A monoclonal antibody to the phosphorylated form of phenylalanine hydroxylase. Definition of the phosphopeptide epitope

Abstract

Monoclonal antibody PH7 has specificity for the phosphorylated form of the human liver phenylalanine hydroxylase and negligible reactivity towards the dephosphorylated form of the native enzyme by enzyme-linked immunoassay. PH7 binds specifically to the phosphorylated form of the liver enzyme after SDS/polyacrylamide-gel electrophoresis and transfer to nitrocellulose. Competitive blocking assays have been applied in conjunction with reversed-phase h.p.l.c. of purified tryptic fragments of human liver phenylalanine hydroxylase to localize the epitope. The major immunoreactive tryptic peptide cross-reacting with PH7 had an amino acid analysis corresponding to the first 41 amino acids of the human liver phenylalanine hydroxylase sequence and included the serine residue that is thought to be the phosphorylation site. The monoclonal antibody recognized the phosphorylated form of the synthetic decapeptide corresponding to the local phosphorylation-site sequence Gly-Leu-Gly-Arg-Lys-Leu-Ser(P)-Asp-Phe-Gly, but not the dephosphodecapeptide. Thermolysin digestion of the peptide demonstrated the monoclonal antibody bound to the pentapeptide Leu-Ser(P)-Asp-Phe-Gly. Monoclonal antibody PH7 recognized the phosphodecapeptide at concentrations 10(3)-fold higher than with phenylalanine hydroxylase, compared with 10(4)-10(7)-fold higher for other phosphopeptides and phosphoproteins. The results demonstrate that monoclonal antibody PH7 has specificity for the phosphorylated form of phenylalanine hydroxylase at the phosphorylation site.