The <i>in vitro</i> polymerization of tubulin from beef brain

Lee, Young C.; Samson, Frederick E.; Houston, L. L.; Himes, Richard H.

doi:10.1002/neu.480050404

Cited by 67 publications

(32 citation statements)

References 19 publications

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“…The viscosity of the tubulin fraction typically showed a transient rise at approximately 5 min followed by a rapid decline to baseline levels. After [20][21][22][23][24][25][26][27][28][29][30] (2) Sedimentation. Following incubation at 37°for 30 min, we assayed 0.5 ml aliquots of the same samples by sedimentation to determine the extent of polymerization.…”

Section: Methodsmentioning

confidence: 99%

“…Purified microtubule protein was obtained from porcine brain tissue by two cycles of a reversible temperature-dependent assembly procedure (10). Protein was prepared at 00 in polymerization medium (PM) [0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes) adjusted with NaOH to pH 6 Since monovalent cation concentrations above 0.2 M inhibit microtubule assembly (16,21), it was necessary to remove the salt from the purified fractions before assaying them for polymerization. When purified tubulin (0.8 M KCI fraction) was desalted and brought up to 370 in PM containing 1.0 mM GTP, viscometry and sedimentation showed that little polymerization occurred at protein concentrations as high as 4.0 mg/ml.…”

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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Section: Methodsmentioning

confidence: 99%

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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“…4b) as the previous control. Cooling to O' will not depolymerize the fixed microtubules, indicating they are chemically crosslinked, since cooling of untreated microtubules readily reverses polymerization (10). After pressure treatment at 5000 lbs./inchl for 45 min, the tubules' appearance suggests that they are more flexible ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Tubulin, the protein subunits of the microtubules, will quickly polymerize to form microtubules within 1-2 min under optimal conditions and can be followed by spectrophotometric changes at 500 nm (10). No Fig.…”

Section: Resultsmentioning

confidence: 99%

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Stability of Neuronal Microtubules to High Pressure In Vivo and In Vitro

O'Connor

Houston

Samson

1974

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

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Neuronal microtubules in a variety of nerve cell types are unaffected by high hydrostatic pressures over a range of 1400-10,000 pounds/inch2 and periods of 10-45 min. Similarly, purified tubulin polymerized to form microtubules in vitro were not depolymerized by the same range of pressures. The depolymerization of microtubules in several types of non-neuronal cells, which has been reported, may have been over-generalized with regard to the direct action of pressure on microtubule stability.Within the past several years the knowledge of the structure and biochemistry of microtubules has advanced rapidly (1-3). In view of their ubiquity and their functional importance to the cell, a better understanding of the forces of interactions between tubulin subunits that result in polymerization-depolymerization will improve our concept of the physiological mechanisms of the control and action of microtubules.The commonly held view is that microtubules are sensitive to hydrostatic pressure (3)(4)(5)(6). This view is supported by reports that the microtubules of the mitotic apparatus are depolymerized by hydrostatic pressure in the range of 3000-3800 lbs./inch2 (7). Further, it was shown that the microtubules of the heliozoan axopodium are sensitive to high hydrostatic pressure (5). That is, pressures ranging from 4000 to 8000 lbs./inch2 caused a rapid disintegration of the microtubules of the axopodia and cytosome of Actinosphaerium nucleofilum. A wide variety of cells tend to become rounded and lose their motility when exposed to pressure at 4000-8000 lbs./inch2, including amoebae, human fibroblasts in tissue culture, various gate the direct effects of pressure on microtubule assembly. It was found that high hydrostatic pressure did not depolymerize either the microtubules of vertebrate neurons or microtubules in vitro, prepared from purified beef brain tubulin. MATERIALS AND METHODSThe pressure chamber used in this study was similar to the one designed by Landau and Thibodeau (8). This chamber permits tissues to be fixed under high pressure without decompression. Basically, it is two chambers in one, with the inner assembly consisting of two compartments separated by a thin cover glass. Living tissue is placed in one of the compartments. This compartment is separated from the compartment containing the fixative by the cover glass. The ends of the two compartments are covered with rubber diaphragms which allow the pressure to be transmitted equally to the tissue and fixative. The inner assembly, containing the tissue and fixative, is placed in a large outer pressure cylinder and the latter is then filled with water.The pressure cylinder was connected to a hand-operated hydraulic pump (American Instrument Co.). This assembly allowed compressions up to 10,000 lbs./inch2, with the pressures reached in less than 5 sec. After compression, the valve to the pressure cylinder was closed and the cylinder was disconnected from the pump. The cylinder with "sealed in" pressure was, after a period of time, inverted and shake...

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The presence of cyclic AMP, and its effects on oral regeneration and in situ ciliary regeneration in stentor coeruleus

Maloney

Burchill

1977

Journal Cellular Physiology

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We have found cyclic AMP in the large, heterotrichous ciliate Stentor coeruleus in amounts per milligram protein similar to those found in another ciliate, Tetrahymena pyriformis. The possible function of cyclic AMP in Stentor was first examined by determining its effects on oral regeneration, the process by which Stentor can replace a missing oral apparatus in eight to ten hours. Once begun (by brief exposure to a 15% sucrose solution, causing shedding of the oral apparatus) regeneration follows eight specific morphological stages visible with the dissecting microscope. Continuous exposure of regenerating cells to either N6, Z'-O-dibutyryl adenosine cyclic 3':5'-monophosphate (DBC) or theophylline begun a t the onset of oral regeneration (stage 0) caused delays in the completion of regeneration. The delays induced by DBC occurred in the early stages prior to stage 5. Regenerating cells exposed to DBC or theophylline at various stages of development were delayed, even at stages 5 and 6. Both DBC and theophylline reversibly bleached the cortical pigment of the cells. Guanosine 3':5'-cyclic monophosphate (cyclic GMP), AMP, GMP, and sodium butyrate neither delayed oral regeneration nor bleached the cortical pigment. Excess extracellular calcium ions alone had no effect on oral regeneration, but 10 mM calcium and DBC caused more delay than DBC alone. Thus, the delay of oral regeneration in Stentor caused by cyclic AMP may involve calcium ions.To determine if cyclic AMP can retard in situ ciliary regeneration by Stentor, as it does in Tetrahymena, a new technique, more accurate than past methods, was developed to monitor ciliary regrowth. Using this procedure we found that both DBC and theophylline significantly delayed the in situ ciliary regeneration by Stentor.

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The in vitro polymerization of tubulin from beef brain

Cited by 67 publications

References 19 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.

Stability of Neuronal Microtubules to High Pressure In Vivo and In Vitro

The presence of cyclic AMP, and its effects on oral regeneration and in situ ciliary regeneration in stentor coeruleus

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