Purification of a HeLa cell high molecular weight action binding protein and its identification in HeLa cell plasma membrane ghosts and intact HeLa cells.

The high molecular weight protein (HMWP) which was previously observed to be a major component of the actin based gels formed by incubating cytoplasmic extracts of HeLa cells at 25 degrees C [Weihing, R. R. (1977) J. Cell Biol. 75, 95-103] has now been purified by gel filtration of 0.6 M KCl extracts of precipitated gels. A few hundred micrograms of HMWP, which is about 90% pure, can be isolated from 4 X 10(9) cells. HMWP can gel muscle actin and cross-link it into filament bundles. Its subunit molecular weight is 250 0000, its Stokes radius is 125 A, and its sedimentation coefficient is 9 S. A native molecular weight of 480 000 was calculated by using the latter two parameters, and therefore the native molecule is a dimer. Its amino acid analysis is nearly indistinguishable from that of macrophage actin binding protein and of mammalian and avian filamins. All of these findings indicate that HMWP is homologous to the latter proteins. However, HeLa cell HMWP and avian filamin must differ in their primary sequences because their partial peptide maps are distinct and because an antiserum against HMWP reacts only weakly with filamin. For studies on the intracellular location of HMWP, a goat antiserum against purified HMWP was prepared and characterized and then used to localize HMWP in suspension grown cells. The technique of immunoblotting revealed that the antiserum reacted virtually exclusively with the high molecular weight polypeptide that comigrates with HMWP in cell lysates and in ZnCl2-stabilized plasma membrane ghosts prepared from HeLa cells [Gruenstein, E., Rich, A., & Weihing, R. R. (1975) J. Cell Biol. 64, 223-234] and that it did not react with rabbit myosin heavy chain, microtubule proteins (MAPS and tubulin) from HeLa cells and calf brain, or the proteins of human erythrocyte ghosts including spectrin. Suspension-grown cells which were stained with the antiserum by the technique of indirect immunofluorescence showed bright fluorescence at the rim of the cells and less intense generalized fluorescence. If preimmune serum or immune serum treated with HMWP was substituted for the immune serum, then staining at the rim was not observed, but the generalized fluorescence was only slightly reduced; unpermeabilized cells were not stained. These results indicate that HMWP is a component of the cortical cytoplasm of HeLa cells. Possible functions of cortical HMWP are discussed briefly.