THE GLU-MODIFICATION OF ALPHA-TUBULIN IN THE FEEDING APPARATUS OF THEPRIMITIVE FLAGELLATE ENTOSIPHON SULCATUM IS ONLY APPARENT AFTER DETERGENT TREATMENT

Using specific monoclonal antibodies, we investigated the distribution of post-translational modified Tyr-and Glu-tubulins during interphase of the primitive flagellate Entosiphon sulcatum. Immunofluorescence s tudies of simultaneously permeabilized and fixed cells revealed that m icrotubular structures comprising Ca2+-labile subpellicular and flagel lar MTs and Ca2+-stable MTs in the siphon complex (feeding organelle) reacted surprisingly unorthodox with antibodies against Tyr-and Glu-tu bulin: Unexpectedly, the siphon complex consisting of Ca2+-stable MTs appeared exclusively Tyr-positive, whereas the Ca2+-labile subpellicul ar and flagellar MTs reacted with the Glu-as well as with the Tyr-anti body. That the siphon MTs were indeed Ca2+-stable and all other MTs ha d become solubilized, was verified by EM-observation. This surprising result contrasting considerably with the permanent nature of the sipho n complex, was reconsidered after preceding lysis and extraction proce dures. Depending on the type of detergent used and on extraction times applied, the MTs of the siphon complex now always showed also Glu-pos itivity, indicating the presence of detyrosinated cr-tubulin as a bioc hemical marker of stabilized MTs. Since saponin, irrespective of subse quent extraction times, always produced a Glu-positive reaction and ul trastructural analysis never gave compelling evidence for a drastic MA P-removal, we conclude that the Glu-epitope became freely accessible d ue to conformational changes in the tubulin polymeres.