Antigenic probes locate a serum-gelsolin-interaction site on the C -terminal part of actin

Scallop muscle arginine kinase binds to F-actin from mollusc and rabbit muscle in vitro. One site of interaction appears to be located in residues 305 -325 of a C-terminal fragment (residues 285 -375) of actin. The binding is hindered in the presence of arginine, Mg2+-ADP and NO;, which form a dead-end complex with the enzyme. F-actin inhibits the enzyme activity non-competitively with respect to Mg2+-ATP. As a function of arginine concentration, the inhibition is of the mixed type, where K,,, is affected more than V,,,,,.

Section: Resultssupporting

confidence: 86%

“…Actin fragments 1-226 and 285-375 were obtained as previously described (Boyer et al, 1985;Lebart et al, 1990). The peptide (residues 355-375) derived from CNBr cleavage of actin was purified by HPLC on a C4 column (Lebart et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

Interaction in vitro of scallop muscle arginine kinase with filamentous actin

Reddy

Houmeida

Benyamin

et al. 1992

“…1). Specific anti-actin antibodies directed against epitopes including the residues Ile76 and Thrl03 {anti-[actin(40-113)] antibodies} (Mkjean et al, 1988), the residue Val201 {anti-[actin(l95-206)] antibodies} (Mkjean et al, 1988) or the residues Met299, Met325 and Met355 (anti-[actin(285 -375)] antibodies} (Boyer et al, 1987) were selectively purified using Sepharose 4B immunoadsorbents. These immunoadsorbents were mhde with the actin fragments actin(40 -11 3), actin(1-226) and actin(285 -373, respectively.…”

Section: Immunological Techniquesmentioning

confidence: 99%

Localization and identification of actin structures involved in the filamin – actin interaction

Méjean

Lebart

Boyer

et al. 1992

The interface between gizzard filamin and skeletal muscle actin was located on the actin monomer. Conserved sequences 105 -120 and 360 -372, in the actin subdomain 1 near the myosin binding sites, were involved in this interaction. The corresponding peptides for these sequences were each found to bind filamin and compete in the actin-filamin interaction. When these two peptides were used together in the presence of filamin and filamentous actin, they dissociated sedimentable complexes formed by these two proteins.

“…The amount of specific antibodies elicited (0.3 mg/ml) remained constant after 20 days of immunization (data not shown). Anti-[actin (285 -375) sequence] antibodies were purified by affinity chromatography as previously reported [20,211 and their specificity determined [22,231. These antibodies were specific for three epitopes in skeletal muscle actin, near residues 305, 325 and 355.…”

Section: Antibodiesmentioning

confidence: 99%

Localization of a vitamin‐D‐binding protein interaction site in the COOH‐terminal sequence of actin

Houmeida

Hanin

Constans

et al. 1992

The serum vitamin D binding protein is the carrier of vitamin D and its derivatives in the plasma. One of the known roles of this protein is to sequester monomeric actin in the blood, therefore implicating this protein in actin elimination. However, its binding site at the surface of actin is poorly delimited. We report here the results of a study which locates, using several actin fragments together with immunological probes, a vitamin D binding protein site near the COOH-terminal extremity.Thus, the interface is delimited by the sequence 360-372 in subdomain I of actin.The vitamin D binding protein (DBP), also called Gc (group component), is a plasma protein found in humans and other mammalian species [l]. It is a monomeric protein with an a2-globulin mobility, secreted by the liver [2, 31 and has a relative molecular mass (M,) of 52000 [3]. The first recognized function of DBP was the plasma transport of vitamin D and other related derivatives [4]. In addition, other previous studies [5] have shown that the serum affords actin-depolymerizing activity. This property was further ascribed to the conjugate effects of gelsolin and DBP [6]. However, whereas gelsolin can cause severing of actin filaments, DBP can sequester actin monomers in a 1 : l complex and thereby leads to actin depolymerization. The in vivo occurrence of a DBP-actin complex was detectcd, at a very low level, in the serum of healthy patients [7]. A drastic increase in the amount of actin and its DBP complex following tissue injury (hepatic necrosis for example) or trauma have also been reported [7, 81. Thus, one of the physiological roles of DBP would be to avoid actin filament formation, which would occur at normal salinity of the serum, and thus accelerate clearing of actin from the circulation [9, 101.In vitru, studies have clearly shown the existence of a very tight G-actin -DBP complex without interaction with F-actin [6]. Other studies have also suggested that the interaction site is restricted to the 227-375 segment of actin [ll], the Cterminal extremity being excluded. Another actin-sequestering protein, profilin, binds with a relatively low affinity at or near the C-terminal extremity of actin [12]. Thus, the two proteins would have different binding sites on actin. In fact, other actin-associated proteins have been shown to be directed to the C-terminal part of the actin sequence. This is true, not only for proteins which prevent actin polymerization, but also for crosslinking proteins such as a-actinin [13], for severing proteins such as gelsolin which interact with the 305 -326 actin sequence [14] and for the myosin subfragment-1 (SI)In view of the potential importance of DBP in the elimination of actin from plasma, we have begun a study aimed at defining the location of the DBP interface in the actin sequence. We located an interaction site of DBP precisely in subdomain I of actin. ~5 1 . MATERIALS AND METHODS Materials7-Chloro-4-nitrobenzo-2-oxa-I ,3,diazole (Nbd-C1) and Niodoacetyl-N-(5-sulfo-l-naphthyl) ethylenediamine (1,5-I-AED...