Skip to main content
SpringerLink
Log in

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

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 β-II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the β-II tubulin fragment, but it showed interaction with the Affigel-conjugated β-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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

PAA:

poly (L-aspartic acid)

HMW-MAPs:

high molecular weight microtubule associated proteins

References

  1. Riederer B, Matus A: Differential expression of distinct microtubule-associated proteins during brain development. Proc Natl Acad Sci USA 82:6006–6009, 1985

    Google Scholar 

  2. Garner C, Garner A, Huber G, Kozak C, Matus A: Molecular cloning of microtubule-associated protein 1 (MAP-1A) and microtubule-associated protein 5 (MAP-1B): Identification of distincts genes and their differential expression in developing brain. J Neurochem 55:146–154, 1990

    Google Scholar 

  3. Crandall JE, Fischer I: Developmental regulation of microtubule-associated protein 2 expression in regions of mouse brain. J Neurochem 53:1910–1917, 1989

    Google Scholar 

  4. Nunez J: Differential expression of microtubule components during brain development. Devl Neurosci 8:421–457, 1986

    Google Scholar 

  5. Kosik K, Orecchio L, Bakalis S, Neve R: Developmental regulated expression of specific tau sequences. Neuron 2:1389–1397, 1989

    Google Scholar 

  6. Maccioni RB, Rivas C, Vera J: Differential interaction of synthetic peptides from carboxyl-terminal regulatory domain of tubulin with microtubule-associated proteins (MAPS). EMBO J 7:1957–1963, 1988

    Google Scholar 

  7. Vallee RB, Bloom G, Luca FC: Differential structure and distribution of the high molecular weight brain microtubule-associated proteins, MAP-1 and MAP-2. In: Soifer D (ed.) Dynamic aspects of microtubule biology. Ann New York Acad Sci, USA v 466:134–147, 1986

    Google Scholar 

  8. Paschal B, Obar R, Vallee R: Interaction of brain citoplasmic dynein and MAP-2 with a common sequence at the C-terminus of tubulin. Nature 342:569–572, 1989

    Google Scholar 

  9. Cross D, Domínguez J, Avila J, Maccioni RB: MAP-1 and MAP-2 binding sites at C-terminus of beta tubulin. Studies with synthetic tubulin peptides. Biochemistry 30:4362–4366, 1991

    Google Scholar 

  10. Herrmann H, Dalton JM, Wiche G: Microheterogenity of microtubule-associated proteins MAP-1 and MAP-2, and differential phosphorylation of individual subcomponents. J Biol Chem 260: 5797–5803, 1985

    Google Scholar 

  11. Fujii T, Nakamura A, Ogoma Y, Yoshiyuki K, Arai T: Selective purification of microtubule associated proteins 1 and 2 from rat brain using poly(L-aspartic acid). Anal Biochem 184:268–273, 1990

    Google Scholar 

  12. Cross D, Vial C, Maccioni RB: A tau-like protein mediates interactions between microtubules and actin filaments in human and rodent cell lines. J Cell Sci 105:51–60, 1993

    Google Scholar 

  13. Vera JC, Rivas C, Maccioni RB: Biochemical dissection of the role of the 1 kDa C-terminal domain of tubulin subunits in the assembly of microtubules. Biochemistry 28:333–339, 1989

    Google Scholar 

  14. Weingarten M, Lockwood A, Hwo S, Kirschner M: A protein factor essential for microtubule assembly. Proc Natl Acad Sci USA 72:1858–1862, 1975

    Google Scholar 

  15. Laemmli UK: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685, 1970

    Google Scholar 

  16. Rivas C, Vera J, Maccioni RB: Anti-idiotypic antibodies reacting with MAPs in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain. Proc Natl Acad Sci USA 85:6092–6096, 1988

    Google Scholar 

  17. Farías G, Vial C, Maccioni RB: Specific macromolecular interactions between tau and the microtubule system. Mol Cell Biochem 112:81–88, 1992

    Google Scholar 

  18. Vera J, Rivas C, Maccioni RB: Heat-stable microtubule protein MAP-1 binds to microtubules and induces microtubule assembly. FEBS Lett 232:159–162, 1988

    Google Scholar 

  19. Nakamura A, Arai T, Kondo Y, Fujii T: Inhibitory effects of poly (L-aspartic acid) on the assembly of brain microtubules and the interaction of microtubule associated protein 2 with F-actinin vitro. J Biochem 106:93–97, 1989

    Google Scholar 

  20. Maccioni RB, Serrano L, Avila J, Cann JR: Characterization and structural aspects of the enhanced assembling capacity of tubulin after removal of its carboxyl-terminal domain. Eur J Biochem 154: 427–435, 1985

    Google Scholar 

  21. Rivas-Berrios A, Hernández M, Domínguez J, Avila J, Maccioni RB: Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau. Biochem Biophys Acta 1040:382–390, 1990

    Google Scholar 

  22. Maccioni RB, Vera J, Domínguez J, Avila J: A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau. Arch Biochem Biophys 275:568–579, 1989

    Google Scholar 

  23. Vera JC, Rivas C, Maccioni RB: Antibodies to synthetic peptides from the tubulin's regulatory domain interact with tubulin and microtubules. Proc Natl Acad Sci USA 85:6763–6767, 1988

    Google Scholar 

  24. Binder LI, Frankfurter A, Kim H, Cáceres A, Payne MR, Rebhun LI: Heterogeneity of microtubule-associated protein 2 during rat brain development. Proc Nat Acad Sci USA 81:5613–5617, 1984

    Google Scholar 

  25. Riederer B, Cohen R, Matus A: MAP-5: A novel microtubule-associated protein under strong developmental regulation. J Neurocytol 15:763–775, 1986

    Google Scholar 

  26. Herzog w, Weber K: Fractionation of brain microtubule-associated proteins. Isolation of two different proteins which stimulate tubulin polymerizationin vitro. Eur J Biochem 92:1–8, 1978

    Google Scholar 

  27. Vallee R: A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). J Cell Biol 92:435–442, 1982

    Google Scholar 

  28. Maccioni RB, Arechaga J: The cytoskeleton in cell differentiation and development. ICSU Press-IRL (Oxford University Press), 1987; pp 397

  29. Matus A: Microtubule-associated proteins and neuronal morphogenesis. J Cell Sci 15:61–67, 1991

    Google Scholar 

  30. Maccioni RB, Vial C, Cross D, González M: Specific macromolecular interactions modulating the cytoskeleton organization. In: Becerra J (ed.) Progress in cell biology. Servicio Editorial Universidad de Málaga, 1992

Download references

Author information

Authors and Affiliations

  1. International Center for Cancer & Developmental Biology (ICC), University of Chile, Las Palmeras 3425, Nuñoa, Santiago, Chile

    Leda Guzman, Rodrigo Bustos & Ricardo B. Maccioni

  2. Laboratory of Cellular & Molecular Biology, Department of Biology, Faculty of Sciences, University of Chile, Las Palmeras 3425, Nuñoa, Santiago, Chile

    Ricardo B. Maccioni

Authors
  1. Leda Guzman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rodrigo Bustos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ricardo B. Maccioni
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guzman, L., Bustos, R. & Maccioni, R.B. Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain. Mol Cell Biochem 131, 105–113 (1994). https://doi.org/10.1007/BF00925946

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00925946

Key words

Search

Navigation