Biomedical subjects

C Roustan

Publications and source records attributed to C Roustan.

At least 37 records · Page 2Linked to original sources

PubMed1995

Sequences of actin implicated in the polymerization process: a simplified mathematical approach to probe the role of these segments.

Regulation of actin polymerization and depolymerization is essential for the functions of actin in non-muscle cells and is mediated by a large number of heterologous actin-binding proteins which questions their true impact on the polymerization process. As a model, we report here the modulating effect of monospecific antibody fragments (Fab) as in vitro effectors on actin polymerization kinetics. Polymerization curves were obtained through fluorescence measurements. They were fitted using analytical equations derived from classical models describing the actin polymerization process with the aim of identifying kinetic steps potentially altered by the effectors. The study was limited to three short segments bore by the 300-328 sequence which is located in actin subdomain 3 and implicated in one of the monomer-monomer interfaces. We observed that antibodies which inhibited actin polymerization reacted with both G- and F-actins, modulated both nucleation and elongation steps, enhanced actin monomer dissociation from the filament and apparently did not act as capping or sequestering proteins. Among the antibody populations specific for a restricted and selected sequence in subdomain 3 of actin (sequence 300-326), only those directed to epitopes located near Met 305 and 325 were effective. In contrast, antibodies directed towards the alpha-helix located between the two preceding epitopes had no effect. All the results analyzed here emphasize the important role of some discrete regions and their conformational state in regulation of the interconversion between monomeric and polymeric actins which could be controlled in different ways by the various actin-binding proteins.

Actins↗

PubMed1995

Isolation and properties of white skeletal muscle alpha-actinin from sea-trout (Salmo trutta) and bass (Dicentrarchus labrax).

Fish alpha-actinin purified from sea-trout and bass white muscle by means of two different extraction procedures was used to investigate the eventual presence of different muscle isoforms in Z-disks. These fish alpha-actinins have the same apparent molecular weight (100 kDa) and the same isoelectric point (pI = 5.6), and also have a total antigenic identity towards anti-bass and anti-chicken alpha-actinin antibodies, suggesting a single molecular species. The role of fish alpha-actinin as an anchorage site for thin actin filaments and elastic titin filaments in Z-bands was studied. Despite conservation of the actin-binding site, fish alpha-actinin has a better actin-binding ability (kD = 0.3 microM) than chicken smooth muscle alpha-actinin (kD = 1.6 microM). Several other structural and functional characteristics of fish alpha-actinin were also studied: conservation of sequence and domain structure, the role of divalent ions (Ca2+, Mg2+) and the dielectric constant of the medium in alpha-actinin-actin interaction. Although the reason for fish white muscle alpha-actinin's close affinity to actin was not clearly established, our results suggested that the physicochemical environment of the Z-filaments in Z-disks might be crucial.

Actinin↗

PubMed1994

Characterization of the actin binding site on smooth muscle filamin.

We have isolated an NH2-terminal fragment of filamin (M(r) = 70,000) after digestion with Staphylococus aureus V8 protease. This fragment was shown to interact with filamentous actin in cosedimentation assays. Using cross-reactive anti-peptides antibodies directed against the strongly conserved 27-mer sequence of alpha-actinin, already implicated as an actin binding site (Kuhlman, P. A., Hemmings, L., and Critchley, D. R. (1992) FEBS Lett. 304, 201-206), we obtained evidence suggesting that the homologous sequence of filamin (121-147 sequence) is the major element in the interaction with actin. In particular, we used enzyme-linked immunosorbent assay experiments, in conjunction with a synthetic peptide approach, and found that the hydrophobic part of the 27-mer peptide (141-147 sequence) is largely involved in actin binding. Thus, the filamin sequence 121-147 (or the alpha-actinin sequence 108-134) and the actin counterpart composed of residues 112-125 and 360-372 (we have already implicated) could constitute the main interface between actin and these cytoskeletal proteins. However, the divergent behavior of filamin and alpha-actinin toward conformational changes of actin argues in favor of distinctive interfaces. Finally, the ionic strength dependence of the filamin-actin interaction, in contrast to that with alpha-actinin, strongly suggests that, besides hydrophobic interactions conferred by the 27-mer sequence, more hydrophilic region(s) of filamin participate(s) in the binding.

