Abstract
We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross- linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC- tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross- linking using glutaraldehyde, dimethyl suberimidate, and bis[2- (succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)
Full Text
The Full Text of this article is available as a PDF (1.2 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bayley P. M., Charlwood P. A., Clark D. C., Martin S. R. Oligomeric species in glycerol-cycled bovine-brain microtubule protein. Analytical ultracentrifugal characterisation. Eur J Biochem. 1982 Jan;121(3):579–585. doi: 10.1111/j.1432-1033.1982.tb05826.x. [DOI] [PubMed] [Google Scholar]
- Ben-Ze'ev A., Farmer S. R., Penman S. Mechanisms of regulating tubulin synthesis in cultured mammalian cells. Cell. 1979 Jun;17(2):319–325. doi: 10.1016/0092-8674(79)90157-0. [DOI] [PubMed] [Google Scholar]
- Bergen L. G., Borisy G. G. Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly. J Cell Biol. 1980 Jan;84(1):141–150. doi: 10.1083/jcb.84.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Borisy G. G., Olmsted J. B., Klugman R. A. In vitro aggregation of cytoplasmic microtubule subunits. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2890–2894. doi: 10.1073/pnas.69.10.2890. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Bryan J. Biochemical properties of microtubules. Fed Proc. 1974 Feb;33(2):152–157. [PubMed] [Google Scholar]
- Carlier M. F. Kinetic evidence for a conformation change of tubulin preceding microtubule assembly. J Biol Chem. 1983 Feb 25;258(4):2415–2420. [PubMed] [Google Scholar]
- Carpenter F. H., Harrington K. T. Intermolecular cross-linking of monomeric proteins and cross-linking of oligomeric proteins as a probe of quaternary structure. Application to leucine aminopeptidase (bovine lens). J Biol Chem. 1972 Sep 10;247(17):5580–5586. [PubMed] [Google Scholar]
- Clark D. C., Martin S. R., Bayley P. M. Conformation and assembly characteristics of tubulin and microtubule protein from bovine brain. Biochemistry. 1981 Mar 31;20(7):1924–1932. doi: 10.1021/bi00510a031. [DOI] [PubMed] [Google Scholar]
- Cleveland D. W., Lopata M. A., Sherline P., Kirschner M. W. Unpolymerized tubulin modulates the level of tubulin mRNAs. Cell. 1981 Aug;25(2):537–546. doi: 10.1016/0092-8674(81)90072-6. [DOI] [PubMed] [Google Scholar]
- Detrich H. W., 3rd, Williams R. C. Reversible dissociation of the alpha beta dimer of tubulin from bovine brain. Biochemistry. 1978 Sep 19;17(19):3900–3907. doi: 10.1021/bi00612a002. [DOI] [PubMed] [Google Scholar]
- Durham A. C., Finch J. T., Klug A. States of aggregation of tobacco mosaic virus protein. Nat New Biol. 1971 Jan 13;229(2):37–42. doi: 10.1038/newbio229037a0. [DOI] [PubMed] [Google Scholar]
- Durham A. C. Structures and roles of the polymorphic forms of tobacco mosaic virus protein. I. Sedimentation studies. J Mol Biol. 1972 Jun 20;67(2):289–305. doi: 10.1016/0022-2836(72)90242-2. [DOI] [PubMed] [Google Scholar]
- Gethner J. S., Flynn G. W., Berne B. J., Gaskin F. Characterization of heterogeneous solutions using laser light scattering: study of the tubulin system. Biochemistry. 1977 Dec 27;16(26):5776–5781. doi: 10.1021/bi00645a020. [DOI] [PubMed] [Google Scholar]
- Gethner J. S., Flynn G. W., Berne B. J., Gaskin F. Equilibrium components of tubulin preparations. Biochemistry. 1977 Dec 27;16(26):5781–5785. doi: 10.1021/bi00645a021. [DOI] [PubMed] [Google Scholar]
