Actin filament capping protein from bovine brain.

Abstract

An actin filament capping protein has been purified from bovine brain. The protein has a native mol. wt. of 63 kilodaltons (kd) with subunits of 36 kd and 31 kd and is globular in shape. It nucleates actin polymerization, inhibits filament elongation and filament interactions, and decreases the steady state viscosity of F‐actin in substoichiometric amounts (molar ration 1:1000). In addition, the protein increases the critical concentration for actin polymerization. Neither Ca2+ nor calmodulin affects it action. All these effects can be explained by the binding of the protein to the ‘barbed’ end of actin filaments leading to a blockade of actin monomer addition at the preferred growing end. This is directly demonstrated by electron microscopy. Concerning the polypeptide composition, Ca2+‐independence, mode, and stoichiometry of actin interaction, the protein is similar to the capping protein, previously isolated from Acanthamoeba.

12-fold difference between the critical monomer concentrations of the two ends of actin filaments in physiological salt conditions.

“…Because the assembly reaction at the barbed end-is fast, a few uncapped filaments are able to consume the monomers released from the pointed ends efficiently (4, 5). These experiments confirm the previously reported depolymerizing effect of molecules capping the barbed end (2,8,13,14). However, they also show that a quantitative statement about the critical concentration of the pointed end 4922 The publication costs of this article were defrayed in part by page charge payment.…”

“…The critical concentration of the pointed end could not be measured directly because association and dissociation at the barbed end occurs much faster than at the pointed end. Therefore, events occurring at the pointed end of filaments are difficult to observe without interfering with the dynamics of the barbed end (5, 6).In this study this problem was overcome by capping the barbed filament end with an actin filament capping protein from bovine brain (7,8). Capped filaments labeled with 7-chloro-4-nitrobenz-2-oxa,1,3-diazole were mixed with various concentrations of unlabeled monomeric actin.…”

FtsZ filament capping by MciZ, a developmental regulator of bacterial division

“…In contrast, cappers block the ability of a subunit to bind at one end but leave the other end available to bind to a filament and block further assembly. Cappers thus tend to exhibit effects even at substoichiometric concentrations (32,33). To study the mechanism of MciZ inhibition, we monitored the effect of various concentrations of MciZ on the polymerization of FtsZ by light scattering.…”

‘Cap 90’, a 90‐kDa Ca2+‐dependent F‐actin‐capping protein from vertebrate brain

“…RESULTS 3.1. The p~r~~~~t~~~ of the 5%klla capping protein from bovine bruin The fractionation on DEAR-cellulose of an extract from bovine brain cortex was performed essentially as in [7]. The fractions of the DEAE column which reduced the viscosity of actin solu-226 tions and eluted with a step-gradient between 75 mM and 150 mM NaCl were pooled, dialyzed against IDE-buffer (20 mM imidazole, pH 7.2, 1 mM EGTA, 0. directly applied to a DNase-I column (1.5 x 6 cm of affi-gel 10, BioRad, to which 200 mg of DNase-I (Sigma D4763) had been covalently coupled) equilibrated with: IDE buffer containing 5 mM CaCl2.…”

“…This may be at least in part due to the fact that in these cells, like in red blood cells, actin exists only as short filaments or even as oligomers which are difficult to visualize. Our search for capping proteins in the nervous system led to the discovery of a first capping protein, which was isolated from bovine brain cortex [7], This protein has a native MX-value of 63 kDa, consists of two polypeptides of 36 kDa and 3 1 kDa and caps the fast-growing end of actin filaments in a Caz+-insensitive manner. Recently, the isolation of a 90-kDa Ca2+-dependent actin fragmenting protein from neuronal tissue was reported [8].…”