The effect of phospholipids on the in vitro polymerization of rat brain tubulin

The association of brain tubulin, as measured by the temperature-dependent development of turbidity at 350 nm, is greatly stimulated by the detergent Nonidet P-40 in crude extracts of rat brain tissue. Stimulation of turbidity development is also obtained with partially purified rat brain tubulin treated with Nonidet or other detergents, or preincubated with phospholipase C or D; treatment with bovine pancreatic phospholipase A₂ produces an inhibition. Exogenous phospholipids, diglycerides, other related derivatives, and lipophilic extracts of tubulin and brain supernatants can also alter the turbidity development. In addition, microtubules arising from tubulin obtained in the presence of Tween-20 or Nonidet P-40 exhibit a 50 and 100% increased specific viscosity, respectively, over that of tubulin prepared in the absence of detergent or in the presence of Kyro or Triton N-101. The effectiveness of these detergents in removing phospholipids from tubulin preparations follows a similar pattern: Nonidet P-40 removes 80%, Tween-20 removes 50%, and Kyro or Triton N-101 removes none. The total mass of microtubule formed, as determined by sedimentation, is the same regardless of the effect of the detergents on the viscosity. The microtubules obtained in the presence of Nonidet P-40 have a normal appearance when examined by electron microscopy, and their composition on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is indistinguishable from that of standard tubulin, especially with regard to the minor protein bands always present in the tubulin preparations. The results obtained suggest that the phospholipids associated to brain tubulin preparations might have a role in determining the association of tubulin and/or the final dimensions of the assembled microtubules.