Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells.

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Abstract
Juxtanuclear birefringent caps (FC) containing 10 nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10 nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as 2 polypeptides of .apprx. 54,000 and 55,000 MW on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10 nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10 nm filaments was achieved by treatment of FC with 6 mM sodium-potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10 nm filaments upon the addition of 0.171 M. NaCl.

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