Purification of Intact Microtubules From Brain

Kirkpatrick, Joel B.; Hyams, Lyon; Thomas, Virginia L.; Howley, Peter M.

doi:10.1083/jcb.47.2.384

Cited by 78 publications

(32 citation statements)

References 41 publications

(30 reference statements)

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“…We refer to these bands collectively as high-molecular-weight components (HMW). Bands of high molecular weight have also been observed by others using a similar assembly procedure (11)(12)(13), an assembly procedure using glycerol (14), and in purified tubule preparations stabilized in hexylene glycol (15). Previous results showed that the HMW components copurified in constant stoichiometry with tubulin through repeated cycles of assembly-disassembly (9,10,14).…”

mentioning

confidence: 53%

“…16 and that of the subunits alone reached only 0.05 after 25 min incubation. The mixture of subunits with fragments, however, polymerized rapidly and by 15 mentation analysis showed that the microtubule growth resulting from the addition of purified tubulin subunits at 2.0 mg/ml was 75% of that which was obtained using unfractionated microtubule protein which had received a similar exposure to high salt. These data indicated that the DEAEpurified tubulin was competent to polymerize onto preexisting microtubule fragments but could not polymerize well on its own.…”

Section: Methodsmentioning

confidence: 99%

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Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

Murphy¹,

Borisy²

1975

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

491

232

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High-molecular-weight components (HMW) specifically associated with microtubule protein purified from porcine brain tissue were separated from tubulin by DEAE-Sephadex ion exchange chromatography. Analysis by viscometry, sedimentation, and electron microscopy of the unfractionated microtubule protein, separated HMW and tubulin fractions, and reconstituted mixtures showed that HMW promoted formation of ring structures at 50 and tubule polymerization at 370. The HMW reassociated with tubulin and was identified in thin sections as 18.9 X 5.6 nm projections attached to the microtubules with a longitudinal periodicity of 32.5 nm. These studies: (1) indicate that the HMW fraction stimulates microtubule assembly by facilitating the formation of ring structures which are apparently intermediates in polymerization, and (2) demonstrate that the HMW associates with microtubules as a structural component projecting from the surface of the microtubule wall.Cytoplasmic microtubules play important roles in cell elongation and motile processes such as mitosis and intracellular transport (1). For some of these functions microtubule-associated elements such as crossbridges have been postulated (2, 3), and structural components attached to microtubules have been observed in neurons (4), myoblasts (5), and the mitotic spindle (6, 7). Although a microtubule sidearm which produces the force for axonemal motility has been isolated from cilia and flagella (8), in no case have the structural components observed on cytoplasmic microtubules been characterized biochemically or had their exact functions defined.Microtubule-associated proteins have been observed in preparations of purified microtubule protein obtained from brain tissue by an in vitro assembly procedure (9, 10). These components account for 15-20% of the total purified material and are typically resolved on 5% acrylamide gels as a pair of closely spaced bands (286,000 and 271,000 MW) together with a minor band of higher molecular weight (345,000 MW). We refer to these bands collectively as high-molecular-weight components (HMW). Bands of high molecular weight have also been observed by others using a similar assembly procedure (11-13), an assembly procedure using glycerol (14), and in purified tubule preparations stabilized in hexylene glycol (15). Previous results showed that the HMW components copurified in constant stoichiometry with tubulin through repeated cycles of assembly-disassembly (9,10,14). Agents that inhibited microtubule assembly also inhibited the sedimentation of the HMW under conditions that sedimented microtubules but not the unpolymerized material (10). Therefore, we concluded that these components were not contaminants but rather were specifically associated with microtubules. The HMW components were therefore further investigated to determine the nature of their association with microtubules and their effect on tubule assembly in vitro. MATERIALS AND METHODSPreparation of HMW and Tubulin Fractions. Purified microtubule protein was obtained from p...

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mentioning

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

Murphy¹,

Borisy²

1975

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

491

232

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“…The possible significance of membrane-bound CB-protein in relation to microtubule structure and function remains a matter for speculation, especially since to our knowledge, most of the evidence that the soluble colchicine-binding protein purified from nerve tissue represents microtubule subunit protein is of indirect nature [2,6]. However, it is perhaps worth noting that extensively purified colchicine-binding protein prepared from soluble extracts of pig brain possesses both an intrinsic cyclic AMP-activated phosphokinase activity [see also 151, as well as the capacity to bind cyclic AMP, as measured by the methods of Tao et al [ 161 (Lagnado, Lyons and Weller, paper in preparation).…”

Section: Discussionmentioning

confidence: 99%

The subcellular distribution of colchicine‐binding protein (‘microtubule protein’) in rat brain

Lagnado¹,

Lyons²,

Wickremasinghe³

1971

FEBS Letters

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Su bcellular frac tiona tionRat brain cortex was homogenised in 9 parts of 0.32 M sucrose containing 1 mM sodium phosphateMg2+ buffer, pH 6.5. Primary fractions (crude nuclear, 254 mitochondrial, microsomal, soluble) and purified nuclei were prepared according to the method of Balazs and Cocks [7]. The purification of nerveending membranes (i.e. synaptic membranes) and vesicles from osmotically disrupted mitochondrial suspensions was carried out following the procedure of Lapetina et al. [B] , as summarized in table I, and the homogenity of the subfractions was checked by electron microscopy. The purity of the nuclear fraction was checked by light microscopy and DNA determinations [9]. All fractions to be assayed were resuspended in, or adjusted to, 10 mM sodium phosphateMg*+ buffer, pH 6.5, and kept on ice (maximum 3 hr) prior to incubation. Assay of colchicine-binding activityBound 3H-colchicine was assayed by the filterdisc (DE81) method of Weisenberg et al.[lo], as modified by Wilson [ II], except that incubations were carried out in 10 mM sodium phosphate-Mg*' buffer pH 6.5, instead of in phosphate-glutamate buffer [ 1 l] . The different subcellular fractions were diluted prior to assay to give a protein concentration of 1 OO-1000 ng/ml reaction mixture, in which range colchicine-binding was found, in preliminary experiments, to be proportional to protein concentration. Protein-bound 3H-colchicine absorbed on DE81 filter discs was counted directly in 5 ml Bray's solution [ 121 in a Packard 3375 spectrometer, at 48% efficiency. Radioactivity measurements were carried out on triplicate samples, and results given are based on the means of at least three separate experiments (maximum variability between experiments f 10%).

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“…4). Treatments were as follows: RNase A, 10 pg/ml; DNase I, 15 ,ug/ml; phospholipase C, 10,ug/ml; trypsin, 8 mg/ml. All treatments were for 10 min at 370C.…”

mentioning

confidence: 99%

Molecules in mammalian brain that interact with the colchicine site on tubulin.

Lockwood¹

1979

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

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Colchicine, a plant alkaloid, is a potent inhibitor of mitosis and other physiological processes that involve microtubules. These effects are mediated by the specific binding of colchicine to a high-affinity receptor site on tubulin, the major protein of microtubules. It seemed possible that the colchicine site on tubulin might also be the receptor for endogenous cellular molecules. We now report that mammalian brain does, in fact, contain a class of molecules that interact with the colchicine site on tubulin. Tubulin-agarose affinity chromatography has been used to isolate factors from soluble extracts of bovine brain that interact with tubulin.

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Purification of Intact Microtubules From Brain

Cited by 78 publications

References 41 publications

Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

The subcellular distribution of colchicine‐binding protein (‘microtubule protein’) in rat brain

Molecules in mammalian brain that interact with the colchicine site on tubulin.

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