DETECTION OF PHOSPHOTYROSINE IN GLUTARALDEHYDE-CROSS-LINKED AND ALKALI-TREATED PHOSPHOPROTEINS FOLLOWING THEIR PARTIAL ACID-HYDROLYSIS IN GELS

Soluble fractions and particulate extracts from human prostate, and extracts from rat-liver membranes were used as a source of kinases to phosphorylate endogenous proteins in the presence of gamma-P-32-labeled ATP. Histone was also added as a substrate in order to compare the direct partial acid hydrolysis of phosphoproteins in gels to an indirect procedure involving partial acid hydrolysis after extraction in sodium dodecyl sulfate followed by precipitation with acetone. These procedures led to recoveries of P-32-labeled material of 90% and 40%, respectively, with a similar proportion of radiolabeled phosphoamino acids. Several P-32-labeled phosphoproteins separated in gels were therefore directly HCl-hydrolyzed and their phosphoamino acids were quantitated either prior to, or after glutaraldehyde crosslinking, with and without alkali treatment. By preventing protein losses occurring in hot alkali, glutaraldehyde crosslinking increased by an average factor of 6.5 the P-32-labeled material available for phosphoamino-acid analyses. For eight phosphoproteins analyzed, the overall effect of combined glutaraldehyde and alkali treatments was a relative decrease in phosphoserine (up to 8-fold), with concomitant relative increases in phosphotyrosine and phosphothreonine (up to 62- and 6-fold, respectively). This method will especially be useful for the detection of pTyr, a less abundant phosphoamino acid, in proteins which suffer from poor transfer efficiency in Western blot, are weakly antigenic towards anti-phosphotyrosine antibodies, can hardly be extracted from a gel and for identification of protein tyrosine kinases renatured in gels.