Identification and purification of calcium dependent modulators of actin polymerization from bovine thyroid

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Abstract
The purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography is described. The 40K actin modulator was purified in 98% homogeneity. It is a single polypeptide chain with a MW of approximately 40,000 and an isoelectric point of 8.1. It s amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. A 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a MW of approximately 80,000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique revealed that both the 91K and 95K proteins bind 125-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.