Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain |
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Authors: | Leda Guzman Rodrigo Bustos Ricardo B Maccioni |
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Institution: | (1) International Center for Cancer & Developmental Biology (ICC), University of Chile, Las Palmeras 3425, Nuñoa, Santiago, Chile;(2) Laboratory of Cellular & Molecular Biology, Department of Biology, Faculty of Sciences, University of Chile, Las Palmeras 3425, Nuñoa, Santiago, Chile |
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Abstract: | 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 highests 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 -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA
poly (L-aspartic acid)
- HMW-MAPs
high molecular weight microtubule associated proteins |
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Keywords: | neonatal rat brains MAPs polyaspartic acid MAP-3 interaction with calmodulin C-terminal tubulin peptides |
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