Kinetic evidence for insertion of actin monomers between the barbed ends of actin filaments and barbed end-bound insertin, a protein purified from smooth muscle

Compare and contrast actin filaments and microtubules.

“…This would lead to movement of polymerized actin away from the site while barbed ends remain attached. The protein insertin has been proposed to hold on to growing barbed ends while allowing subunit insertion (Ruhnau et al, 1989). Neither the function nor location of this protein in cells is yet known.…”

Actin and Phosphoinositide Binding by the ActA Protein of the Bacterial Pathogen Listeria monocytogenes

“…After centrifugation in the presence or absence of 4 M F-actin for 15 min at 245,000 ϫ g at 4°C, the pellets were resuspended in 100 l of 50 mM HEPES, pH 7.0, and equal amounts of supernatants and resuspended pellets were analyzed on a 12% SDS gel. Actin polymerization assays were performed as described (26). Briefly, 2 M G-actin, of which 5% was labeled with 7-chloro-4-nitro-2,1,3-benzoxadiazol at lysine 373, was polymerized in 100 mM KCl, 20 mM NaCl, 1 mM MgCl 2 , 0.16 mM CaCl 2 , 0.4 mM ATP, 2.4 mM NaN 3 , 6.6 mM sodium phosphate, and 4 mM triethanolamine/HCl, pH 7.2, in the presence or absence of 0.2 or 2 M ActA.…”

Derivation of insertin

“…Investigations on the mode of action of tensin and fragments of tensin, in particular of the insertin domain, can provide a hint of the significance of the proteolytic fragmentation of tensin. In this respect, it is interesting that for insertin domain preparations, both actin monomer insertion activity and capping activity have been reported [Ruhnau et al, 1989;Lo et al, 1994]. It is conceivable that the mode of action of tensin fragments and insertin domains is modified by proteolytic processing.…”

“…It retards actin polymerization but does not inhibit actin polymerization like capping proteins. According to kinetic and equilibrium analysis, two insertin molecules bind to the barbed end of an actin filament and allow incorporation of actin molecules [Ruhnau et al, 1989;Gaertner and Wegner, 1991]. Amino acid sequence analysis of insertin showed that the primary structure of this molecule is almost completely identical to amino acids (aa) 862-1292 of tensin [Weigt et al, 1992], an actin-binding protein that occurs in focal adhesion sites and that plays an important in role in the early events of association of microfilaments with membranes [Miyamoto et al, 1995].…”

“…This protein has been shown to bind to the barbed ends of actin filaments [Ruhnau et al, 1989]. It retards actin polymerization but does not inhibit actin polymerization like capping proteins.…”