Characterization of microtubule assembly in porcine brain extracts by viscometry

Olmsted, J.B.; Borisy, Gary G.

doi:10.1021/bi00745a037

Cited by 319 publications

(141 citation statements)

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“…2 the specific viscosity of the fragments alone remained unchanged at 0. 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.…”

Section: Methodsmentioning

confidence: 86%

“…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%

See 1 more Smart Citation

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: 86%

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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“…calcium ions are essential for flagellar regeneration in Chlamydomonas (25) . However, it is known that calcium ions will inhibit microtubule assembly "in vitro" (20) . This suggests that the stimulatory effect of calcium on the regeneration is probably not caused by a direct stimulation of microtubule assembly, but by some other unknown mechanism.…”

Section: Discussionmentioning

confidence: 99%

On the possible role of serotonin in the regulation of regeneration of cilia.

Rodríguez¹,

Renaud²

1980

The Journal of Cell Biology

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A study was made of the interrelationship of serotonin, cAMP, and calcium ions in the regulation of regeneration of cilia by Tetrahymena pyriformis. All these compounds stimulated the regeneration, whereas a blocker of serotonin synthesis, p-chlorophenylalanine, and a calcium chelator, EGTA, inhibited the process . This inhibition could be overcome by the addition of any of the stimulatory compounds .cAMP was also found to be inhibitory at high concentrations . The intracellular concentration of this nucleotide was found to increase during the regeneration, and this increase occurred precociously in the presence of serotonin . It was concluded that serotonin may regulate ciliary regeneration by a mechanism involving cAMP and calcium ions, but that the causal relationships among these compounds still need to be established .Serotonin is a neurotransmitter in invertebrate nervous systems and a putative neurotransmitter in vertebrates (7) . However, its biological role is not necessarily restricted to the transmission of chemical signals by nerve cells, because it has also been reported in a wide variety of non-neural systems . For example, it has been found in unfertilized vertebrate and invertebrate eggs (3-5, 9), in protozoa (16), and in blood platelets (22) . However, the information we have about its mechanism of action is very limited . In most systems studied so far, it seems to involve both cyclic nucleotides and divalent cations . For example, the stimulation of fly salivary gland secretion by serotonin has been studied in detail and is known to involve cAMP and calcium ions (23) . An increase in the level of cAMP has also been found during ciliary movement (10), but in the case of blood platelet activation (1), serotonin causes an increase in cGMP but no changes in cAMP . Calcium ions have also been implicated in some of these systems ; for example, it is known that a calcium ionophore will activate both blood platelets and salivary gland secretion (18,24). On the other hand, the role of serotonin in unfertilized eggs and protozoan cells is even more obscure . These systems are particularly intriguing because, being unicellular, the target of the neurohormone would be the secretory cell itself. Buznikov et al . speculated that serotonin may act as a regulator of cell division in the sea urchin egg, as they found that antiserotonin agents caused delays in the first division (4) . In addition, Gustafson and Toneby (14) found that p-chlorophenylalanine (PCPA), a specific inhibitor of serotonin synthesis, caused developmental abnormalities in sea urchin embryos. The effects of this drug could be overcome by the addition of 5-hydroxytryptophane (5-HTP), the immediate precursor of serotonin.In Tetrahymena pyriformis, serotonin has been reported to be present in stationary-phase cultures (2), but its specific biological role in this organism J . CELL BIOLOGY

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“…Immediately prior to use, an aliquot of the stock solution of tubulin was chromatographed on Sephadex G-25 (coarse), which had been equilibrated at 4°C with elution buffer containing 5 mM MES, 50 mM KCl, 1 mM ATP and 0.5 mM MgClz (pH 6.6), in order to remove glycerol and EGTA. Tubulin recovered in the void volume was reassembled at 37°C for 30 min in the absence of EGTA and glycerol as reported by Olmsted and Borisy [2], and the sediment obtained after centrifugation at 59 000 g for 30 min at 30°C was resuspended at 37°C in an appropriate volume of the elution buffer to give a favorable concentration of protein of about 4-8 mg/ml for calcium binding assay. To get disassembled tubulin, tubulin reassembled in the elution buffer was immersed in an ice-bath for 30 min, then centrifuged at 105 000 4 for 60 min at 4°C.…”

Section: Preparation Of Tubandinmentioning

confidence: 99%