Separation and characterization of microtubule proteins from calf brain

Berkowitz, Steven A.; Katagiri, Joseph; Binder, Helen K.; Williams, Robley C.

doi:10.1021/bi00644a035

Cited by 208 publications

(140 citation statements)

References 55 publications

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“…In addition to this evidence for intracellular association between microtubules and neurofilaments, there is evidence of association in vitro. Neurofilaments adhere to microtubules and cosediment with them under the conditions employed in microtubule preparation (14)(15)(16). These considerations prompted the deliberate search, reported here, for a microtubule-neurofilament complex.…”

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confidence: 94%

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ATP-induced formation of an associated complex between microtubules and neurofilaments.

Runge¹,

Laue²,

Yphantis³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Neurofilaments (also called 10-nm filaments or intermediate filaments) from bovine brain were incubated with microtubule protein at 37"C in the presence or absence of 1 mM ATP and in a buffer that allowed microtubule assembly. Fallingball viscometry revealed that the (non-Newtonian) apparent viscosity of the ATP-containing mixtures is 5-20 times greater than that of the mixtures prepared without ATP. A larger ATP-dependent increase in viscosity (approximately 100-fold) was seen when purified tubulin replaced microtubule protein. The (1,4,5), and occasional observations of highly ordered arrays of neurofilaments surrounding microtubules (6) suggest together that the two structures are connected in vivo as part of an axoplasmic filamentous network. Neurofilaments are transported in the slow component of axonal transport (7,8), and it has been suggested (7) Neurofilaments, Tubulin, and Microtubule-Associated Proteins (MAPs). Neurofilaments were prepared from bovine brain as described (17). Microtubule protein referred to as "threetimes-cycled" was prepared by three cycles of the assembly/ disassembly method of Shelanski and coworkers (14, 18). Phosphocellulose (PC)-purified tubulin was prepared from twicecycled microtubule protein (19,20). The fraction that was initially retained by the PC column was eluted in buffer A containing 0.8 M NaCl and centrifuged for 60 min at 95,500 X g to remove any neurofilaments (see ref. 15). This material is referred to as "whole MAPs. " Aliquots of the whole MAP fraction were heated to 100°C for 5 min and then were centrifuged at 4°C for 20 min at 25,000 X g to remove flocculated protein (21). This material is referred to as "heat-treated MAPs." Purified MAP2 was prepared as described by Kim et al. (22). Upon NaDodSO4/polyacrylamide gel electrophoresis by the method of Laemmli (23), each of these five preparations appeared quite similar in protein composition to those described in the references given. The tubulin polymerized in buffer A at protein concentrations greater than about 2.7 mg/ml. Each preparation was dialyzed against buffer A and then stored in liquid N2. Aliquots were thawed, centrifuged for 10 min in a clinical centrifuge at 4°C, and used within 6 hr.Viscometry. Falling-ball viscometers were constructed and employed exactly as described by . Protein solutions were mixed at 4°C, and appropriate additions were made of nucleotide triphosphate solutions or of equal volumes of buffer in the cases of the controls. The cold solutions were immediately drawn into the viscometers, which were then immersed in a Tamson water bath controlled to +0.010C at 37. 0°C. The mixing and loading procedure required Abbreviations: MAP(s), microtubule-associated protein(s); PC, phosphocellulose; AdoPP[NH]P, adenyl-5'-yl imidodiphosphate. t Present address:

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confidence: 94%

“…Neurofilaments were prepared from bovine brain as described (17). Microtubule protein referred to as "threetimes-cycled" was prepared by three cycles of the assembly/ disassembly method of Shelanski and coworkers (14,18). Phosphocellulose (PC)-purified tubulin was prepared from twicecycled microtubule protein (19,20).…”

mentioning

confidence: 99%

ATP-induced formation of an associated complex between microtubules and neurofilaments.

