphate, and adenosine monophosphate protected the enzyme against the inactivation by succinylation while adenosine triphosphate-Mg2+ did not. The maleylation and succinylation led to the dissociation of phosphofructokinase to protein derivatives with a molecular weight of approximately 80,000. The reaction with pyridoxal 5′-phosphate resulted in the formation of a Shiff base and revealed the existence of three types of amino groups which differ in the reactivity toward the reagent. At relatively low concentrations of pyridoxal 5′-phosphate the phosphofructokinase was modified such that the degree of its inhibition by adenosine triphosphate was altered. The enzyme was dissociated to a 7S protein by the treatment with a higher concentration of pyridoxal 5′-phosphate. It was found that the antibody against the native phosphofructokinase reacted to form a precipitin with higher molecular weight maleyl, succinyl, and 5′-phosphopyridoxyl enzymes, but did not form precipitin with the derivatives with molecular weight of 80,000.
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