Identification of G actin-binding proteins in rat tissues using a gel ovarlay technique

Abstract

Actin-binding proteins were assayed in various tissues using an 125I-actin overlay procedure. Four major G actin-binding proteins of 90 000, 65 000, 58 000 and 40 000 M r have been identified. The 90K protein is present in all tissues and binds labelled actin in a calcium-sensitive manner with binding increasing 3–4-fold in the presence of Ca 2+. The distribution of the 58K and 65K protein which are not Ca 2+-sensitive was more variable. These proteins were present in different ratios in different tissues. 125I-actin binding to all four actin-binding proteins is specific and can be displaced by preincubation of the gels with unlabelled actin. The interaction of actin with these proteins does not appear to involve ionic forces, since binding is not diminished by varying the salt concentration. Skeletal muscle glycolytic enzymes, the lens crystallins and the histones also bind 125I-actin. This binding cannot be displaced by preincubation with unlabelled actin and is presumably non-specific. The calcium sensitivity of two highly purified actin-binding proteins, the 90K human platelet protein and villin was compared using 125I-actin. The platelet 90K protein binds actin at less than 10 −7 M free calcium, but detectable binding to villin does not occur below 10 −6 M free calcium. The ubiquity of these actin-binding proteins is clear and we conclude that the calcium-sensitive 90K actin-binding protein in all of these tissues is the same as the platelet protein.