Identification of the AMP binding sites of rabbit phosphofructo-1-kinase isozymes B and C

Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo.