Network formation by neurofilament-induced polymerization of tubulin: 200K subunit of neurofilament triplet promotes nucleation of tubulin polymerization and enhances microtubule assembly

Abstract

The previous paper (Minami, Y., et al. (1982) J. Biochem. 92, 889-898) demonstrated that the neurofilament preparation obtained from porcine brain promotes tubulin polymerization, thereby leading to network formation in vitro. Since this preparation fractionated by gel filtration and centrifugation was contaminated mainly by tubulin, the neurofilaments thus prepared were solubilized in a 6 M urea solution, further purified by hydroxyapatite column chromatography, and reconstituted into neurofilaments. This highly purified neurofilament was found to retain the ability to stimulate microtubule assembly and to cause gelation. Next we separated the purified neurofilament into the individual triplet subunits, referred to as 200K, 150K, and 70K proteins, by DEAE-cellulose (DE-52) column chromatography in the presence of 6 M urea. By measuring viscosity and turbidity changes, it has been found that the activity to stimulate polymerization of tubulin is due to the 200K polypeptide, while the 150K protein was less active to promote tubulin polymerization and the 70K protein was totally inactive.