A reevaluation of the structure of purified tubulin in solution: evidence for the prevalence of oligomers over dimers at room temperature.

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Abstract
The molecular form of tubulin in solution was studied by ultrafiltration, nondenaturing electrophoresis and chemical cross-linking. The results are not consistent with the generally-held belief that tubulin in solution is a 110,000-MW dimer. Tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. The filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC-tubulin) was compared with aldolase (142,000 MW). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperture 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. The average effective MW of tubulin was determined from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective MW of .apprx. 300,000. This MW was significantly reduced at 10.degree. C, indicating that oligomers dissociated a low temperatures. Oligomers were also demonstrated by chemical cross-linking using glutaraldehyde, dimethyl suberimidate and bis[2-(succinimidooxycarbonyoxy)ethyl] sulfone. PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE [polyacrylimide gel electrophoresis] 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 (MAP) 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 MAP 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 < 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. The differences between these results and earlier results obtained by other methods are discussed with particular emphasis on the potential contribution of cold and hydrostatic pressure to the dissociation of oligomers. The implications of oligomer formation for microtubule assembly are discussed.