The cytoskeletal components, macrophage actin-binding protein and filamin, were dried from glycerol and examined by low-angle rotary shadowing electron microscopy . Both are elongate, flexible molecules whose general morphology is similar to that of erythrocyte spectrin . Neither actin-binding protein nor filamin binds to spectrin-depleted erythrocyte membranes .Proteins that bind and cross-link actin filaments have been identified . Several of these proteins, including filamin (21), macrophage actin-binding protein (10), and spectrin (5, 12, 13), share certain properties . The three molecules are composed of high molecular weight subunits that can self-associate to form dimers ; they have relatively low sedimentation coefficients, large Stokes's radii, and high frictional ratios. These properties have suggested that the three molecules may exist in solution as prolate ellipsoids or as flexible rods . Electron microscopy of filamin (7) and actin-binding protein (15) with the use of negative stain techniques has tended to support the assignment of a prolate ellipsoid shape to these molecules . However, examination of spectrin after low-angle rotary shadowing indicates that this molecule forms an extended, flexible rod in solution (14) . Because of the striking biophysical similarities between the three proteins, and in the light of our experience with spectrin which binds with high affinity to membranes, we have reexamined the structure and membrane-binding properties of filamin and actin-binding protein .
MATERIALS AND METHODS
Purification of Human SpectrinSpectrin was prepared by well-established methods (I, 2, 3, 18). Low ionic strength extraction of ghosts in l mM Ntris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) 0.1 mM EDTA, pH 7.6 was followed by column chromatography on Sepharose 4B in a buffer consisting of 5 mM NaP0 4 , I MM EDTA, 20 mM KCI, 0.2 mM dithiothreitol, pH 7.6 . For lowangle rotary shadowing, purified spectrin was sprayed onto mica in a buffer containing I mM NaPO,, 0.2 mM EDTA, 20 mM KCI, 0.2 mM dithiothreitol, pH 7.6, and 60% glycerol .Filamin from chicken gizzards was prepared by the method of Wang (20) with some modifications. Diisopropyl fiuorophosphate (iPr2FP) (0 .015% vol/vol) was used in place of phenylmethylsulfonyi fluoride as an inhibitor of serine proteases. Chromatography was carried out on Sepharose 4B (2 .5 x 90 cm column) rather than on Bio-Gel A-15m (Bio-Rod Laboratories, Richmond . Calif.) . DEAF cellulose (DE52, Whatman) chromatography of peak fractions from Sepharose 4B yielded essentially pure filamin, free from contamination by low molecular weight polypeptides. Purified filamin was sprayed for rotary shadowing in a buffer containing 5 mM Tris acetate, 50 mM KCI, pH 7.6, and 60% glycerol.Actin-binding protein prepared from rabbit macrophages by the method of Hartwig and Stossel (10) was a gift from T. Stossel and J. Hartwig (Harvard Medical School). For rotary shadowing, the protein was sprayed in a buffer containing 2 mM Tris acetate, 30 mM KCI, 0.1 ...