- Jeffrey P. D. Polymerization behavior of bovine zinc-insulin at neutral pH. Molecular weight of the subunit and the effect of glucose. Biochemistry. 1974 Oct 8;13(21):4441–4447. doi: 10.1021/bi00718a029. [DOI] [PubMed] [Google Scholar]
- Johnson K. A., Borisy G. G. Kinetic analysis of microtubule self-assembly in vitro. J Mol Biol. 1977 Nov 25;117(1):1–31. doi: 10.1016/0022-2836(77)90020-1. [DOI] [PubMed] [Google Scholar]
- Keith C. H., Feramisco J. R., Shelanski M. Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts. J Cell Biol. 1981 Jan;88(1):234–240. doi: 10.1083/jcb.88.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lauffer M. A., Stevens C. L. Structure of the tobacco mosaic virus particle; polymerization of tobacco mosaic virus protein. Adv Virus Res. 1968;13:1–63. doi: 10.1016/s0065-3527(08)60250-x. [DOI] [PubMed] [Google Scholar]
- Lee J. C., Frigon R. P., Timasheff S. N. The chemical characterization of calf brain microtubule protein subunits. J Biol Chem. 1973 Oct 25;248(20):7253–7262. [PubMed] [Google Scholar]
- Lee J. C., Timasheff S. N. In vitro reconstitution of calf brain microtubules: effects of solution variables. Biochemistry. 1977 Apr 19;16(8):1754–1764. doi: 10.1021/bi00627a037. [DOI] [PubMed] [Google Scholar]
- Ludueńa R. F., Shooter E. M., Wilson L. Structure of the tubulin dimer. J Biol Chem. 1977 Oct 25;252(20):7006–7014. [PubMed] [Google Scholar]
- MacLean-Fletcher S. D., Pollard T. D. Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors. J Cell Biol. 1980 May;85(2):414–428. doi: 10.1083/jcb.85.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mandelkow E. M., Harmsen A., Mandelkow E., Bordas J. X-ray kinetic studies of microtubule assembly using synchrotron radiation. Nature. 1980 Oct 16;287(5783):595–599. doi: 10.1038/287595a0. [DOI] [PubMed] [Google Scholar]
- Marcum J. M., Borisy G. G. Characterization of microtubule protein oligomers by analytical ultracentrifugation. J Biol Chem. 1978 Apr 25;253(8):2825–2833. [PubMed] [Google Scholar]
- Marcum J. M., Borisy G. G. Sedimentation velocity analyses of the effect of hydrostatic pressure on the 30 S microtubule protein oligomer. J Biol Chem. 1978 Apr 25;253(8):2852–2857. [PubMed] [Google Scholar]
- Margolis R. L., Wilson L. Regulation of the microtubule steady state in vitro by ATP. Cell. 1979 Nov;18(3):673–679. doi: 10.1016/0092-8674(79)90122-3. [DOI] [PubMed] [Google Scholar]
- Morrow J. S., Marchesi V. T. Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton. J Cell Biol. 1981 Feb;88(2):463–468. doi: 10.1083/jcb.88.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morrow J. S., Speicher D. W., Knowles W. J., Hsu C. J., Marchesi V. T. Identification of functional domains of human erythrocyte spectrin. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6592–6596. doi: 10.1073/pnas.77.11.6592. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Palmer G. R., Clark D. C., Bayley P. M., Sattelle D. B. A quasi-elastic laser light scattering study of tubulin and microtubule protein from bovine brain. J Mol Biol. 1982 Oct 5;160(4):641–658. doi: 10.1016/0022-2836(82)90320-5. [DOI] [PubMed] [Google Scholar]
- Pantaloni D., Carlier M. F., Simon C., Batelier G. Mechanism of tubulin assembly: role of rings in the nucleation process and of associated proteins in the stabilization of microtubules. Biochemistry. 1981 Aug 4;20(16):4709–4716. doi: 10.1021/bi00519a029. [DOI] [PubMed] [Google Scholar]
