Phosphotyrosine in proteins. Stability and quantification.
The acid and base stability of the phosphoryl bond of phosphotyrosine (Tyr-P) was studied using conditions for rapid and complete hydrolysis of protein peptide bonds. A method was developed for the quantification of Tyr-P in proteins using rapid base hydrolysis and an amino acid analyzer equipped with a fluorometric detection system. The recovery of [32P]Tyr-P from base digests of radiolabeled samples of phosphotyrosyl glutamine synthetase, transforming protein of Rous sarcoma virus, casein, and rabbit anti-sarcoma IgG was 80 +/- 2%. Phosphotyrosine could not be detected in several commercial histone samples, but Tyr-P was detected in phosvitin samples. The putative Tyr-P from the phosvitin hydrolysate was separated from normal amino acids by Dowex 50-H+ chromatography. Treatment of the partially purified Tyr-P with bacterial alkaline phosphatase produced tyrosine in near equivalent quantities to the measured level of Tyr-P. These results show that basic hydrolysis of phosphotyrosyl proteins yields Tyr-P in constant and good yields which can be quantified in amounts greater than or equal to 100 pmol or radiochemically detected in smaller amounts with an amino acid analyzer.
D000912:Antibodies, Neoplasm / Q000032:analysis; D002364:Caseins / Q000032:analysis; D004355:Drug Stability; D005974:Glutamate-Ammonia Ligase / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000032:analysis; D010455:Peptides / Q000032:analysis; D019000:Phosphotyrosine; D010774:Phosvitin / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012509:Sarcoma / Q000276:immunology; D015291:Transforming Growth Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D000912:Antibodies, Neoplasm; D002364:Caseins; D007074:Immunoglobulin G; D010455:Peptides; D011506:Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D015291:Transforming Growth Factors; D010774:Phosvitin; D005974:Glutamate-Ammonia Ligase