Identification of a new phosphorylation site in cardiac muscle phosphofructokinase

The novel phosphorylation site (Ser376) that we discovered during in vitro studies of the troponin C- or calmodulin-induced phosphorylation of rabbit muscle phosphofructokinase [Zhao, Z., Malencik, D.A., and Anderson, S. R. (1991) Biochemistry 30, 2204] also undergoes phosphorylation in the epinephrine-stimulated rabbit heart. Reversed-phase HPLC and/or iron chelate affinity chromatography performed on CNBr digests of phosphofructokinase that had been isolated from epinephrine-treated hearts yields a largely phosphorylated peptide corresponding to amino acid residues 371-378 of the enzyme. Mass spectrometry, gas phase sequencing, and amino acid analyses establish the structure and phosphorylation state of the peptide.