- Prakash V., Timasheff S. N. The interaction of vincristine with calf brain tubulin. J Biol Chem. 1983 Feb 10;258(3):1689–1697. [PubMed] [Google Scholar]
- Regula C. S., Pfeiffer J. R., Berlin R. D. Microtubule assembly and disassembly at alkaline pH. J Cell Biol. 1981 Apr;89(1):45–53. doi: 10.1083/jcb.89.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rodbard D., Chrambach A. Estimation of molecular radius, free mobility, and valence using polyacylamide gel electrophoresis. Anal Biochem. 1971 Mar;40(1):95–134. doi: 10.1016/0003-2697(71)90086-8. [DOI] [PubMed] [Google Scholar]
- Salisbury J. L., Condeelis J. S., Maihle N. J., Satir P. Calmodulin localization during capping and receptor-mediated endocytosis. Nature. 1981 Nov 12;294(5837):163–166. doi: 10.1038/294163a0. [DOI] [PubMed] [Google Scholar]
- Salmon E. D. Pressure-induced depolymerization of spindle microtubules. II. Thermodynamics of in vivo spindle assembly. J Cell Biol. 1975 Jul;66(1):114–127. doi: 10.1083/jcb.66.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scheele R. B., Borisy G. G. Electron microscopy of metal-shadowed and negatively stained microtubule protein. Structure of the 30 S oligomer. J Biol Chem. 1978 Apr 25;253(8):2846–2851. [PubMed] [Google Scholar]
- Shizuta Y., Shizuta H., Gallo M., Davies P., Pastan I. Purification and properties of filamin, and actin binding protein from chicken gizzard. J Biol Chem. 1976 Nov 10;251(21):6562–6567. [PubMed] [Google Scholar]
- Siezen R. J. Interactions of lens proteins. Ultrafiltration is unsuitable to detect self- or mixed-association. Biophys Chem. 1984 Jan;19(1):49–55. doi: 10.1016/0301-4622(84)85005-x. [DOI] [PubMed] [Google Scholar]
- Stearns M. E., Brown D. L. Purification of a microtubule-associated protein based on its preferential association with tubulin during microtubule initiation. FEBS Lett. 1979 May 1;101(1):15–20. [PubMed] [Google Scholar]
- Stephens R. E. High-resolution preparative SDS-polyacrylamide gel electrophoresis: fluorescent visualization and electrophoretic elution-concentration of protein bands. Anal Biochem. 1975 May 12;65(1-2):369–379. doi: 10.1016/0003-2697(75)90521-7. [DOI] [PubMed] [Google Scholar]
- Stossel T. P., Hartwig J. H. Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin. J Biol Chem. 1975 Jul 25;250(14):5706–5712. [PubMed] [Google Scholar]
- Tilney L. G. Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments. J Cell Biol. 1975 Feb;64(2):289–310. doi: 10.1083/jcb.64.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weisenberg R. C., Borisy G. G., Taylor E. W. The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry. 1968 Dec;7(12):4466–4479. doi: 10.1021/bi00852a043. [DOI] [PubMed] [Google Scholar]
- Weisenberg R. C. Role of co-operative interactions, microtubule-associated proteins and guanosine triphosphate in microtubule assembly: a model. J Mol Biol. 1980 Jun 5;139(4):660–677. [PubMed] [Google Scholar]
- Weisenberg R. C., Timasheff S. N. Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine and vinblastine. Biochemistry. 1970 Oct 13;9(21):4110–4116. doi: 10.1021/bi00823a012. [DOI] [PubMed] [Google Scholar]
- Zarling D. A., Watson A., Bach F. H. Mapping of lymphocyte surface polypeptide antigens by chemical cross-linking with BSOCOES. J Immunol. 1980 Feb;124(2):913–920. [PubMed] [Google Scholar]