End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin.
End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin.
复制标题
末端标签指纹图谱显示肌动蛋白的 N 端和 C 端位于与凝溶胶蛋白的接触位点。
DOI：
10.1021/bi00438a052
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Yin,HL
中科院分区：
文献类型：
--
作者：
Sutoh,K;Yin,HL
Hematology-Oncology Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 Received July 26, 1988; Revised Manuscript Received February 8, 1989 abstract: Gelsolin was cleaved by chymotrypsin or thermolysin into an N-terminal Mr 45 000 fragment (45N) and a C-terminal Mr 38 000 fragment (38C). The N-terminal half was further cleaved into two fragments with Mt 17 000 (17N) and Mr 28000 (28N). These fragments were complexed with actin and cross-linked with l-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) to introduce covalent bonds into their contact sites. The location of these bonds was mapped along the actin sequence by end-label fin-gerprinting with highly sensitive probes for the N-and C-termini of actin. The mapping studies revealed that two gelsolin N-terminal fragments (17N and 28N) were cross-linked with the actin N-terminal segment, while the gelsolin C-terminal fragment (38C) was cross-linked with the actinC-terminal segment. The result indicates that the actin N-and C-terminal segments are in the binding site of gelsolin.(jTelsolin is a Ca2+-and poly (phosphoinositide)-modulated actin binding protein found in the cytoplasm of a large variety of cells and in a variant form in blood plasma (plasma gelsolin or brevin). It severs actin filaments, nucleates actin assembly, and caps the “barbed” end of actin filaments. The multiple actions of gelsolin appear to involve different actin binding domains, and three such domains have been identified thus far by use of proteolytic cleavage [see Matsudaira and Janmey (1988) as a review for the actin-severing proteins]. In thepresence of Ca2+, gelsolin is rapidly cleaved by a variety of enzymes into halves (Kwiatkowski et al., 1985; Chaponnier et al., 1986; Bryan & Hwo, 1986). For example, chymotrypsin cleaves human plasma gelsolin between residues 406 and 407 to generate an N-terminal 45-kDa fragment (CT45N) and an C-terminal 38-kDa fragment (CT38C). 1 CT45N can be further cleaved between residues 149 and 150 to generate CT17N and CT28N. Thermolysin digestion also generates similar fragments, and they have been referred to as TL45N, TL17N, TL28N, and TL38C, respectively. The N-half of gelsolin (eg, CT45N) severs actin filaments as effectively as intact gelsolin, although neither of its subfrag-ments (CT17N or CT28N) does so in isolation. Therefore, severing appears to require a cooperative interaction between the two N-terminal actin binding domains. On the basis of the finding that CT28N can bind stoichiometrically to actin protomers within a filament and its binding is inhibited by poly (phosphoinositides) with a dose-response similar to that observed for severing, it is proposed that severing requires binding of gelsolin through a site within CT28N to the side of actin filaments initially and subsequently binding of another site within CT17N (Yin et al., 1988). A third actin binding domain is located in the C-terminal half of gelsolin (CT38C or TL38C). This site requires Ca2+ to bind actin and does