Runge¹,

Laue²,

Yphantis³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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“…When such preparations are visualized with the electron microscope, MAP2 appears as regularly spaced filamentous sidearms decorating the surface of microtubules (1), reminiscent of the appearance of microtubules in situ (4,5). In addition to its interaction with microtubules, MAP2 interacts with neurofilaments and actin filaments (3,(6)(7)(8)(9)(10). This suggests that MAP2 may act as a crosslinker mediating the interaction between microtubules, other cytoskeletal elements, and cytoplasmic organelles.…”

Section: Introductionmentioning

confidence: 99%

“…It is one of a number of high molecular weight polypeptides associated with in vitro assembled brain microtubules, collectively referred to as HMW MAPs (3). NaDodSO4/urea/polyacrylamide gel electrophoresis separates MAP2 into two closely migrating polypeptides, which we designate MAP2a and MAP2b for the slower and faster migrating species, respectively.…”

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confidence: 99%

Heterogeneity of microtubule-associated protein 2 during rat brain development.

Binder¹,

Frankfurter²,

Kim³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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The electrophoretic pattern of the large microtubule-associated protein, MAP2, changes during rat brain development. Immunoblots of NaDodSO4 extracts obtained from the cerebral cortex, cerebellum, and thalamus at 10-15 days after birth reveal only a single electrophoretic species when probed with any of three MAP2 monoclonal antibodies. By contrast, adult MAP2 contains two immunoreactive species, MAP2a and MAP2b. The single band of MAP2 from immature brain electrophoretically comigrates with adult MAP2b. Between postnatal days 17 and 18, immature MAP2 simultaneously resolves into two species in both the cerebellum and cerebral cortex. Immunoblots of NaDodSO4 extracts from spinal cord demonstrate the adult complement of MAP2 by day 10, indicating that MAP2 does not change coordinately throughout the entire central nervous system. In vitro cAMPdependent phosphorylation of immature MAP2 causes a band split reminiscent of that seen during brain development in vivo. The possibility that the developmentally regulated changes observed in MAP2 during brain maturation are due to timed phosphorylation events is discussed.Microtubule-associated protein 2 (MAP2) is a heat-stable phosphoprotein (Mr 300,000) that copurifies with brain microtubules in vitro and maintains a constant stoichiometry to tubulin through several cycles of temperature-dependent assembly and disassembly (1, 2). It is one of a number of high molecular weight polypeptides associated with in vitro assembled brain microtubules, collectively referred to as HMW MAPs (3). NaDodSO4/urea/polyacrylamide gel electrophoresis separates MAP2 into two closely migrating polypeptides, which we designate MAP2a and MAP2b for the slower and faster migrating species, respectively. In vitro, MAP2 will stimulate the assembly of purified tubulin, lowering the critical concentration for tubulin assembly (1). When such preparations are visualized with the electron microscope, MAP2 appears as regularly spaced filamentous sidearms decorating the surface of microtubules (1), reminiscent of the appearance of microtubules in situ (4,5). In addition to its interaction with microtubules, MAP2 interacts with neurofilaments and actin filaments (3,(6)(7)(8)(9)(10). This suggests that MAP2 may act as a crosslinker mediating the interaction between microtubules, other cytoskeletal elements, and cytoplasmic organelles. Evidence is accumulating which suggests that the relative affinity of MAP2 for different cytoskeletal structures may be controlled by its degree of phosphorylation (10-12).Radioimmunoassay of a variety of tissues suggests that only brain contains significant amounts of MAP2 (13). Fluorescence, light, and immunoelectron microscopic studies using monoclonal antibodies have localized MAP2 in neurons but not glia (13,14). Biochemical and morphological studies further indicate that within nerve cells more of this protein is present in dendrites and cell bodies than in axons (14-16). Furthermore, MAP2 from neonatal rat brain was shown to be biochemically distinct from its a...

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“…Bulk separation of MAP 1 from MAP 2 components under non-denaturing conditions revealed that brain MAP 1, like MAP 2, stimulates in vitro polymerization of microtubules (Kuznetzov et al, 1981;Vallee & Davis, 1983). It was also found that lowMr components, referred to as light chains 1-3 (Berkowitz et al, 1977;Vallee & Davis, 1983;Kuznetzov et al, 1986;Kuznetzov & Gelfand, 1987) (Furtner & Wiche 1987). In addition, MAP 1 possesses a binding site for the cytomatrix protein plectin .…”

Section: Other Proceduresmentioning

confidence: 99%