Microtubules from chicken erythrocytes and brain tissue contain beta tubulin variants that can be biochemically distinguished by differences in electrophoretic properties and in primary structure as revealed by peptide mapping. These differences are reflected by differences in polypeptide composition, assembly properties, solubility and stability of microtubules. It is possible that the beta tubulin variants are different gene products. We propose to examine this possibility and determine how differences in tubulin assembly and function are related to differences in tubulin structure. The unique properties of erythrocyte tubulin and the finding that erythroblasts may not contain the beta tubulin variant will also allow us to use antibodies to examine differentiating blood cells to examine gene switching, protein sorting, and differential utilization of tubulin variants in development. Three areas of investigation are proposed. (1) Peptide mapping of beta tubulin subunits and amino acid sequencing of specific peptides will be done to explain the shift in isoelectric points (0.3 pH unit) and establish if the tubulin variants are different gene products. Particular attention will be given to the acidic carboxyl-terminal peptide since variation in the beta tubulin sequence is predicted to occur in this position. (2) Surface domains involved in interactions among tubulin subunits and MAPs will be identified by 'zero-length crosslinking"""""""" with a water-soluble carbodiimide together with partial digestion with proteases and SDS gel electrophoretic analysis (Sutoh, 1982). (3) Rabbit antibodies to """"""""total tubulin"""""""" and to """"""""erythrocyte beta tubulin"""""""" will be used to examine the time of appearance (tubulin gene switching) and sites of incorporation (protein sorting and compartmentalization) of tubulin variants during chicken red cell differentiation. By immuno-electron microscopy we will determine if protein sorting occurs at the molecular level, preventing the formation of copolymers of the tubulin variants.
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