Microheterogeneity of Micro tubule‐Associated τ Proteins Is Due to Differences in Phosphorylation

We have studied the heterogeneity of the microtu‐bule‐associated τ proteins using τ‐specific antibodies and two‐dimensional electrophoresis. Both monoclonal and polyclonal antibodies to τ proteins recognize five bands in cow brain microtubule proteins run on sodium dodecyl sul‐fate (SDS)‐polyacrylamide gels, with apparent molecular weights between 56 ,000 and 66 ,000. Immunoblots of cow brain microtubules separated on two‐dimensional gels, using nonequilibrium pH gradient electrophoresis in the first dimension and SDS‐gel electrophoresis in the second, reveal that >30 isoforms of τ exist. The T proteins vary in pI from 6.5 to 8.5, with the higher‐molecular‐weight forms being more acidic. The microheterogeneity of τ is not induced by cycling of microtubules, because two‐dimensional immunoblots of τ from total brain are almost identical to those of τ from cycled tubules. Adult rat brain τ, which appears as three doublet bands on SDS gels, also exhibits considerable isoelectric heterogeneity, as does τ from 7‐day‐old rats, which appears as only one band on SDS gels. After dephos‐phorylation of cow brain τ with alkaline phosphatase, the highest‐molecular‐weight band disappears on SDS gels. On two‐dimensional gels, the number of τ variants decreases by more than half after dephosphorylation, and the more basic species increase greatly in intensity. Preliminary experiments with T labeled in vivo with 32 PO 4 also indicate that the more acidic τ proteins are the more highly phosphorylated forms. Thus, isoelectric heterogeneity of τ proteins exists at all ages and is due, at least in part, to differences in the state of phosphorylation of τ isoforms.