PURIFICATION AND CHARACTERIZATION OF THE HIGH-MOLECULAR-WEIGHT MICROTUBULE-ASSOCIATED PROTEINS FROM NEONATAL RAT-BRAIN

The changes in the levels of microtubule-associated proteins (MAPs) du ring advanced embryonic stages, neonatal and adult organisms reflect t he importance of these cytoskeletal proteins in relation to the morpho genesis of the central nervous system. MAP-1B is found in prenatal bra ins and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast p rocedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal r at brains was designed, based on the differential capacity of poly L-a spartic acid to release MAPs during temperature-dependent cycles of mi crotubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular prote in polymerization in the presence of low concentrations of polyasparti c acid. Instead, MAP-2 and a 180 kDa protein with characteristics of M AP3 remained associated to the polymer after the assembly. Further pur ification of MAP-1B was attained after phosphocellulose chromatography . Isolation of MAP-2 isoforms together with MAP3 was achieved on the b asis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide beta-II (422- 434) derivatized on an Affigel matrix. However, MAP-1B did not interac t with the beta-II tubulin fragment, but it showed interaction with th e Affigel-conjugated beta-I (431-444) tubulin peptide. The different M APs componentes were characterized by western blots using specific mon oclonal antibodies. A salient feature of neonatal rat brain MAP3 was i ts interactions with site-directed antibodies that recognize binding e pitopes on the repetitive sequences of tau and MAP-2. However, these s ite-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.