Antibodies and radioimmunoassays for phosphoserine, phosphothreonine and phosphotyrosine. Serologic specificities and levels of the phosphoamino acids in cytoplasmic fractions of rat tissues.
For antibody production, the O-phosphorylated derivative of tyrosine, threonine, or serine was covalently linked to succinylated bovine albumin via the carbodiimide reaction. Each conjugate was then complexed with methylated bovine albumin for immunization of rabbits. To determine binding, the corresponding O-phosphorylated [3H]amino acids were chemically synthesized. In addition, these 3H-phosphorylated derivatives were acylated (with succinic or acetic anhydride) to obtain ligands whose structures resemble those present in the immunogen. The acylated ligands bound to their respective antibodies more effectively: in some cases binding was about three orders of magnitude greater than their non-acylated counterparts. Radioimmunoassays were therefore developed using the N-succinyl-[3H]phosphoamino acids. When the unlabeled N-succinyl-phosphorylated amino acids were used as inhibitors in the homologous immune systems, 50% displacement of the labeled ligand was found with 0.06, 0.27 or 0.8 pmol of the tyrosine, threonine, or serine derivative, respectively. The antibodies were highly specific for the homologous hapten; the requirement for the phosphate group on the acylated amino acid was essentially absolute. Antibody content (expressed as mg/ml serum) and apparent binding constants for the N-succinyl derivatives in individual bleedings of immune sera were 1.9 and 1 X 10(10) M-1 for phosphotyrosine, 0.825 and 6 X 10(8) M-1 for phosphothreonine, and 0.150 and 2 X 10(8) M-1 for phosphoserine. The radioimmunoassays were used to quantitate the phosphoamino acids in cytoplasmic fractions of rat tissue extracts. The production of antibodies to phosphorylated O-tyrosine has been reported previously, but to our knowledge, this represents the first report of antibodies specific for O-phosphorylated serine and threonine residues.
Levine
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Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Gjika
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HB
Van Vunakis
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H
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000276:immunology; D010768:Phosphoserine / Q000032:analysis*; D010769:Phosphothreonine / Q000032:analysis*; D019000:Phosphotyrosine; D011817:Rabbits; D011863:Radioimmunoassay; D051381:Rats; D011916:Rats, Inbred F344; D012694:Serine / Q000031:analogs & derivatives*; D012710:Serum Albumin, Bovine; D013912:Threonine / Q000031:analogs & derivatives*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D000906:Antibodies; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D012710:Serum Albumin, Bovine; D013912:Threonine; D014443:Tyrosine; D012694:Serine