Actinin↗

PubMed1993

Definition of the EGTA-independent interface involved in the serum gelsolin-actin complex.

The gelsolin-actin complex in the presence of Ca2+ revealed at least three interacting sites on the gelsolin molecule located in the S1, S2-3, and S4-6 domains. In the presence of EGTA, the N-terminal domain of gelsolin is known to be involved. However, the corresponding site on the surface of actin is poorly defined. The present result locates the Ca(2+)-independent plasma gelsolin-binding site on the actin surface. Natural and synthetic actin peptides were tested for their possible interaction with gelsolin and monitored by fluorescence anisotropy measurements and e.l.i.s.a. The interface was thus located within the 360-372 actin sequence near the C-terminal extremity. In addition, we used a chymotryptic digest of gelsolin and determined that its N-terminal domain (S1) was implicated in this interface. We conclude that the interaction of the 41-126 region of plasma gelsolin is the counterpart of the 360-372 sequence in subdomain 1 of actin.

Actins↗

PubMed1993

Localization of two myosin-subfragment-1 binding contacts in the 96-132 region of actin subdomain-1.

Many direct observations and indirect experimental approaches have pin-pointed two segments (sequences 1-28 and 360-372) in actin subdomain-1 which bind to myosin subfragment-1. In a previous investigation [Labbé, J. P., Méjean, C., Benyamin, Y. & Roustan, C. (1990) Biochem. J. 271, 407-413], we have observed competition between myosin subfragment-1 and anti-actin antibodies specific to epitopes including Thr103. A multisite interface model has also been proposed to take into account myosin-head binding to the N-terminal and C-terminal regions and to more central 40-113 sequence of actin. In the present study, two limited actin segments encompassing residues 96-103 and 112-125 were identified as myosin-head-binding sites. Myosin subfragment-1 competed for monomeric actin with the antibodies directed against sequences 96-105 and 114-120 and its binding to the tryptic 96-113 and synthetic 112-125 actin peptides was prevented by magnesium pyrophosphate but not by calcium pyrophosphate. In the presence of ATP-Mg2+, myosin subfragment-1 was dissociated by filamin from its complex with monomeric actin or with peptide 105-120. Contact points of filamin on actin were previously located in the 105-120 and 360-372 actin sequences [Méjean, C., Lebart, M. C., Boyer, M., Roustan, C. & Benyamin, Y. (1992) Eur. J. Biochem. 209, 555-562]. The in vitro inhibitory effect of filamin on actin-activated Mg2+-ATPase would thus be explained by this competition. Furthermore, the (27-kDa-50-kDa-20-kDa) trypsin-split myosin subfragment-1 which could no longer be activated by actin, did not bind at all to the two sites located in the 96-125 region, but it still interacted with the 360-372 segment. Our results regarding the position of the myosin head on actin monomers in rigor conditions provide evidence on the presence of two topologically independent contact points in the myosin-head/actin interface. One group exposed residues in the 1-7, 21-29, 77-95 and 96-103 actin segment, another, on the opposite side of subdomain-1, included residues from 112-125 and 360-372 sequences.

Actins↗

PubMed1993

Effects of different enzymic treatments on the release of titin fragments from rabbit skeletal myofibrils. Purification of an 800 kDa titin polypeptide.

In myofibrils, titin (also called connectin) molecules span from Z line to M line and constitute a third filament system containing an elastic domain in the I band. This giant protein is particularly sensitive to proteolysis in situ. Treatment of rabbit skeletal myofibrils with exogenous proteinases induces a release of titin fragments, which are detected in the soluble myofibrillar fraction. The cleavage of titin occurs at specific points localized at the proximity of Z line and could lead to a concomitant release of alpha-actinin.

