Divalent cation stimulation of in vitro fibre assembly from epidermal keratin protein

Abstract

Keratin was extracted from purified cornified cells of newborn rats in Tris-HCl-buffered 8 M urea containing beta-mercaptoethanol. Microfilaments were assembled in vitro by reducing the ionic strength of buffer and the urea concentration. One millimolar concentration of KCl and NaCl did not affect filament formation, but the same concentration of divalent cations greatly altered this process. CaCl2 and MgCl2 induced gelation of keratin by formation of bundles of birefringent macrofilaments. ZnCl2, CuSO4 and HgCl2 formed greater numbers of macrofilaments and the protein aggregated.

Factors modulating filament formation by bovine glial fibrillary acidic protein, the intermediate filament component of astroglial cells

“…Cu2+ showed an extreme ability to induce GFAP aggregation (Figure 4b) indicated by formation of a white, flocculent precipitate rather than the opalescent turbidity observed in a filament suspension. A similar effect of Cu2+ on keratin was described by Fukuyama et al (1978). The turbidity increases produced by Mn2+, as well as its location in the periodic table , were between those of copper and calcium.…”

“…Under physiological conditions, intermediate filaments are insoluble and must be extracted from detergent-insoluble pellets with denaturing reagents such as 8 M urea (Cook, 1976; Fukuyama et al, 1978;Rueger et al, 1979). Their subunits show remarkable propensities for assembly.…”

“…Upon removal of denaturing reagents by dialysis, the polypeptide subunits assemble into filaments which biochemically and morphologically are very similar to those formed in vivo (Steinert et al, 1976;Osborn et al, 1977;Sun & Green, 1978;Rueger et al, 1979). High ionic strength and low pH favor filament assembly except for keratin filaments which are particularly prone to aggregate even in urea solution (Baden et al, 1973;Steinert, 1975;Fukuyama et al, 1978;Rueger et al, 1979;Moon et al, 1981;Jones et al, 1982).…”

“…filaments are soluble, but increases in ionic strength promote filament assembly (Steinert et al, 1976(Steinert et al, , 1981Rueger et al, 1979;Huiatt et al, 1980;Yang & Babitch, 1988). Divalent cations may act through different mechanisms from monovalent cations in accelerating filament formation (Fukuyama et al, 1978).…”

Molecular Insight into the Regulation of Vimentin by Cysteine Modifications and Zinc Binding

“…These low affinity carriers allow the cell to sense zinc deficiency early [95], in contrast to high affinity binding proteins, which will remain in their zinc-bound form in situations of mild zinc deficiency. Of note, within the intermediate filament family, keratins have long been known to bind zinc [96,97], although the precise site(s) of interaction, to the best of our knowledge, has not been elucidated. Moreover, Cys328 is not conserved in keratins.…”