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Isolation of a Ca2+-protease resistant high Mr microtubule binding protein from mammalian brain: Characterization of properties partially expected for a dynein-like molecule

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Summary

A novel microtubule binding protein was isolated from cell extracts of pig brain after selective destruction of high Mr MAPs by Ca2+-activation of endogenous proteases. The protein had an apparent Mr of 300,000, but several criteria, including peptide maps, immunological crossreactivities, resistance to Ca2+-activated proteolysis, and inability to induce microtubule assembly, distinguished this protein from the major high Mr microtubule associated proteins MAP-1 and MAP-2. Certain molecular properties of the protein resembled those of dynein: its size under denaturing conditions, its crossreactivity with antibodies toTetrahymena dynein, the ATP-dependence of its microtubule binding, the erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA)—sensitivity of its ATPase activity and its resistance to Ca2+-activated proteolysis. However, in peptide maps and the insensitivity of its ATPase activity to vanadate the protein could be differentiated fromTetrahymena dynein heavy chain polypeptides. Based on the properties characterized so far, the protein seems qualified to function as a mechanochemical enzyme in the cytoplasm of mammalian brain cells and could represent a cytoplasmic dynein variant.

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Abbreviations

EGTA:

ethylene glycol bis([3-aminoethylether)N,N,N',N'-tetraacetic acid

MAP:

microtubule associated protein

Mr:

molecular weight

SDS:

sodium dodecyl sulfate

References

  • Allen RD, Weiss DG, Hayden DG, Brown DT, Fujiwake H, Simpson M (1985) Gliding movements of and bidirectional transport along single native microtubules from squid axoplasm: Evidence for an active role of microtubules in cytoplasmic transport. J Cell Biol 100: 1736–1752

    PubMed  Google Scholar 

  • Asai DJ, Leslie RJ, Wilson L (1986) Dynein-like cytoplasmic microtubule translocators. Ann NY Acad Sci 466: 275–291

    PubMed  Google Scholar 

  • Brady ST (1985) A novel brain ATPase with properties expected for the fast axonal transport motor. Nature (London) 317: 73–75

    Google Scholar 

  • Burns RG, Pollard TD (1974) A dynein like protein from brain. FEBS Letters 40: 274–280

    PubMed  Google Scholar 

  • Gibbons IR, Cosson MP, Evans JA, Gibbons BH, Houck B, Martinson KH, Sale WS, Tang WY (1978) Potent inhibition of dynein adenosine triphosphatase and of motility of cilia and sperm flagella by vanadate. Proc Natl Acad Sci 75: 2220–2224

    PubMed  Google Scholar 

  • Haimo LT, Telzer BR, Rosenbaum JL (1979) Dynein binds to and crossbridges cytoplasmic microtubules. Proc Natl Acad Sci 76: 5759–5763

    PubMed  Google Scholar 

  • Herrmann H, Pytela R, Dalton JM, Wiche G (1984) Structural homology of microtubule associated proteins 1 and 2 demonstrated by peptide mapping and immunoreactivity. J Biol Chem 259: 612–617

    PubMed  Google Scholar 

  • —,Dalton JM, Wiche G (1985) Microheterogeneity of microtubule-associated proteins, MAP-1 and MAP-2, and differential phosphorylation of individual subcomponents. J Biol Chem 260: 5797–5803

    PubMed  Google Scholar 

  • Hollenbeck PJ, Suprynowicz F, Cande WZ (1984) Cytoplasmic dynein-like ATPase crosslinks microtubules in an ATP-sensitive manner. J Cell Biol 99: 1251–1258

    PubMed  Google Scholar 

  • Karr TL, White HD, Purich DL (1979) Characterization of brain microtubule proteins prepared by selective removal of mitochondrial and synaptosomal components. J Biol Chem 254: 6107–6111

    PubMed  Google Scholar 

  • Koszka C, Leichtfried F, Wiche G (1985) Identification and spatial arrangement of high molecular weight proteins (Mr=300,000–330,000) co-assembling with microtubules from a cultured cell line (rat glioma C6). Eur J Cell Biol 38: 149–156

    PubMed  Google Scholar 

  • Lacal JC, Srivastana SK, Anderson PS, Aaronson SA (1986) Ras p21 proteins with high or low GTPase activity can efficiently transform NIH/3T3 cells. Cell 44: 609–617

    PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680–685

    Google Scholar 

  • Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275

    PubMed  Google Scholar 

  • Murachi T (1983) Calpain and calpastatin. Trends Biochem Sci 8: 167–169

    Google Scholar 

  • Penningroth SM, Rose PR, Cheung A, Peterson DD, Rothacker DQ, Bershak P (1985) An EHNA-sensitive ATPase in unfertilized sea urchin eggs. Cell Motil 5: 61–75

    Google Scholar 

  • Pratt MM (1980) The identification of a dynein ATPase in unfertilized sea urchin eggs. Dev Biol 74: 364–378

