Actin-fragmin interactions as revealed by chemical cross-linking

Sutoh, Kazuo; Hatano, Sadashi

doi:10.1021/bi00350a024

Cited by 53 publications

(36 citation statements)

References 23 publications

(58 reference statements)

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“…The conclusion is consistent with the obscrvations that CT 17N showed high-affinity Ca-insensitive binding to G-actin [S] and that CT 17N retains Caresistance to chelation by EGTA upon binding to actin [27]. Other barbed end-capping proteins such as severin [28], Acmrlwnweba profilin [29] and fragmin [30] are also shown to bind at the carboxyl-terminal area. Rcgarding the sequence similarities at actin binding regions among the capping proteins, including probably adseverin [31], it is tempting to generalize that the capping proteins bind to the carboxy!-terminal segmer?t of the actin molecule topologically equivalent to the EGTA-resistant actin between the two actin molecules found at the barbed end of an actin filament.…”

Section: Discussionsupporting

confidence: 77%

The amino‐terminal fragment of gelsolin is cross‐linked to Cys‐374 of actin in the EGTA‐resistant actin‐gelsolin complex

1992

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Section: Discussionsupporting

confidence: 77%

The amino‐terminal fragment of gelsolin is cross‐linked to Cys‐374 of actin in the EGTA‐resistant actin‐gelsolin complex

1992

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“…Sutoh and Hatano [18] suggest that the unusual amino acid sequence made this segment a favorable anchoring site for various types of actin-binding proteins. These include myosin [19], depactin [20], cofilin [36] and fragmin [18]. Now we can add to this list plasma gelsolin and probably cytoplasmic gelsolin.…”

Section: Discussionmentioning

confidence: 99%

“…Since the first step of severing action by plasma gelsolin may start with the binding of a plasma gelsolin molecule to F-actin filaments at their peripheries, it is possible that the crosslinking reaction by EDC fixes the initial actin -plasmagelsolin complex, which is directly associated with the severing action of plasma gelsolin. It is pertinent to note that the NH2-terminal segment is thought to be situated outside the F-actin filaments in view of its involvement in binding myosin [18].…”

Section: Discussionmentioning

confidence: 99%

Plasma-gelsolin-binding sites on the actin sequence

1987

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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...

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“…12,13) In addition, the 42-merP may contain a binding site for actin-binding proteins and regulate actin functions. [14][15][16] These observations suggest that the truncated actin may impair neutrophil function. In addition, the 42-merP itself may have adverse effects on neutrophil function.…”

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confidence: 86%