Reaction of phosphofructokinase 2/fructose 2,6-bisphosphatase with monoclonal antibodies. A proof of the bifunctionality of the enzyme

Abstract

Monoclonal antibodies were derived from mice immunized against homogeneous chicken liver phosphofructokinase 2/fructose 2,6-bisphosphatase. Of 112 clones, 30 were found to secrete antibodies that specifically reacted with the antigen in enzyme-linked immunoabsorbant assay (ELISA) while 17, which were ELISA-negative, produced antibodies that affected the enzymic activity of the antigen. Four clones were subcloned and used for an extensive investigation of the reaction of the corresponding antibodies with the supposedly bifunctional enzyme. A definite proof of the bifunctionality of the enzyme was obtained from the two following observations. First, the two activities were similarly retained by the four antibodies that had been coupled to Sepharose. Second, one of the antibodies inhibited both activities with the same efficiency. Furthermore, the antigen-antibody reaction led to the formation of aggregates with an apparent molecular mass of several megadaltons, showing that the two subunits of the antigen reacted with the same antibody and were therefore identical. The four monoclonal antibodies affected the activity of phosphofructokinase 2. This effect was seen as an up to 17-fold activation as well as an up to 85% inhibition. Only one of the four antibodies (antibody 10) had inhibitory effects on fructose 2,6-bisphosphatase, an effect which was in part explained by a decrease in the rate of formation of the intermediary phosphoenzyme. All the effects described above were obtained on both the chicken liver and the pigeon muscle enzymes but with lower doses of antibody in the case of the former enzyme. Antibody 10 was also shown to react with mouse liver phosphofructokinase 2/fructose 2,6-bisphosphatase, and with phosphofructokinase 2 from chicken brain, heart and testis and from frog skeletal muscle and liver. None of the four antibodies cross-reacted with phosphofructokinase 2 from Saccharomyces cerevisiae or from spinach leaves.