Ionic and nucleotide requirements for microtubule polymerization in vitro. Export

@article{citeulike:4498894, abstract = {The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (piperazine-N,N'-bis(2-ethanesulfonic acid)); increasing ionic strength by addition of NaCl to samples prepared in lower buffer concentrations did not result in an equivalent level of polymerization. Both Na-+ and K-+ inhibited microtubule formation at levels greater than 240 mM, withmaximal assembly occurring at physiological concentrations of 150 mM. Maximal extent of assembly occurred at pH 6.8 and optimal rate at pH 6.6. Inhibition of polymerization was half-maximal at added calcium concentrations of 1.0 mM and magnesium concentrations of 10.0 mM. EGTA (ethylene glycol bis(beta-aminoethyl ether)tetraacetic acid), which chelates Ca-2+, had no effect on polymerization over a concentration range of 0.01-10.0 mM. In contrast, EDTA (ethylenediaminetetraacetic acid), which chelates both Mg-2+ and Ca-2+, inhibited assemble half-maximally at 0.25 mM and totally at 2.0 mM. As determined from experiments using Mg-2+-EDTA buffers, magnesium was required for polymerization. Magnesium promoted the maximal extent of assembly at substoichiometric levels relative to tubulin, but was maximal for both rate and extent at stoichiometric concentrations. Elemental analyses indicated that approximately 1 mol of magnesium was tightly bound/mol of tubulin dimer. Viscosity development was dependent upon hydrolyzable nucleoside triphosphate, and stoichiometric levels of GTP were sufficient for maximal polymerization. The effect of magnesium in increasing the rate of GTP-dependent polymerization suggests that a Mg-2+-GTP complex is the substrate required for a step in assembly.}, author = {Olmsted, J. B. and Borisy, G. G.}, citeulike-article-id = {4498894}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/238580}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=238580}, comment = {Andy Maloney found this classic paper which is basically the origin of the BRB80 buffer used in microtubule protocols. See Andy's summary here: http://openwetware.org/wiki/User:Andy\_Maloney/Notebook/Lab\_Notebook\_of\_Andy\_Maloney/2009/05/08/Ionic\_and\_nucleotide\_requirements\_for\_microtubule\_polymerization\_in\_vitro\_review The paper has been cited about 250 times. It's a very nice paper, demonstrating the need for Mg++ and GTP to polymerize MTs, and finding the optimium pH and ionic strength for polymerization. Having read Parsegian and Rau, I now see that osmotic pressure is glaringly missing from any of the discussion, especially when they're comparing with in vivo conditions. It's understandable, though, since osmotic pressure is rarely talked about. I wonder if a statistically designed experiment (DOE) (factorial design) could optimize the tubulin polymerization protocol much better. As great as this paper is, I think it's highly likely that the "one factor at a time" analysis did not find the true maximum. Or even then, we are not using the same proteins that they were. So, use of BRB80 all the time may not always be optimal. I do need to read the more recent papers that cite this paper, though.}, issn = {0006-2960}, journal = {Biochemistry}, keywords = {brb80, calcium, classic, divalent-cations, dtra, gtp, magnesium, microtubules, polymerization, potassium, sodium, tubulin}, month = {July}, number = {13}, pages = {2996--3005}, posted-at = {2009-05-10 05:26:22}, priority = {0}, title = {Ionic and nucleotide requirements for microtubule polymerization in vitro.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/238580}, volume = {14}, year = {1975} }

TY - JOUR ID - citeulike:4498894 L3 - citeulike-article-id:4498894 N2 - The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (piperazine-N,N'-bis(2-ethanesulfonic acid)); increasing ionic strength by addition of NaCl to samples prepared in lower buffer concentrations did not result in an equivalent level of polymerization. Both Na-+ and K-+ inhibited microtubule formation at levels greater than 240 mM, withmaximal assembly occurring at physiological concentrations of 150 mM. Maximal extent of assembly occurred at pH 6.8 and optimal rate at pH 6.6. Inhibition of polymerization was half-maximal at added calcium concentrations of 1.0 mM and magnesium concentrations of 10.0 mM. EGTA (ethylene glycol bis(beta-aminoethyl ether)tetraacetic acid), which chelates Ca-2+, had no effect on polymerization over a concentration range of 0.01-10.0 mM. In contrast, EDTA (ethylenediaminetetraacetic acid), which chelates both Mg-2+ and Ca-2+, inhibited assemble half-maximally at 0.25 mM and totally at 2.0 mM. As determined from experiments using Mg-2+-EDTA buffers, magnesium was required for polymerization. Magnesium promoted the maximal extent of assembly at substoichiometric levels relative to tubulin, but was maximal for both rate and extent at stoichiometric concentrations. Elemental analyses indicated that approximately 1 mol of magnesium was tightly bound/mol of tubulin dimer. Viscosity development was dependent upon hydrolyzable nucleoside triphosphate, and stoichiometric levels of GTP were sufficient for maximal polymerization. The effect of magnesium in increasing the rate of GTP-dependent polymerization suggests that a Mg-2+-GTP complex is the substrate required for a step in assembly. IS - 13 JF - Biochemistry SN - 0006-2960 EP - 3005 TI - Ionic and nucleotide requirements for microtubule polymerization in vitro. VL - 14 SP - 2996 KW - brb80 KW - calcium KW - classic KW - divalent-cations KW - dtra KW - gtp KW - magnesium KW - microtubules KW - polymerization KW - potassium KW - sodium KW - tubulin N1 - Andy Maloney found this classic paper which is basically the origin of the BRB80 buffer used in microtubule protocols. See Andy's summary here: http://openwetware.org/wiki/User:Andy_Maloney/Notebook/Lab_Notebook_of_Andy_Maloney/2009/05/08/Ionic_and_nucleotide_requirements_for_microtubule_polymerization_in_vitro_review The paper has been cited about 250 times. It's a very nice paper, demonstrating the need for Mg++ and GTP to polymerize MTs, and finding the optimium pH and ionic strength for polymerization. Having read Parsegian and Rau, I now see that osmotic pressure is glaringly missing from any of the discussion, especially when they're comparing with in vivo conditions. It's understandable, though, since osmotic pressure is rarely talked about. I wonder if a statistically designed experiment (DOE) (factorial design) could optimize the tubulin polymerization protocol much better. As great as this paper is, I think it's highly likely that the "one factor at a time" analysis did not find the true maximum. Or even then, we are not using the same proteins that they were. So, use of BRB80 all the time may not always be optimal. I do need to read the more recent papers that cite this paper, though. AU - Olmsted, JB AU - Borisy, GG PY - 1975/07// UR - http://view.ncbi.nlm.nih.gov/pubmed/238580 ER -