Plasma-gelsolin-binding sites on the actin sequence

Abstract

Plasma gelsolin was cross-linked to fluorescently labeled actin in order to identify plasma-gelsolin-binding sites on the primary sequence of actin. Plasma gelsolin can be cross-linked to actin with l-ethyl-3- [3-(dimethylamino)propyl]carbodiimide (EDC), resulting in the formation of 1 : 1 and 1 : 2 plasma gelsolin: actin cross-linked complexes with apparent molecular masses of 130 kDa and 180 kDa respectively. The cross-linked complexes were isolated separately, partially digested with cyanogen bromide and subjected to sodium dodecyl sulfate gel electrophoresis to analyze fluorescent fragments. The electrophoretic pattern showed that fluorescent CNBr fragments obtained from a free actin molecule were all found in those obtained from both the complexes. Since the fluorescent probe was attached to an actin molecule through the penultimate cysteine residue (Cys-374), the agreement in the fluorescent patterns indicated that the NH2-terminal CNBr fragments of both the actin molecules were involved in cross-linking with plasma gelsolin. This was also suggested by hydroxylamine cleavage of the two complexes as the cleavage gave the fluorescent 41-kDa fragment which could not be produced unless plasma gelsolin was cross-linked at the NH,-tenninal segment comprising 12 amino acids. Since EDC cross-links an amino group with a carboxyl group only when they are direct contact, the characteristic acidic amino acid residues at the NH2 terminus of actin are suggested to participate in binding plasma gelsolin.As one of the major components of the cytoskeleton, actin plays important roles in cell motility and its spatial organization. In non-muscle cells, actin plays these roles through the process of polymerization and depolymerization, which appears to be regulated by various actin-binding proteins [l]. Plasma gelsolin belongs to a group of the actinbinding proteins that bind to the fast growing (or 'barbed') end of actin filaments [2-41. Its functional and structural similarities to cytoplasmic gelsolin have been demonstrated [5]. Plasma gelsolin is also called actin-depolymerizing factor [6] or brevin [7]. The effect of plasma gelsolin on actin is Ca2 N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide. myosin S-1 binding sites on the primary sequence of actin [19]. He used fluorescent actin labeled selectively at the penultimate amino acid residue (Cys-374) for cross-linking with S-1, digested the isolated cross-linked actin-S-1 complex by CNBr, and subsequently analyzed the fluorescent products by one-dimensional peptide mapping to identify the binding sites for myosin on the actin sequence. The same cross-linking reagent was successfully used for identifying the binding sites of various types of actin-binding proteins showed that plasma gelsolin and actin were crosslinked by EDC to form 1 : 1 and 1 : 2 plasma gelsolin: actin cross-linked complexes. She indicated that the extent of crosslinking was much reduced at low calcium concentration [21]. The formation of 1 : 1 and 1 : 2 cross-linked complexes was also demonstra...

Functional sequences of the myosin head

“…When actin was covalently crosslinked with gelsolin (93kDa) using EDC, the association of one molecule of each protein yielded a product that appeared as a 130-kDa band on an SDS gel at the standard polyacrylamide concentration, and two actin monomers associated with one gelsolin appeared as a 180-kDa product (Rouayrenc et al, 1986;Doi et al, 1987). In these experiments, there is practically complete agreement between the apparent molecular weight obtained after electrophoretic migration and the combined molecular weights of all the proteins.…”

The Arabidopsis Microtubule-Associated Protein AtMAP65-1: Molecular Analysis of Its Microtubule Bundling Activity

“…Cross-linking was performed using EDC (Doi et al, 1987). EDC was added up to the final concentration of 5 mM in a 20-mM solution of AtMAP65-1 in MTSB and incubated at room temperature for 1 h. The reaction was stopped by addition of SDS-PAGE sample buffer, and then the mixture was boiled for 3 min and the sample separated on a 7.5% acrylamide gel.…”

Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase

“…Of note is that very exceptionally, mature protein N termini start with an acidic amino acid, as acidic residues normally retain their initiator methionine. Actins from most eukaryotic species however undergo a unique N-terminal protein modification process, which has been shown to play important roles in the interaction of actin with several actin-binding proteins (63)(64)(65)(66)(67)(68). Nterminal actin modifications, include the post-translational removal of the acetylated initiator methionine, in the case of ␤-and ␥-actin by an unconventional MAP, and the cotranslational removal of the initiator methionine and post-translational removal of the N-terminal acetylated cysteine in the case of ␣-actin, leaving all of them with an acidic residue at their newly exposed N terminus (69 -71).…”