CONCENTRATED TRIS SOLUTIONS FOR THE PREPARATION, DEPOLYMERIZATION, AND ASSAY OF ACTIN - APPLICATION TO ERYTHROID ACTIN

High concentrations of Tris are effective in dissociating actin-contai ning complexes, such as the red cell membrane cytoskeleton. A preparat ive procedure for red cell actin is based on the dissociation of the m embrane skeletal complex in a buffer containing 1 M Tris hydrochloride , followed by gel filtration chromatography in the same medium. The ac tin is recovered as the monomer and is fully native, as judged by its critical concentration of polymerization, inhibition of DNase I, stimu lation of myosin ATPase, and the appearance in the electron microscope of filaments, both bare and decorated with heavy meromyosin, and of m agnesium ion-induced paracrystals. The Tris solution causes rapid depo lymerization of F-actin with no denaturation, and the solution of mono meric actin in this medium is stable for many weeks in the cold; conce ntrated Tris is more reliable than guanidinium chloride for the depoly merization of F-actin in the estimation of total actin concentration b y the DNase I inhibition assay. (C) 1995 Academic Press,Inc.