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 ex tracts from rat-liver membranes were used as a source of kinases to ph osphorylate endogenous proteins in the presence of gamma-P-32-labeled ATP. Histone was also added as a substrate in order to compare the dir ect 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 procedu res led to recoveries of P-32-labeled material of 90% and 40%, respect ively, with a similar proportion of radiolabeled phosphoamino acids. S everal P-32-labeled phosphoproteins separated in gels were therefore d irectly HCl-hydrolyzed and their phosphoamino acids were quantitated e ither prior to, or after glutaraldehyde crosslinking, with and without alkali treatment. By preventing protein losses occurring in hot alkal i, glutaraldehyde crosslinking increased by an average factor of 6.5 t he P-32-labeled material available for phosphoamino-acid analyses. For eight phosphoproteins analyzed, the overall effect of combined glutar aldehyde and alkali treatments was a relative decrease in phosphoserin e (up to 8-fold), with concomitant relative increases in phosphotyrosi ne and phosphothreonine (up to 62- and 6-fold, respectively). This met hod will especially be useful for the detection of pTyr, a less abunda nt phosphoamino acid, in proteins which suffer from poor transfer effi ciency in Western blot, are weakly antigenic towards anti-phosphotyros ine antibodies, can hardly be extracted from a gel and for identificat ion of protein tyrosine kinases renatured in gels.