    PubMed  Google Scholar 

  • — (1986) Homology of egg and flagellar dynein. J Biol Chem 261: 956–964

    PubMed  Google Scholar 

  • Sandoval IV, Weber K (1978) Calcium-induced interaction of microtubule formation in brain extracts. Eur J Biochem 92: 463–470

    PubMed  Google Scholar 

  • Schmidt W,Seyfert HM (1987) Antibodies against axonemal dynein of the ciliateTetrahymena decorate nuclear spindle microtubules from mammalian cells but not fromTetrahymena. Exp Cell Res submitted

  • Scholey JM, Neighbours B, McIntosh JR, Salomon ED (1984) Isolation of microtubules and dynein-like Mg ATPase from unfertilized sea urchin eggs. J Biol Chem 259: 6516–6525

    PubMed  Google Scholar 

  • —,Porter ME, Grissom PM, McIntosh JR(1985) Identification of kinesin in sea urchin eggs, and evidence for its localization in the mitotic spindle. Nature (London) 318: 483–486

    Google Scholar 

  • Murphy DB, Borisy GG (1975) Association of high-molecular weight proteins with microtubules and their possible role in microtubule assembly in vitro. Proc Natl Acad Sci 72: 2696–2700

    PubMed  Google Scholar 

  • —,Hiebsch RR, Wallis KT (1983 a) Identity and origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles. J Cell Biol 96: 1298–1305

    PubMed  Google Scholar 

  • —,Wallis KT, Hiebsch RR (1983 b) Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000 dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria. J Cell Biol 96: 1306–1315

    PubMed  Google Scholar 

  • Nasr A, Satir P (1985) Alloaffmity filtration: A general approach to the purification of dynein and dynein-like molecules. Anal Biochem 151: 97–108

    PubMed  Google Scholar 

  • Siman R, Baudry M, Lynch G (1985) Regulation of glutamate receptor binding by the cytoskeletal protein fodrin. Nature (London) 313: 225–228

    Google Scholar 

  • Sloboda RD, Rudolph SA, Rosenbaum JL, Greengard R (1975) Cyclic AMP-dependent endogeneous phosphorylation of a microtubule associated protein. Proc Natl Acad Sci 72: 177–181

    PubMed  Google Scholar 

  • Tack BF, Dean J, Eilat D, Lorenz PE, Schechter AN (1980) Tritium labelling of proteins to high specific radioactivity by reductive methylation. J Biol Chem 255: 8842–8847

    PubMed  Google Scholar 

  • Vale RD, Reese TS, Sheetz MP (1985 a) Identification of a novel force-generating protein, kinesin, involved in microtubule based motility. Cell 42: 39–50

    PubMed  Google Scholar 

  • —,Schnapp BJ, Mitchison T, Steurer E, Reese TS, Sheetz MP (1985 b) Different axoplasmic proteins generate movement in opposite directions along microtubules in vitro. Cell 43: 623–632

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • —,Bloom GS (1983) Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies. Proc Natl Acad Sci 80: 6259–6263

    PubMed  Google Scholar 

  • Weingarten MD, Lockwood AH, Hwo SY, Kirschner MW (1975) A protein factor essential for microtubule assembly. Proc Natl Acad Sci 72: 1858–1862

    PubMed  Google Scholar 

  • Wiche G, Briones E, Hirt H, Krepler R, Artlieb U, Denk H (1983) Differential distribution of microtubule-associated proteins MAP-1 and MAP-2 in neurons of rat brain and association of MAP-1 with microtubules of neuroblastoma cells (clone N2A). EMBO J 2: 1915–1920

    PubMed  Google Scholar 

  • — —,Koszka C, Artlieb U, Krepler R (1984) Widespread occurence of polypeptides related to neurotubule associated proteins (MAP-1 and MAP-2) in non-neuronal cells and tissues. EMBO J 3: 991–998

    PubMed  Google Scholar 

  • —, (1985) High-molecular-weight microtubule-associated proteins (MAPs): a ubiquitous family of cytoskeletal-connecting links. Trends Biochem Sci 10: 67–70

    Google Scholar 

  • Yoshimura N, Tsukahara I, Murachi T (1984) Calpain and calpastatin in porcine retina. Biochem J 223: 47–51

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Institute of Biochemistry, University of Vienna, Waehringer Strasse 17, A-1090, Vienna, Austria

    Ch. Koszka, R. Foisner & G. Wiche

  2. Institut für Tierphysiologie, Justus-Liebig-Universität, Giessen

    H. M. Seyfert

Authors
  1. Ch. Koszka
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  2. R. Foisner
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  3. H. M. Seyfert
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  4. G. Wiche
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Koszka, C., Foisner, R., Seyfert, H.M. et al. Isolation of a Ca2+-protease resistant high Mr microtubule binding protein from mammalian brain: Characterization of properties partially expected for a dynein-like molecule. Protoplasma 138, 54–61 (1987). https://doi.org/10.1007/BF01281185

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  • DOI: https://doi.org/10.1007/BF01281185

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