Animals↗

PubMed1993

Further characterization of the alpha-actinin-actin interface and comparison with filamin-binding sites on actin.

The interaction between alpha-actinin and actin was further characterized using natural and synthetic peptides of actin together with anti-actin antibodies of known specificity. We demonstrated that two alpha-actinin binding sequences on actin are located within residues 112-125 and 360-372. Each peptide was shown to directly bind alpha-actinin and was able to dissociate the alpha-actinin-actin complex using solid phase binding assays and cosedimentation experiments. Taking into account the three-dimensional structure of actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37-44), we postulate that these two segments, proximal in the actin structure, are part of the same site. In addition, we compared these two segments with those recently found for filamin (Méjean, C., Lebart, M. C., Boyer, M., Roustan, C., and Benyamin, Y. (1992) Eur. J. Biochem. 209, 555-562), Egan, S., Stewart, M., Stossel, T. P., Kwiatkowski, D. J., and Hartwig, J. H. (1990) J. Cell Biol. 111, 1089-1105), and concluded that the two actin-binding proteins interact with closely spaced or overlapping but not identical sequences of actin subdomain 1.

Actinin↗

PubMed1992

Localization and identification of actin structures involved in the filamin-actin interaction.

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.

Actins↗

PubMed1992

Localization of a myosin subfragment-1 interaction site on the C-terminal part of actin.

The actin-myosin head complex in the rigor state reveals several high-affinity sites on the actin molecule in sequences 18-28 and 40-113. In the presence of Mg(2+)-ATP, participation of the actin N-terminal 1-7 sequence is known to occur. The proximity of the C-terminal region of actin to the A1 light chain of the myosin head [S-1(A1)] (where S-1 is myosin subfragment-1) was described previously. We observed that C-terminal antigenic structures located near Met-305, Met-325 and Met-355 and the C-terminal end (Cys-374) of actin are markedly modified in the presence of S-1(A1), S-1(A2) and scallop S-1 and in the absence of Mg(2+)-ATP. This seems to rule out any important specific involvement of the A1 light chain in the described conformational changes. An S-1-binding site was located in this actin C-terminal region by testing the tryptic CB9 peptide (360-372 sequence) previously implicated in the A1 light chain interaction. This peptide was able to bind well to S-1(A1), S-1(A2) and scallop S-1, but not in the presence of Mg(2+)-pyrophosphate. These results strengthen the hypothesis of a multisite interface between S-1 and actin located in the actin subdomain I.

Actins↗

PubMed1992

Interaction in vitro of scallop muscle arginine kinase with filamentous 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, Mg(2+)-ADP and NO3-, which form a dead-end complex with the enzyme. F-actin inhibits the enzyme activity non-competitively with respect to Mg(2+)-ATP. As a function of arginine concentration, the inhibition is of the mixed type, where Km is affected more than Vmax.

Actins↗

PubMed1992

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

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.

Actins↗

PubMed1991

Definition of a Ca2(+)-sensitive interface in the plasma gelsolin-actin complex.

Gelsolin is a Ca2(+)-dependent protein which severs actin filaments, caps their fast-growing ends and promotes nucleation. We report here results that delimit one of the interfaces between serum gelsolin and actin monomer. An actin-derived synthetic peptide (amino acids 305-326 of actin) coupled to a hydrophilic resin was tested for its possible interaction with gelsolin. We selected this sequence because it corresponds to a region implicated in the gelsolin-actin complex in a previous work [Boyer, Feinberg, Hue, Capony, Benyamin & Roustan (1987) Biochem. J. 248, 359-364]. We showed that this actin sequence is located at the surface of the actin molecule and observed a Ca2(+)-sensitive binding of gelsolin to this actin-derived peptide. In addition, by using a chymotryptic digest of gelsolin, we reported that only the C-terminal half of gelsolin interacts with the actin-(305-326)-peptide. These results that the Ca2(+)-sensitive interface includes both amino acids 305-326 of actin and probably amino acids 660-738 of gelsolin.

Actins↗

PubMed1991

Purification and properties of two molecular forms of arginine kinase from the adductor muscle of the scallop, Pecten maximus.

