Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane‐Bound Polysomes

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Abstract
Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[35S]methionine. The translation products were analyzed by 2-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least 4 major tubulin subunits (.alpha.1, .alpha.2, .beta.1 and .beta.2) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly 1 protein (MB) in the tubulin region of the 2-dimensional gel. MB has a MW and isoelectric point similar to .alpha.-tubulin. Only trace amounts of .alpha.- and .beta.-tubulin and actin were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after 2 cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and .alpha.- and .beta.-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of .alpha.- and .beta.-tubulin; MB yielded peptides not found in tubulin. Membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to .alpha.-tubulin synthesized by free polysomes on the basis of MW isoelectric point and peptide analysis.