CHARACTERIZATION AND LOCALIZATION OF TROPOMYOSIN PROTEINS IN XENOPUS EMBRYOS WITH SPECIFIC ANTIBODIES

In the process of monoclonal antibody (mAb) production against the 38 kDa protein which is lacking in the gastrula arrested mutant embryos i n Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) prote ins in Xenopus embryos. The characterization of the corresponding anti gens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescen t microscopy. By 2-D immunoblots with those mAb, three distinct protei n spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa sp ot with a pi of approximately 4.8 reacted with both antibodies in embr yos at stages later than the mid-tailbud (stage 28) and two 30 kDa spo ts, which are probably isomers, with a pi of approximately 4.8 were de tected with 2D10 antibody in embryos at stages extending from the fert ilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their pr ecursor cells. In contrast, 2D10 antibody stained the cytoplasm of alm ost all cells in embryos at stages from the fertilized to the tadpole. Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.