Purification and characterization of the high molecule weight microtubule associated proteins from neonatal rat brain.
The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highest levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis 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 interact with the beta-II tubulin fragment, but it showed interaction with the Affigel-conjugated beta-I (431-444) tubulin peptide. The different MAPs components were characterized by western blots using specific monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
Guzman
L
L
International Center for Cancer & Developmental Biology ICC, Nuñoa, Santiago, Chile.
Bustos
R
R
Maccioni
R B
RB
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D001921:Brain / Q000254:growth & development / Q000378:metabolism; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats
D008869:Microtubule-Associated Proteins