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

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)

Slow transport of unpolymerized tubulin and polymerized neurofilament in the squid giant axon

“…This conclusion is also compatible with our ability to visualize by electron microscopy short filaments Ϸ10 nm in diameter in negatively stained samples of neurofilament injectate and with our inability to visualize formed microtubules in the tubulin injectate. The presumption that the tubulin was unpolymerized at the time of injection is also consistent with previous reports that tubulin exists primarily as dimers and small oligomers at room temperature (36).…”

Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli

“…We calculated the ratio of Stokes radius between two different oligomers of CS6 to understand the surface area of each oligomeric form. The calculated value of the ratio of Stokes radii of CS6 oligomeric species suggested a spherical shape and a molar specific volume of 0.74 cm 3 g 21 mol 21 (Rodbard & Chrambach, 1971;Kravit et al, 1984). The molecular dimensions were consistent with the Mw(s) for a series of CS6 oligomers.…”

“…A standard curve was constructed by plotting the logarithm of calibrant relative mobility (log 10 R f ) as a function of total polyacrylamide concentration. Retardation coefficients (K R ) were then computed from the plot slope and graphed versus molecular mass (K R plot) (Kravit et al, 1984). CS6 samples were processed in the same way as the calibrants and the molecular masses were finally calculated by K R plot interpolation.…”

“…To resolve the stoichiometry of the CS6 oligomers in solution, we determined the Mw of the CS6 oligomers and protomer by comparing the relative electrophoretic mobility (R f ) with standard proteins of known Mw(s) at different acrylamide concentrations in native-PAGE (Rodbard & Chrambach, 1971;Kravit et al, 1984). We found that the Mw of the fastest migrating band of CS6 containing both CssA and CssB subunits was approximately 32+2.7 kDa (Fig.…”

Electrophoresis and orientation of F-actin in agarose gels

“…In spite of the difficulties associated with the electrophoresis of native proteins due to their complex shapes and small charges, they can be fractionated by electrophoresis (or by isoelectric focusing; for review see Chrambach, 1980). In particular, native gel electrophoresis allows one to measure (during the fractionation) the catalytic activity of various enzymes (Siciliano et al, 1976;Moody and Dailey, 1983;Nagy and Simon, 1983), to observe the degree of polymerization of native polymers (e.g., tubulin;Kravit et al, 1984) and to fractionate isoenzymes (e.g., myosin; Hoh et al, 1976;d'Albis et al, 1979;Takano-Ohmuro and Kohama, 1987). If it were possible to carry out the electrophoresis of native F-actin (and of a complex of F-actin with myosin fragments), one could likewise measure the activity of acto-heavy meromyosin (HMM)' (or acto-S-1 ) during the electrophoresis and one could estimate the weight (or number) distribution of F-actin sizes.…”