1. Two molecular forms of arginine kinase, AK1 and AK2 have been purified from the adductor muscle of the scallop, Pecten maximus. AK2 was retained on a DEAE-cellulose column at pH 7.5, but AK1 was not. 2. Both forms were monomeric (mol. wt. approximately 42,000) and showed the same pH optimum (7.5-8.0) in the direction of phosphoarginine synthesis. 3. AK1 had slower electrophoretic mobility at pH 8.3 towards the anode, higher lysine content, lower glutamate content, lower Km for L-arginine and higher Km for Mg(2+)-ATP than AK2. Unlike AK1, AK2 was strongly inhibited at high concentrations of Mg(2+)-ATP. 4. Both molecular forms cross-reacted with antisera raised against native as well as performic acid-oxidized lobster muscle arginine kinase. However, AK1 showed a greater affinity than AK2 to anti-lobster arginine kinase antibodies, particularly to those raised against the native enzyme.

Amino Acids↗

PubMed1991

Sarcomeric disorganization in post-mortem fish muscles.

1. The post-mortem evolution of protein pattern in fish striated muscle was followed by SDS-PAGE, after different conditions of storage time and temperature. 2. Sarcoplasmic and sarcomeric fractions were analyzed respectively by low and high ionic strength extractions of fish muscle samples. 3. No evident modification of electrophoretic patterns was observed during the pre-rigor mortis period. 4. The high mol. wt proteins titin and nebulin were highly sensitive to proteolysis during the rigor mortis period. 5. Myosin extraction was predominantly influenced by the storage temperature. The myosin content of the extracts decreased during the rigor mortis period at storage temperatures greater than 8 degrees C. 6. alpha-Actinin was very resistant to proteolysis, but could be released from Z-disc structure during post-mortem aging.

Actinin↗

PubMed1990

Localization of a new alpha-actinin binding site in the COOH-terminal part of actin sequence.

The interaction of filamentous actin with alpha-actinin, an actin cross-linking protein, is well established. On the other hand, monomeric actin-alpha-actinin interaction has been a subject of controversy. In this report, we have characterized the interaction of monomeric actin, coated on plastic plates under conditions of non-polymerization, with alpha-actinin in presence of magnesium. Using specific polyclonal anti-actin antibodies, with the whole molecule or purified peptides, we have localized two sites of interaction on action molecule: one near Thr-103 and a new one in the twenty last amino acids.

Actinin↗

PubMed1990

Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes.

Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction.

Actins↗

PubMed1990

Immunological comparison between albumins of three species of mice (genus Mus).

Three closely related species of short-tailed mice (Mus musculus musculus, M. spretoides and M. spicilegus) were tentatively discriminated using immunological techniques based on albumin cross-reactivity. Different fractionations of crude albumin antisera allowed the recovery of antibody populations specific to the M. m. musculus albumin, whereas antibody population differences do not seem to exist between M. spicilegus and M. spretoides. Moreover, immunoreactivities tested with native and S-carboxymethylated albumins revealed that species-specific antibodies correspond to antigenic determinants depending on the amino acid sequence (sequential determinants). The observed immunological differences are related to species divergence and albumin sequences.

Albumins↗

PubMed1989

Antigenic probes locate binding sites for the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase on the actin monomer in microfilaments.

The topology of the interfaces between actin monomers in microfilaments and three glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase) was investigated using several specific antibodies directed against precisely located sequences in actin. A major contact area for glyceraldehyde-3-phosphate dehydrogenase was characterized in a region near residue 103. This interaction altered, by long-range conformational changes, the reactivity of antigenic epitopes in the C-terminal part of actin. The interface between actin and aldolase appeared to involve a sequence around residue 299 in the C-terminal region of actin. The interaction of phosphofructokinase, in contrast, modified the reactivity of all antibodies tested. Finally, the phosphagen kinases arginine kinase and creatine kinase showed no interaction with the microfilament.

Actin Cytoskeleton↗