PHOSPHOAMINO ACIDS IN PROTEASOME SUBUNITS

Proteasomes, the major catalysts of the non-lysosomal proteolytic path way in eukaryotic cells, were analyzed for their content of phosphoami no acids using polyacrylamide gel electrophoresis and subsequent detec tion on Western blots by phosphoamino acid antibodies. No specific bin ding to proteasome subunits was observed with phosphoserine or phospho threonine antibodies, whereas phosphotyrosine antibodies were bound by a single proteasome subunit, which was identified in rat as well as i n human proteasomes as subunit C7-I. Since dephosphorylation of the su bunit by phosphatases was not possible, analysis of phosphoamino acid content of all proteasome subunits was performed using another method. All proteasome subunits were isolated from PD-polyacrylamide gels and subjected to partial acid hydrolysis. Phosphoamino acids were subsequ ently detected by capillary electrophoresis after their derivatization with phenylisothiocyanate. This analysis revealed no phosphorylated a mino acid in subunit C7-I, however, subunit C3 contained phosphotyrosi ne and phosphothreonine, and phosphoserine was detected in subunits ze ta, C5, C8 and C9.