In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells

R. V. Zackroff, R. D. Goldman

Research output: Contribution to journalArticlepeer-review

95 Scopus citations

Abstract

Intermediate filaments (IF) from baby hamster kidney (BHK-21)cells can be disassembled at low ionic strength and reassembled upon addition of salt. Turbidimetric analyses show that reassembled IF exhibit the light scattering properties of long rods under physiological conditions (5 mM Na+/K+ phosphate, pH 7.2/170 mM NaCl at 21°C). IF weight concentration, determined by centrifugation, is directly proportional to the optical density at 300 nm. Thus, turbidity can be used as a quantitative asay for IF assembly. Turbidimetric and centrifugation analyses both indicate that IF assembly exhibits a critical protein concentration of 0.05-0.15 mg/ml. Above the critical concentration, IF weight concentration at steady-state is a linear function of the total protein concentration. Negative stain observations at early stages of the assembly process suggest lateral association of protofilaments to form short IF. This lateral association is accompanied by a rapid turbidity increase which is then followed by IF elongation and a slower turbidity increase to plateau. Further purification of IF by low/high-NaCl-induced cycles of disassembly-reassembly results in retention of 54- and 55-kilodalton (decamin) polypeptides. These results constitute a quantitative description of in vitro reassembly of IF from homogeneous cultures of nonkeratinizing cells and establish conditions for further studies on the regulation of IF assembly.

Original languageEnglish (US)
Pages (from-to)6226-6230
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume76
Issue number12
DOIs
StatePublished - 1979

ASJC Scopus subject areas

  • General

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