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We have previously described a protein called “insertin” that binds strongly to barbed ends of actin filaments and permits polymerization of actin filaments by insertion of actin monomers between the barbed ends and barbed end-bound insertin. We determined the amino acid sequence of insertin and found that the primary structure of insertin is almost identical to amino acid residues 862 to 1212 of the actin-binding protein tensin.

Binding of biological phosphate compounds to actin was investigated by the effect of these compounds on the critical concentration of the pointed ends of gelsolin-capped actin filaments. According to this assay millimolar concentrations of glucose 6-phosphate and the bisphosphorylated sugars fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, glucose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate and 2,3-bisphosphoglycerate were found to associate with actin. Glycerophosphoinositol phosphates bound to actin if they were present in millimolar concentrations, and if carbon atom 4 of the inositol ring was phosphorylated and carbon atom 5 was free of phosphate. Also phosphoserine and phosphotyrosine were found to interact with actin. Most of the actin-binding compounds stabilized actin filaments by decreasing the critical concentration suggesting that these compounds had a higher affinity for the subunits along actin filaments than for actin monomers. However, 2,3-bisphosphoglycerate and fructose 2,6-bisphosphate increased the critical concentration probably because these sugar phosphates bound to actin monomers thereby inhibiting actin polymerization.

Insertin, a protein purified from chicken gizzard smooth muscle, has been shown to retard but not to inhibit actin polymerization at the barbed ends of actin filaments. This effect has been explained by a model in which insertin remains bound to the barbed ends of actin filaments and new actin molecules are inserted into filaments between the barbed ends and barbed end-bound insertin molecules. In this paper we discuss the mechanism of the insertion reaction on a molecular level. A number of simple models were devised and were judged by their agreement with available experimental data. In one class of models insertin was assumed to dissociate from filament ends and to re-associate with the ends. Actin monomers would then bind to a filament end between a dissociation and an association reaction of insertin. In one of the two proposed models in this class insertin binds to an ATP-containing terminal subunit with higher affinity than to an ADP-containing terminal subunit. Dissociation of insertin is brought about by ATP hydrolysis at the terminal filament subunit. Insertion was then thought to re-associate with a filament end following binding of an ATP-containing actin monomer to the filament end. In the other of the two models' insertin was assumed to occur in two conformations which bind to filament ends with different affinities. Association and dissociation of insertin is caused by interconversion between the two forms of insertin. Both models turned out to be incompatible with experimental data. All types of models in which retardation of actin polymerization is brought about by dissociation and re-association of insertin with filament ends can be excluded by a common argument. As 10 nM insertin retards polymerization of 2 microns monomeric actin with maximal efficiency, the rate constant of binding of insertin to a filament end must be considerably higher (greater than 2 microM/10 nM = 200-fold). As the rate of association of actin with a barbed end is almost diffusion-controlled, assembly of insertin with a filament end would have to exceed the rate of a diffusion-controlled reaction. In the other class of models it was assumed that insertin remains permanently bound to filament ends during association or dissociation of an actin molecule and to move towards the terminal subunit of filaments. These models are compatible with experimental data. Thus, models are favoured where insertin remains bound to filament ends during polymerization and depolymerization of actin.

An actin polymerization-retarding protein was isolated from chicken gizzard smooth muscle. This protein copurified with vinculin on DEAE-cellulose and gel filtration columns. The polymerization-retarding protein could be separated from vinculin by hydroxylapatite chromatography. The isolated polymerization-retarding protein lost its activity within a few days, but was stable for weeks when it was not separated from vinculin. We termed the polymerization-retarding protein “insertin”. Because of the instability of the isolated insertin, we investigated the effect of insertin-vinculin on actin polymerization. Insertin-vinculin retarded nucleated actin polymerization maximally fivefold. Polymerization at the pointed ends of gelsolin-capped actin filaments was not affected by insertin-vinculin, suggesting that insertin-vinculin binds to the barbed ends, but not to the pointed ends, of actin filaments. Retarded polymerization was observed even if the actin monomer concentration was between the critical concentrations of the ends of treadmilling actin filaments. As at this low monomer concentration the pointed ends depolymerize, monomers appeared to be inserted at the barbed ends between the terminal subunit and barbed end-bound insertin molecules. Insertin-vinculin was found not to increase the actin monomer concentration to the value of the pointed ends. These observations support the conclusion that insertin is not a barbed end-capping protein but an actin monomer-inserting protein. According to a quantitative analysis of the kinetic data, all observations could be explained by a model in which two insertin molecules were assumed to bind co-operatively to the barbed ends of actin filaments. Actin monomers were found to be inserted between the barbed ends and barbed end-bound insertin molecules at a rate of about 1 × 106 m −1 s −1. Insertin may be an essential part of the machinery of molecules that permit treadmilling of actin filaments in living cells by insertion of actin molecules between membranes and actin filaments.