Participation of 200K or 150K subunit of neurofilament in construction of the filament core with 70K subunit and promotion of tubulin polymerization by incorporated 200K subunit.

J-STAGE
  • MINAMI Yasufumi
    Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo
  • ENDO Sachiko
    Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo
  • SAKAI Hikoichi
    Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo

抄録

We have already reported that neurofilaments are capable of stimulating microtubule assembly and causing gelation. After separation of each of the triplet proteins of neurofilaments it was demonstrated that only the 200 K subunit shows the activity to promote tubulin polymerization (Minami, Y. & Sakai, H. (1983) J. Biochem. 94, 2023-2033). The separation of each subunit protein led us to attempt the reconstitution of filaments from the 200 K and 70 K subunits or from the 150 K and 70 K subunits. It was found that both the 200 K and 150 K subunits independently contribute to the formation of intermediate-sized filaments, provided that each subunit was combined with the 70 K subunit before removing urea by dialysis for reconstitution. On the other hand, the 200 K subunit alone formed a very short thread-like structure after removal of urea, and the 150 K subunit formed a filamentous structure, both incapable of being incorporated into filaments made of the 70 K subunit alone. These observations suggest that the 200 K and 150 K subunits are not peripherally attached to a filament core made of 70 K protein, but they take part in the formation of the core. Moreover, both proteins can co-polymerize with the 70 K protein at a weight ratio of about 1:1 at least, which is in excess of that of the intact neurofilament. We investigated whether or not the 200 K subunit incorporated with the 70 K subunit into filaments could also stimulate tubulin polymerization. Low-shear viscometry measurements suggested that the 200 K subunit retains the activity to initiate tubulin polymerization. This was confirmed by measuring viscosity changes with an Ostwald-type viscometer. In contrast, filaments reconstituted from the 70 K and 150 K proteins were incapable of increasing low-shear viscosity when mixed with tubulin. These observations suggest that the domain of the 200 K protein embedded in the core of intermediatesized filament is separate from the site responsible for promotion of tubulin polymerization.

収録刊行物

詳細情報 詳細情報について

問題の指摘

ページトップへ