A novel approach for evaluating tyrosine kinase activity based on the radioimmunological determination of phosphotyrosine.
A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide.
Shisheva
A
A
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Leitner
O
O
Shechter
Y
Y
D000818:Animals; D000906:Antibodies / Q000032:analysis; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D010455:Peptides; D011506:Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases