In vitro assembly and disassembly of 14-nm filament from Tetrahymena pyriformis. The protein component of 14-nm filament is a 49,000-dalton protein.
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Conditions for <i>in vitro</i> assembly and disassembly of Tetrahymena 14-nm filaments were investigated electron-microscopically by using a crude extract of acetone powder of the cells. The assembly conditions established are: incubation of a protein sample (2mg/ml) in 5mM 2-(<i>N</i>-morpholino) ethanesulfonic aicd (MES) buffer (pH 6.6) containing 0.1mM <i>N</i><sup>α</sup>-tosyl-L-lysyl-chloromethane hydrochloride (TLCK), 50mM KCl, 0.6mM ATP, and 1.2mM CaCl<sub>2</sub> at 30&C for 30min. The disassembly conditions established are: dialysis of the 14-nm filament suspension (3mg protein/ml) against Tris-acetate buffer (pH 8.2) containing 5mM 2-mercaptoethanol, 1mM ethylene glycol bis(β-aminoethyl ether)-<i>N</i>, <i>N'</i>-tetraacetic acid (EGTA), and 0.05mM TLCK at 4&C for 24 h. The assembly and disassembly were repeatable, and resulted in the exclusive retention of the 49, 000-dalton protein. This clearly shows that the previously reported protein component (38, 000-dalton protein: FFP-38) of the 14-nm filament is incorrect and the actual component is indeed a 49, 000-dalton protein.<br> The present research also showed that the <i>Tetrahymena</i> 14-nm filament bore a strong resemblance to 'intermediate filaments' of mammalian cells with respect to molecular weight, amino-acid composition of the protein component, and size and conditions for assembly and disassembly of the filament.
- J Biochem (Tokyo)
J Biochem (Tokyo) 91(5), 1563-1573, 1982
The Japanese Biochemical Society