The effect of solution composition on microtubule dynamic instability.
The exchange of tubulin dimer into steady-state microtubules was studied over a range of solution conditions, in order to assess the effects of various common buffer components on the dynamic instability of microtubules. In comparison with standard buffer conditions (100 mM-Pipes buffer, pH 6.5, containing 0.1 mM-EGTA, 1.8 mM-MgC12 and 1 M-glycerol), the rate and extent of exchange, and thus of dynamic instability, are suppressed by increasing the concentration of glycerol above 2 M. Exchange is enhanced by the addition of further Mg2+ (up to 17 mM) or by the addition of Ca2+ (up to 0.4 mM). Phosphate ion (150 mM) has relatively little effect on the dynamic behaviour of microtubules, as judged by the exchange method. The findings are interpreted within the framework of the Lateral Cap model for microtubule dynamic instability, in terms of the effects of these changes on the intrinsic rate constants of the system. By contrast, the extent of tubulin exchange depends selectively on the value of the dissociation rate constant for tubulin-GDP. A decrease in the extent of exchange, and hence in dynamic activity, is associated with a decreased value for this rate constant, and vice versa. The results also show good agreement of predictions of the model in treating the observed variations in the dynamic properties of individual microtubules, induced by different solution conditions.
Division of Physical Biochemistry, National Institute for Medical Research, London, U.K.
M J
MJ
Schilstra
P M
PM
Bayley
S R
SR
Martin
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D005990:Glycerol / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008870:Microtubules / Q000378:metabolism*; D017239:Paclitaxel; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology; D012996:Solutions; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D000470:Alkaloids; D002021:Buffers; D010710:Phosphates; D012996:Solutions; D014404:Tubulin; D012965:Sodium Chloride; D008274:Magnesium; D017239:Paclitaxel; D005990:Glycerol; C018279:sodium phosphate; D002118:Calcium