Selective Extraction With Sarkosyl And Repolymerization In Vitro Of Cytoskeleton Proteins From Giardia

Open Access
Publisher Website
Google Scholar
Abstract
Sarkosyl has been used to dissociate structures in cytoskeletons isolated from Giardia lambda. Results from sodium dodecyl sulphate/polyacrylamide gel electrophoresis and from electron microscopy of insoluble residues show that the solubilization of components is partly selective. At low concentrations of detergent (<0·3%), microribbons and microtubules of the disc cytoskeleton disappear, but doublet microtubules from axonemes resist extraction. Consequently, the microribbon protein giardin is extracted into solution more completely than tubulin. Soluble proteins in 0·l % Sarkosyl have been fractionated by gel filtration chromatography in Bio-Gel P300. Giardin elutes in two positions: as a low molecular weight subunit, and in early fractions corresponding to a larger particle size in which subunits might be forming oligomers. Supernatants prepared in 0·5 % Sarkosyl were diluted with 0·1 M-KC1 or 0· 1 mM-MgCl2 to bring about reaggregation of the cytoskeleton proteins. Reassembled structures seen in negatively stained preparations were polymorphic. Some tubulin ribbons of 5nm protofilaments were identifiable; also there were large fibres and some flat sheets of very thin filaments. Electron micrographs of sheets have been analysed by optical diffraction. The transforms show that the lateral separation of the fine filaments is about 2·5 nm. Axial periodicities from features spaced along filaments were weak. A 3·75 nm layer-line has been detected, corresponding to a similar periodicity found earlier in microribbons.